Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens

Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens Saleha Hafeez, Muhammad Asghar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8369029/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted 13 You are reading this latest preprint version Abstract Background Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells (RBCs) are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To help overcome this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs, thereby conferring universal compatibility similar to O-negative RBCs. Results In this study, two combinations based on the placement of the anti-Rh(D) variable chain at the C- or N-terminus were generated, and fusion proteins from the first combination produced the closed (C1) and open (O1) triabodies. Intramolecular cooperative binding affinities of the selected triabody-C1 were predicted computationally using blood group antigens A, B, and Rh(D), with no significant changes observed in binding free energies. The triabody-C1 was expressed in Escherichia coli BL21(DE3) using a two-plasmid system and purified through a three-step process, yielding two fractions, AE3-B1 and AE3-B2. The hemagglutination potential of the triabody was evaluated both visually and microscopically through immunocytochemistry. Visually, no hemagglutination was observed, while microscopically, mixed-field hemagglutination occurred when AB+ and AB − RBCs were incubated with the triabody in fraction AE3-B1, but not when A+ or B+ RBCs were tested. No hemagglutination of AB+ RBCs was detected with the triabody in fraction AE3-B2. ELISA-based cooperative binding assays using free antigens showed that the triabody’s monomers functioned independently, with no changes in binding parameters K D or B max . In contrast, assays with RBC-bound antigens revealed increased K D and decreased B max across successive binding events, particularly when Rh(D) antigens were engaged first. Hemagglutination assays confirmed that triabody-coated RBCs exhibited a complete absence of hemagglutination with anti-A, anti-B, and anti-Rh(D) IgM antibodies, confirming effective antigen blocking. Conclusions The trispecific triabody effectively blocks A, B, and Rh(D) antigens, rendering RBCs with O-negative like universal compatibility and offering a promising strategy to expand the supply of universally transfusable blood, particularly in emergency and resource-limited settings. Trispecific triabody Universal red blood cells Binding cooperativity Antigen blocking Hemagglutination Transfusion compatibility ELISA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Transfusion medicine has long been an integral part of modern medicine, which relies heavily on blood products received from voluntary blood donors. A blood transfusion frequently represents the difference between life and death during medical emergencies, critical surgical procedures, and for patients with long-term medical conditions. A critical factor in achieving a successful transfusion is ensuring blood type compatibility, as mismatches can elicit severe and potentially fatal immune responses [ 1 ]. This compatibility is governed by the presence of specific antigens on the surface of red blood cells (RBCs) and the corresponding antibodies found in the plasma [ 2 ]. RBCs clump together in the presence of these antibodies through a process called agglutination [ 3 ]. The effectiveness of hemagglutination mainly depends on the type of antibody involved i.e., immunoglobulin IgG or IgM. The IgM antibody, a large pentameric antibody, is capable of binding ten antigens [ 4 ]. Its multivalency and considerable size make it highly effective at bridging the distance between multiple RBCs. In particular, its large molecular structure enables it to overcome the natural repulsive forces that keep RBCs apart. The anti-A and anti-B antibodies belonging to the major ABO blood group system are typically IgM. Consequently, transfusions related to ABO blood type incompatibility lead to a rapid and immediate hemagglutination reaction, which can result in a life-threatening acute hemolytic transfusion reaction [ 5 , 6 ]. In contrast, IgG antibodies are smaller and bivalent (able to bind only two antigens) in nature. Due to their compact size, IgG antibodies can sensitize the RBCs but are inefficient at bridging the gap between RBCs to overcome the negative zeta potential [ 7 , 6 ]. Consequently, IgG-mediated hemagglutination is not directly visible and requires specialized techniques such as antiglobulin testing, also known as the Coombs test, which utilizes anti-human antibodies (secondary antibodies) to enable visible clumping of cells [ 8 ]. Antibodies belonging to the Rh blood group system are predominantly IgG in nature. An initial exposure of an Rh(D)-negative individual to Rh(D)-positive blood can lead to the development of anti-Rh(D) antibodies. A subsequent blood transfusion of Rh(D)-positive blood can lead to a delayed hemolytic transfusion reaction [ 9 ]. The combination of the ABO and Rh blood group systems gives us various blood types. Among these, O-negative RBCs hold a special position as "universal donors" due to the absence of A, B and Rh(D) antigens. The lack of immunogenic antigens makes them invaluable in time-sensitive situations where patient’s blood type is unknown, or their true blood type has been masked by a recent blood transfusion [ 10 , 11 , 12 ]. Despite their indispensable role, the supply of O-negative blood is limited and often faces significant depletion due to the constant high demand. This persistent need for a constant supply of blood highlights the need for readily available universally compatible blood [ 13 , 14 ]. This challenge is more prominent and acute in blood deserts. Blood deserts are geographical regions where 75% of blood transfusion cases are deprived of timely and affordable blood components [ 15 ]. The urgent requirement for universal blood has driven the research into alternative solutions, including the development of blood substitutes such as perfluorocarbons (PFCs), hemoglobin-based oxygen carriers HBOCs and RBC substitutes [ 16 , 17 ], enzymatic conversion of A and B blood types to O type blood [ 18 ], masking or blocking the surface antigens to increase compatibility [ 13 ] and the generation of cultured RBCs from stem cells [ 19 ]. While these strategies hold promise, no such alternative solution has been approved for blood transfusion yet. Given the advantages of O-negative blood and its persistent demand, the present study introduces a uniquely designed trispecific triabody as a blocking fragment to address situations where immediate cross-matching is not available and to increase the pool of usable blood for emergency use. Building upon previous antigen blocking strategies [ 13 , 20 ], our research introduces a uniquely designed trispecific triabody to block the A, B and Rh(D) blood group antigens. Besides its blocking capabilities, the triabody’s small and compact size is an important feature that allows it to effectively block antigens without causing hemagglutination, thus ensuring a safe method for making RBCs universally compatible. This could drastically improve the outcomes in underserved regions by providing readily available blood products, especially packed red blood cells (pRBCs) for transfusion when compatible blood is unavailable. Here, we computationally designed and modeled all possible structures of the triabody. Molecular docking was performed between the selected structure of the triabody and blood group antigens A, B and Rh(D), followed by computational prediction of intramolecular cooperative binding affinities. Next, triabody was produced from E.coli BL21(DE3) using a two-plasmid system and purified through a three-step process. The immunocytochemistry (ICC) technique was then performed to microscopically determine its hemagglutination capabilities. To determine intramolecular binding cooperativity, ELISA-based experiments were conducted using both free and RBC-bound antigens. Finally, RBC-bound antigens were blocked by the triabody, and a hemagglutination assay was performed to evaluate its effectiveness as a blocking fragment in preventing RBC hemagglutination. 2. Materials and methods 2.1 Software, web servers and chemicals All the software, web servers, chemicals and molecular biology products used in this study are mentioned in the supplementary materials. 2.2 Protein designing, modeling and refinement The sequences of anti-A and anti-B fragment variables (Fvs) were taken from PDB: 1JV5 [ 21 ], and research article published by Santos-Esteban and Curiel‐Quesada (2001) [ 22 ] respectively. The sequences of anti-Rh(D) variable light (VL) and heavy (VH) chains were taken from accession nos AAC13488.1 (anti-Rh(D) VL chain) and AAC13447.1 (anti-Rh(D) VH chain) respectively [ 23 ]. Both chains of anti-A and anti-B Fvs were joined from VH to VL via a (G 4 S) 3 flexible linker (VH-Linker-VL). Interdomain disulfide bonds were added into each Fv at the site described by Hafeez and Zaidi (2024) [ 13 ] and Zhao et al. (2010) [ 24 ]. After the addition of disulfide bonds, the sequences were arranged into two combinations with two fusion proteins in each, as shown in detail in Fig. 1 . The complete disulfide-stabilized single chain fragment variable (ds-scFv) in each fusion protein of both combinations was joined to the variable chain of the other Fv through the hinge region (EPKSCDKTHTCPPCPAPELLGGP) of the IgG1 antibody [ 25 ]. All proteins were modeled through the I-TASSER web server [ 26 ]. Models with correct domain locations and orientations of VH-VL were joined together through molecular docking using LZerD web server [ 27 ] to form a triabody. Best structures were selected on the basis of the accessibility of the binding site, i.e., all structures in which the binding site was not easily accessible for interaction were discarded. PyMOL 3.1 was used to measure all the molecular dimensions [ 28 ]. Theoretical molecular weights were predicted through the ProtParam tool [ 29 ]. The selected structures were protonated to pH 7.4 using the PrepareProtein tool of the PlayMolecule web server [ 30 ]. The protonation states of amino acid residues (histidine, aspartic acid, and glutamic acid) with pKa values close to pH 7.4 were manually assigned. Intramolecular disulfide bonds were preserved while assigning protonation states. For intermolecular disulfide bonds, hydrogen atoms were removed from sulfur group of cysteine residues and bonds were formed via pdb2gmx with (-ss) and (-merge all) flags on GROMACS 2024.2 software [ 31 ]. Molecular dynamics (MD) simulations were conducted to refine the structures of the triabodies. Simulations were performed for 100 ns using the Amber force field ff99SB at a temperature of 298 K in an explicit water environment, using a TIP3P model. Each protein was solvated in a cubic box and neutralized by adding Na + and Cl − ions at concentration of 0.154 M. 2.3 Modeling of antigens and molecular docking The structures of the trisaccharide antigens (A-trisaccharide and B-trisaccharide) were built using the carbohydrate building tool of the Glycam web server [ 32 ]. The protein sequence of the antigen Rh(D) was taken from a research article published by Conroy et al. (2005) [ 33 ] and modeled using the I-TASSER web server. Molecular docking was performed to study the interaction between blood group antigens and the triabody. The SwissDock web server [ 34 ] was used to study the interaction of trisaccharide antigens with triabody. The grid boxes were focused on the respective antigen binding sites for each trisaccharide. For protein-protein docking, the antigen Rh(D) was first protonated to pH 7.4 as mentioned above in section 2.2 and then docked using the LZerD web server. 2.4 MD simulations For MD simulations, best docked structures in the case of trisaccharide antigens were selected on the basis of a single criterion: the terminal monosaccharides should interact. The selection of the best protein-protein docked structure was based on the location of the complex formation. MD simulations were performed to study the stability of the interactions of each antigen with the triabody under physiological conditions (Temp: 310 K, NaCl: 0.154 M and pH 7.4). Simulations were performed for 150 ns on GROMACS 2024.2 software using the Amber force field ff99SB for proteins. For trisaccharide, the Glycam-06h force field was used to generate topology using the ACPYPE tool of the AmberTools24 [ 35 ]. 2.5 Binding free energy calculations Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations were performed to estimate the binding free energy (ΔG Bind ) of the triabody-antigen complexes using the MmPbAaStat.py script within the g_mmpbsa software tool [ 36 ]. The final 10 ns of MD simulations for each monomer within the triabody was considered the equilibrium phase and was used for energy calculations. To computationally predict the intramolecular cooperative binding affinities, blood group antigens A, B and Rh(D) were sequentially docked in all possible orders: A→B→Rh(D), A→Rh(D)→B, B→A→Rh(D), B→Rh(D)→A, Rh(D)→A→B and Rh(D)→B→A. MD simulations were performed after each docking step as mentioned above in section 2.4 . Binding free energies were then calculated through MMPBSA to determine a trend in cooperative binding affinities. 2.6 Production and purification Triabody molecule was expressed using two expression plasmids: pET-28a (+) and pET-21 (+). These plasmids harbor kanamycin and ampicillin resistance genes respectively. The designs of both recombinant fusion protein constructs are shown in Fig. 2 . For expression in pET-28a (+), the first fusion protein consisting of genes encoding anti-A ds-scFv and the VH chain of anti-Rh(D) dsFv was modified to include the genes encoding the OmpA signal peptide at the N-terminus and a 6X histidine tag at the C-terminus. This gene construct was then inserted between the NcoI and XhoI restriction sites. Similarly, the gene construct of the second fusion protein of the triabody consisting of genes encoding anti-B ds-scFv and the VL chain of anti-Rh(D) dsFv, was placed between the BamHI and XhoI restriction sites of the pET-21 (+) plasmid after the genes encoding the ribosomal binding site (RBS) and OmpA signal peptide at the N-terminal region were added. Both recombinant plasmids were obtained from Twist Bioscience (USA) and subsequently cotransformed into E. coli BL21(DE3) via the heat shock method. [ 37 ]. For large-scale production, a 100 ml overnight culture of E.coli BL21 (DE3) was used to inoculate 5 L of LB media supplemented with (25 µg/ml) kanamycin, (50 µg/ml) ampicillin and (1%) glucose. Incubation was performed at 37°C under constant shaking at 200 rpm until an OD 600 of 0.6 was reached. The culture was cooled to 18°C before slow induction was carried out with 0.25 mM IPTG at 18°C for 20 hours. The cells were harvested following induction and total proteins were extracted via sonication as described by Hafeez and Zaidi (2024) [ 13 ]. The crude extract was collected and filtered through a 0.45 µm syringe filter. Western blotting was performed for the analysis of protein samples. A trispecific triabody was purified via a three-step process. Initially, nickel affinity chromatography (Ni 2+ -NTA) was used to isolate the his-tagged triabodies. This was followed by extraction of different-sized triabodies from the Native PAGE gel and finally by acid-glycine elution to separate the functional triabodies. In the second step the Ni 2+ -NTA purified product was loaded onto a 10% Native PAGE gel. After the run, the protein ladder was used as a reference and the gel was divided into sections corresponding to molecular weights ranging from approximately 70–84 kDa, and 85–100 kDa. Each gel piece was placed in a 2 ml Eppendorf tube containing 0.5 ml Tris-buffered saline (TBS) (50 mM Tris, 154 mM NaCl, pH 7.4) and crushed with a Teflon pestle. The tubes were incubated at 37°C with shaking for 24 hours, followed by centrifugation and resuspension every 3 hours. After 24 hours, the tubes were centrifuged at 5,000 rpm, and the supernatant was analyzed via the western blotting. The third purification step involved the sequential purification of triabodies using blood type A−, B − and O + RBCs. The previously purified product (0.5 ml) was incubated with blood type A RBCs (10 µl) for 30 mins to purify triabodies with functional A binding sites. After incubation, unbound triabodies were removed by washing with TBS buffer A (pH 7.4). The bound triabodies were then detached by using 0.25 ml of Glycine-HCl buffer B (50 mM Glycine, pH 3.5). The final eluate-A was neutralized by using 1 ml of TBS buffer C (pH 8.5). Eluate-A was subsequently used to purify triabodies with functional Rh(D) binding sites. The resulting eluate-Rh(D) eluate served as the starting material for the purification of triabodies with functional B binding sites. The final product was lyophilized and stored at − 70°C. 2.7 Immunocytochemistry (ICC) Immunocytochemistry (ICC) was performed to microscopically determine the ability of the triabody to cause hemagglutination of RBCs. Prior to ICC, RBCs from the AB+, AB−, A + and B + blood types were also visibly checked for hemagglutination reactions before being analyzed microscopically. Briefly, 5 µl of RBCs were incubated for 30 mins under gentle shaking in 0.5 ml of 250 µM triabody solution. To prevent hemagglutination due to primary and secondary antibodies, triabody-coated RBCs were first diluted in TBS buffer (pH 7.4) and then smeared onto glass slides. The smears were air dried and fixed with methanol for 1 min. Next, primary rabbit anti-his tag polyclonal antibodies (1:1000 dilution) were added, and the samples were incubated at room temperature for one hour. Following incubation, the slides were washed with TBS buffer (pH 7.4) before being incubated with a secondary HRP-conjugated caprine anti-rabbit IgG polyclonal antibody (1:10000 dilution). After incubation, the slides were washed with TBS buffer (pH 7.4), and staining was performed via 3,3′,5,5′-Tetramethylbenzidine (TMB) staining method as described by Woiszwillo (1991) [ 38 ]. To prevent hemolysis, the smears were exposed to the working TMB precipitating solution (2 mM TMB, 0.1% H 2 O 2 and precipitating polymers: 0.1% alginic acid, 0.1% methyl vinyl ether/ maleic anhydride copolymer, 0.1% dextran sulfate and 0.3% carrageenan) for just 5 secs and then washed with TBS buffer (pH 7.4). This process was repeated until sufficient color was developed for optical microscopy. Any triabody molecule sample that caused hemagglutination of RBCs was excluded from further studies. 2.8 Determination of intramolecular binding cooperativity of the triabody via free blood group antigens Intramolecular binding cooperativity of the triabody was further investigated through specialized ELISA-based experiments. The aim was to understand how the binding of one antigen affects the binding of other antigens. The experimental sequence began with immobilizing anti-his tag IgG antibodies (10 µg/ml) on protein A/G precoated plates, followed by the addition of purified his-tagged triabodies in twofold serial dilutions ranging from 0.2 nM to 100 µM. In studying the binding order A→B→Rh(D), the first binding event involved saturating the A binding sites with the A-trisaccharide, followed by saturation of the B binding sites with the B-trisaccharide (second binding event), and finally, the Rh(D) binding site was saturated with the antigen Rh(D) (third binding event). An overview of the process is given in Supplementary Figure S1 . The concentrations of A-trisaccharide, B-trisaccharide and antigen Rh(D) used for determining the binding cooperativity of triabody are given in Supplementary Table S1 . The binding parameters, including the dissociation constant K D ​ and maximal binding B max of the B and Rh(D) binding sites were determined via indirect ELISA as described by Syedbasha et al. (2016) [ 39 ] using unbound B-trisaccharide and the antigen Rh(D). To determine the changes in the binding parameters of the B binding site, a neoglycoconjugation process was used in which unbound B-trisaccharide was covalently coupled to bovine serum albumin (BSA) via a homobifunctional ethylenediamine (EDA) linker. This procedure proceeded with the coating of BSA (20 µg/ml dissolved in 0.1 M carbonate-bicarbonate buffer pH 9.6) onto 96-well plates overnight at 4°C. The next day, wells were washed several times with 200 µl of 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6). A freshly prepared solution of EDC and Sulfo-NHS (5 mM each, 1:1 molar ratio) in 0.1 M MES buffer was then added (100 µl per well) and incubated for 30 mins at room temperature with mild shaking. Following incubation, the wells were washed three times with 0.1 M MES buffer (pH 6). For the coupling of EDA to BSA, an excess concentration of EDA (100 mM) was prepared in 0.1 M MES buffer (pH 6). Following the washing step, 200 µl of EDA solution was added to each well and incubated overnight at 4°C. Immediately after incubation, the wells were washed three times with a 0.5 M solution of sodium borate Na 2 B 4 O 7 (pH 8.5). A reductive amination reaction was performed to facilitate the attachment of B-trisaccharide to the BSA-EDA conjugates. The plates were incubated for 96 hours at 56°C with 100 µl of the unbound B-trisaccharide solution (from second binding). Simultaneously, a 50 µl solution, composed of 0.5 M sodium borate Na 2 B 4 O (pH 9), 1.5 M sodium sulfate Na 2 SO 4 , and 0.75 M sodium cyanoborohydride NaBH 3 CN was added. Following incubation, the wells were washed rigorously three times with distilled water. An indirect ELISA was performed in which 100 µL of 10 mg/ml his-tagged triabody was used to detect B-trisaccharide. This was followed by sequential incubation with 100 µl of primary anti-his tagged (1:1000 dilution) and secondary HRP-conjugated antibodies (1:10000 dilution). The TMB substrate reaction was performed for 15 mins and stopped with 0.5 M H 2 SO 4 . Absorbance readings were taken at 450 nm, and the binding parameters K D and B max were determined via nonlinear regression curve fitting using GraphPad Prism Version 9.5.1 [ 40 ] and compared with other binding events. Following the determination of the binding parameters K D and B max of the B-trisaccharide, the antigen Rh(D) was introduced and allowed to saturate the Rh(D) binding sites (third binding event). Unbound antigen Rh(D) were collected and coated on ELISA plates. An indirect ELISA was performed as described above (using triabody), and the binding parameters K D and B max were determined and compared with those of the other binding events. Similarly, the intramolecular binding cooperativity of triabody was studied in other orders A→Rh(D)→B and B→A→Rh(D), B→Rh(D)→A, Rh(D)→A→B and Rh(D)→B→A. 2.9 Determination of intramolecular binding cooperativity of the triabody via RBC-bound blood group antigens To determine the intramolecular binding cooperativity of a triabody designed to block blood group antigens on RBCs, an ELISA-based experiment was performed. In this experiment the CDR-grafted humanized nanobodies (VHH) of anti-A, anti-B and anti-Rh(D) were designed and used as blocking fragments of antigens A, B and Rh(D) on RBCs. To make the CDR-grafted humanized nanobodies, the CDR sequences of the anti-A, anti-B and anti-Rh(D) scFvs were taken and grafted onto the framework sequence of the camelid nanobody (Supplementary Figure S2). After that, the whole sequence was humanized via llamanade web server [ 41 ]. Nanobodies were then produced from E.coli BL21(DE3) and the effect of changes in pH on the interaction of complexes of both triabody with antigens and nanobodies with antigens was determined. The criteria for the selection of nanobodies as blocking fragments were as follows: 1) the first blocking nanobody must be sensitive to slight fluctuations in pH compared with the second blocking nanobody, and 2) pH-dependent dissociation must occur at a different pH from that of the triabody. The pH values required for the complete dissociation of several blood group nanobodies are given in Supplementary Table S2. The selected blocking nanobodies (Supplementary Table S3) were then resuspended in TBS buffer (pH 7.4) in a concentration of 10 mg/ml. To study the order, A→B→Rh(D) (an overview of the process is given in Supplementary Figure S3), AB + RBCs (1 µl) were immobilized on ELISA plates using 0.3% glutaraldehyde as previously described by Koganei (2007) [ 42 ] and incubated in 100 µl of a solution of anti-B VHH (first blocking) for one hour under slow shaking. A similar blocking step was followed for anti-Rh(D) (second blocking) with one modification: an anti-Rh(D) scFv was used instead of a nanobody in the binding orders of A→B→Rh(D) and B→A→Rh(D). In other binding orders anti-Rh(D) VHH was used. Unbound blocking fragments were removed by washing several times with TBS buffer (pH 7.4). After blocking antigens B and Rh(D), the A binding sites of triabody were allowed to interact with antigens A on the RBC by incubating 100 µl of triabody (100 µM in TBS buffer, pH 7.4) for 30 mins under slow shaking. After incubation, the wells were washed three times with TBS buffer (pH 7.4). Following washing, first blocking fragment i.e., anti-B VHH was removed by washing three times with TBS (pH 7.83) buffer. After washing, 100 µl of TBS buffer (pH 7.4) was added, and the triabody’s B binding sites were allowed to interact with antigens B on the RBC surface. After antigen B was saturated, 100 µl of his-tagged anti-B ds-scFv (the concentrations and dilutions of all three ds-scFvs were the same as those of the triabody, as provided in Supplementary Table S1 ), which had the same sequence as the triabody’s anti-B ds-scFv, was added, and allowed to interact with unbound antigen B or to displace weakly bound B binding sites for 30 min under constant shaking. Unbound anti-B ds-scFv was taken and indirect ELISA was performed using primary and secondary antibodies as mentioned above in section 2.8 . The binding parameters K D and B max were determined via nonlinear regression curve fitting using GraphPad Prism Version 9.5.1 and compared with the binding parameters of same antigen B in other orders. The same procedure was followed to dissociate the second blocking fragment anti-Rh(D) scFv. The binding parameters K D and B max were determined by measuring the unbound his-tagged anti-Rh(D) ds-scFv via indirect ELISA and compared with the binding parameters of the same antigen in other orders. A similar process was used to measure the intramolecular binding cooperativity for other binding orders, such as A→Rh(D)→B, B→A→Rh(D), B→Rh(D)→A, Rh(D)→A→B, and Rh(D)→B→A. 2.10 Coating of RBCs with a triabody and hemagglutination assay The trispecific triabody was reconstituted in TBS buffer (pH 7.4) and then divided into 100 µl aliquots at a concentration of 100 µM. Approximately 10 µl of RBCs from ABO and Rh(D) blood types (A+, B+, O+, AB+, A−, B − and AB−) were incubated in 100 µl aliquots of triabody at 37°C for 30 mins. Following incubation, the RBCs were washed with TBS buffer (pH 7.4), and this process was repeated with fresh aliquots of triabody. The remaining triabody in the supernatant was quantified via indirect ELISA by using free antigens as described in section 2.8 . The incubation of RBCs with the triabody was repeated until no further changes in the triabody concentration in the supernatant was observed. An immediate hemagglutination assay was performed to evaluate both the effectiveness of the triabody coating and its ability to block all blood group antigens. This involved incubating 1 µl of triabody-coated RBCs with 100 µl of each of the following reagents: anti-A IgM, anti-B IgM, and anti-Rh(D) IgM antibodies for 10 mins at room temperature. 3. Results 3.1 Structures of triabody On the basis of the placement of variable chains of anti-Rh(D), two combinations were used to model the molecules of the triabody (Fig. 1 ). As shown in Fig. 3 , the fusion proteins FP-1a and FP-1b generated from combination one showed correct pairing of VH chains of both anti-A and anti-B ds-scFv with their respective VL chains. On the other hand, three types of fusion proteins (FP-2a, FP-2b and FP-2c) were observed from combination two. Like combination one, the fusion proteins FP-2a and FP-2b in combination two exhibited correct pairing of variable chains. However, the fusion protein FP-2c exhibited the interaction of VL chain of anti-Rh(D) with the VH chain of anti-B ds-scFv. For triabodies to effectively block blood group antigens, all three of their antigen binding sites must be freely accessible. Two types of triabody structures were produced from both combinations. Depending on the location of the disulfide bond formed these structures were in closed and open forms with intramolecular and intermolecular disulfide bonds respectively. The docking of two fusion proteins from combination one produced a closed triabody-C1 form with the antigen binding sites of all three Fv facing outward and an open triabody-O1 form where all the binding sites were easily accessible. Docking of two fusion proteins from combination two with correct pairing (FP-2a and FP-2b) produced an open triabody-O2 form similar to triabody-O1 of combination one and a closed triabody-C2 form with antigen binding sites of anti-A and anti-B ds-scFv facing outward and antigen binding site of anti-Rh(D) facing inward (blocked by intermolecular disulfide bond). Overall, the best structures of triabodies were obtained from the fusion proteins of combination one. The selected triabodies had ten disulfide bonds (eight intramolecular and two intermolecular) in the closed C1 form and eleven disulfide bonds (ten intramolecular and one intermolecular) in the open O1 form. The predicted theoretical weight of both triabodies was 83.3 kDa. Figure 4 shows the comparison of triabodies with the IgG1 antibody structure PDB: 1IGY [ 43 ]. The overall distance between the binding sites of triabody-C1 is half the distance between the binding sites of the IgG1 antibody. The length of triabody-O1 (14.3 nm) is almost the same as the distance between the binding sites (14.5 nm) of an IgG1 antibody. Additional computational studies such as molecular docking and MMPBSA were conducted on closed structure i.e., triabody-C1. 3.2 Functional analysis MMPBSA calculations were performed to predict the intramolecular cooperative binding affinities of the triabody. The results shown in Table 1 revealed no significant changes in the binding free energies of the complexes formed between the triabody and each blood group antigen, regardless of the binding order. For instance, the binding free energy of the triabody–antigen A complex remained consistently around − 13.20 kcal/mol, irrespective of whether antigen A was the first, second, or third to bind. Similarly, the binding free energies of the triabody–antigen B and triabody–antigen Rh(D) complexes were approximately − 12.6 kcal/mol and − 49 kcal/mol, respectively. These results indicate a lack of intramolecular cooperativity, meaning that the binding of one antigen is independent of the binding of the others. Supplementary Figure S4 shows snapshots from the MD trajectories of the triabody complexes with all three blood group antigens at the beginning and end of the 150 ns simulation following the binding sequence A→B→Rh(D). Table 1 Binding free energies (ΔG Bind ) kcal/mol computed by MMPBSA. Binding Sequence First binding Second binding Third binding A→B→ Rh(D) A −13.20 B −12.59 Rh(D) −48.9 A→ Rh(D)→B A −13.18 Rh(D) −49.1 B −12.61 B→A→ Rh(D) B −12.60 A −13.19 Rh(D) −48.8 B→ Rh(D)→A B −12.62 Rh(D) −49.1 A −13.21 Rh(D)→A→B Rh(D) −48.4 A −13.20 B −12.59 Rh(D)→B→A Rh(D) −49.0 B −12.60 A −13.22 3.3 Three-step purification Triabody was produced under slow induction conditions (18°C and 0.25 mM IPTG) to allow proper folding of the protein. His-tagged proteins were purified via Ni 2+ -NTA chromatography (first purification step) (Fig. 5 A) and analyzed via western blotting. As shown in Fig. 5 A, in the Ni 2+ -NTA elution profile E1 (lane 7), the molecular weight of the triabody (~ 83 kDa) was approximately the same as the theoretical weight predicted by the ProtParam tool. In addition to the predicted triabody, a larger triabody of ~ 87 kDa and a half triabody of ~ 45 kDa were also observed. Protein extraction from Native PAGE gel was a second step in separating individual triabody species. The Ni 2+ -NTA elution profile E1 was taken, and Native PAGE was carried out. Subsequently, each individual triabody species was extracted from the gel. As shown in Fig. 5 A, a larger triabody (lane 8) and desired-sized triabody (lane 9) were successfully isolated from a mixture of different-sized his-tagged proteins. Purification through acid-glycine elution was the final crucial step in obtaining a trifunctional triabody. Following Native PAGE purification, the elution profiles for the N1 fractions were AE1-A1, AE2-RhD1, and AE3-B1, whereas the elution profiles for the N2 were AE1-A2, AE2-RhD2, and AE3-B2. As shown in Fig. 5 A, the elution profiles AE1-A1 (lane 10) and AE1-A2 (lane 13) show bands at ~ 87 kDa and ~ 83 kDa respectively. These bands represent the purification of triabodies with functional A binding sites. Similarly, the bands observed in the elution profiles of AE2-RhD1 (lane 11) and AE2-RhD2 (lane 14) represent the purified bifunctional triabodies with functional A and Rh(D) binding sites. Finally, bands in elution profiles AE3-B1 (lane 12) and AE3-B2 (lane 15) show purified trifunctional triabodies (~ 87 kDa and ~ 83 kDa, respectively) with functional A, Rh(D) and B binding sites. Figures 5 B, 5 C, and 5 D depict the expected purified proteins in each step. 3.4 Immunocytochemistry (ICC) Immunocytochemistry (ICC) was performed to observe the hemagglutinating properties of the triabodies. For this purpose, RBCs with at least two blood group antigens were taken and coated with triabodies. As shown in Fig. 6 A no visible hemagglutination of RBCs was observed when these RBCs were incubated with triabodies from either the AE3-B1 (wells 2–5) or the AE3-B2 (well 6) fraction. However, when examined microscopically, mixed-field hemagglutination was observed in AB+ (Fig. 6 C) and AB− (Fig. 6 D) RBCs that were incubated with triabody from the AE3-B1 fraction. In contrast, no hemagglutination was observed in A+ (Fig. 6 E) and B+ (Fig. 6 F) RBCs incubated with the same triabody. Similarly, no hemagglutination was observed in AB + RBCs (Fig. 6 G) incubated with triabody from the AE3-B2 fraction. 3.5 Determination of intramolecular binding cooperativity of the triabody via free and RBC-bound blood group antigens To understand how the binding of one antigen affects the binding of others, the triabody’s intramolecular binding cooperativity was determined via ELISA-based experiments. Free antigens were used to assess whether the changes in binding patterns observed with RBC-bound antigens were a result of the triabody’s small size during initial and subsequent binding events. As shown in Fig. 7 , there were no significant overall changes in the binding parameters K D and B max , suggesting non-cooperative binding, i.e., subsequent binding was not influenced by prior binding events. The K D ​ values for the anti-A, anti-B, and anti-Rh(D) binding sites of the triabody, when binding individually, were 0.790, 0.842, and 0.353 µM, respectively. For the binding orders where A-trisaccharide (Fig. 7 A) was involved in the second binding event (B→A→Rh(D) and Rh(D)→A→B), the K D ​ values were 0.808 and 0.797 µM respectively. Similarly, in the third binding event involving A-trisaccharide (B→Rh(D)→A and Rh(D)→B→A), the K D ​ values were 0.801 and 0.815 µM respectively. The B max values of A-trisaccharide for both the second and third binding events remained close to the B max value of the initial binding event (1.36), with values of 1.39 and 1.35 for the second binding events (B→A→Rh(D) and Rh(D)→A→B), and 1.38 and 1.37 values for the third binding events (B→Rh(D)→A and Rh(D)→B→A). In the case where B-trisaccharide (Fig. 7 B) was involved in the second binding event, the K D ​ values were 0.832 µM and 0.836 µM for the orders A→B→Rh(D) and Rh(D)→B→A, respectively. In the third binding event, the K D ​ values were 0.839 µM and 0.847 µM for the orders A→Rh(D)→B and Rh(D)→A→B, respectively. Similar to the A-trisaccharide, the B max values for the B-trisaccharide were close to the initial value of 1.30. The second binding events yielded B max values of 1.25 and 1.27 for the A→B→Rh(D) and Rh(D)→B→A binding orders, respectively. The third binding events produced B max values of 1.28 for the A→Rh(D)→B order and 1.31 for the Rh(D)→A→B order. For the second binding events in the orders A→Rh(D)→B and B→Rh(D)→A, which involve the antigen Rh(D) Fig. 7 C), the K D values were 0.330 and 0.351 µM, respectively. A similar case was observed for the other binding orders, A→B→Rh(D) and B→A→Rh(D), which involve the antigen Rh(D) in the third binding position, with K D values of 0.337 and 0.348 µM, respectively. The changes in B max values for antigen Rh(D) were similar to those observed for A-trisaccharide and B-trisaccharide, i.e., the B max values for the second and third binding events were closer to the initial value (0.940). The B max values for the binding order A→Rh(D)→B and B→Rh(D)→A, involving Rh(D) in the second binding event, were 0.931 and 0.937, respectively. For the third binding events A→B→Rh(D) and B→A→Rh(D), the B max values were 0.935 and 0.938, respectively. The intramolecular binding cooperativity of the triabody with RBC-bound antigens was indirectly assessed by measuring changes in the binding parameters K D and B max of the anti-A, anti-B, and anti-Rh(D) ds-scFvs. This approach was crucial for understanding the observed cooperativity and how it is influenced by the physical environment of the antigen and the small size of the triabody. As shown in Fig. 8 A, antigen A exhibited higher K D values for both the second and third binding events compared with its initial binding (0.618 µM). When antigen B was bound first, the K D values for antigen A increased to 0.724 µM and 0.730 µM in the sequences B→A→Rh(D) and B→Rh(D)→A, respectively. A further increase was noted when antigen Rh(D) served as the first binding partner, with K D values reaching 0.748 µM and 0.755 µM for Rh(D)→A→B and Rh(D)→B→A, respectively. After initial binding to antigen B, the B max value for antigen A decreased from 1.33 to 1.27 and 1.24, whereas even lower values of 1.14 and 1.12 were observed when antigen Rh(D) was bound first. These results suggest that binding of antigen A is more affected when antigen Rh(D) binds first, showing negative cooperativity. Also, when antigen A was involved the third binding event, K D value was higher and B max value was lower compared to the second binding event. In contrast to antigen A, the K D values for the second and third binding events involving antigen B were found to remain comparable to those observed when antigen B was the first antigen to bind (0.517 µM) as shown in Fig. 8 B. When antigen A was bound first, the K D values for antigen B were 0.520 µM and 0.522 µM for the sequences A→B→Rh(D) and A→Rh(D)→B, respectively. In comparison, when antigen Rh(D) was involved in the initial binding event, higher K D values were observed, i.e., 0.533 µM and 0.540 µM for the sequences Rh(D)→B→A and Rh(D)→A→B, respectively. Following the initial binding of antigen A, the B max values for antigen B were observed to remain close to its initial value (0.954), at 0.941 and 0.926 for the sequences A→B→Rh(D) and A→Rh(D)→B, respectively. However, B max values were significantly reduced to 0.837 and 0.830 when antigen Rh(D) was the first antigen to bind, as observed in the sequences Rh(D)→B→A and Rh(D)→A→B (Fig. 8 B), suggesting that negative cooperativity was introduced when antigen Rh(D) bound first. When the second and third binding events of antigen B were compared, higher K D and lower B max values were observed in the third binding event. Unlike antigens A and B, the binding of antigen Rh(D) remained largely unaffected by prior binding events. As shown in Fig. 8 C when antigen Rh(D) was involved in the second binding event, the K D values were 0.292 µM and 0.291 µM for the sequences A→Rh(D)→B and B→Rh(D)→A, respectively, which were comparable to its initial binding value of 0.289 µM. The B max value was 0.751, which was consistently maintained in second binding events close to the initial binding value of 0.753. However, when Rh(D) was the third antigen to bind, the K D values increased slightly to 0.298 µM and 0.301 µM for the sequences A→B→Rh(D) and B→A→Rh(D), respectively, with corresponding B max values of 0.744. The minimal increase in K D values and slight decrease in B max values suggest that the binding of Rh(D) is only marginally influenced by prior antigen interactions, indicating largely non-cooperative binding. 3.6 Coating of RBCs with triabody and hemagglutination assay To make RBCs from various blood groups mimic O-negative RBCs, the triabody must block the maximum number of blood group antigens A, B and Rh(D). To achieve this, a repeated exposure protocol was used, which involved incubating RBCs in concentrated aliquots of a triabody solution. Supplementary Figure S5 shows that for all blood types of RBCs, a significant decrease in the concentration of triabody was observed in the first aliquot after 30 mins of incubation. However, no further reduction in concentration was observed in aliquots 2 and 3. The overall results (Fig. 9 ) of the hemagglutination assay were negative, indicating that RBCs coated with trispecific triabodies purified in fraction AE3-B2 (Fig. 9 B) did not hemagglutinate in the presence of associated anti-A IgM, anti-B IgM and anti-Rh(D) IgM antibodies. The control wells in Fig. 9 A show positive hemagglutination of uncoated RBCs of different blood types in the presence of associated blood group antibodies. 4. Discussion The main aim of this research was to design a novel trispecific triabody capable of acting as a blocking fragment for blood group antigens A, B, and Rh(D). By blocking these key antigens, any blood type RBCs could mimic universal donor O-negative RBCs, allowing for safe transfusions into any individual regardless of their native blood type. The design of the triabody was inspired by the compact, Y-shaped structure of IgG antibodies. Unlike IgM antibodies, IgG molecules are well known to rarely induce direct hemagglutination of RBCs due to their smaller size [ 44 ]. To further minimize the potential for hemagglutination, the triabody was intentionally designed to be even smaller than an IgG antibody. Furthermore, in addition to its trivalent binding capability, triabody was designed to be trispecific. These combined features would allow the triabody to efficiently block multiple blood group antigens without causing the hemagglutination of RBCs. To design a compact Y-shaped triabody, the hinge region of an IgG1 antibody was used. The hinge region, similar to a glycine-serine (GS) linker, provides flexibility to protein structures. However, unlike GS linker, the flexibility and movement of the hinge region can be controlled by the presence of cysteine residues. These cysteine residues are capable of forming intermolecular disulfide bonds between two hinge regions, thereby stabilizing the overall structure [ 45 , 46 , 47 ]. As shown in Fig. 3 , the addition of hinge regions in the triabodies offered a distinct advantage over GS linkers by producing compact Y-shaped structures (triabody-C1 and C2) that closely mimic the native IgG structure. Such structural compactness would not have been achieved if a GS linker had been used instead. In general, scFvs possess a well-conserved framework and exhibit a remarkable functional diversity. Certain residues within the framework of a scFv are highly conserved and act as essential building blocks for the basic architecture of the molecule. These residues, through various interactions, provide shape and stability to the scFv e.g., cysteines are involved in intradomain disulfide bonds. Functional diversity arises from the hypervariable regions, which provide binding specificity to scFvs [ 48 ]. As shown in Fig. 3 B, incorrect pairing was observed in the fusion protein FP-2c. This mispairing could be due to two reasons: 1) similar frameworks of both chains and 2) the close proximity of the VH chain of anti-B to the VL chain of anti-Rh(D). The VH and VL frameworks of the Fvs used in this study share a high degree of similarity, with the only significant difference located in the sequence of complementarity-determining regions (CDRs) responsible for binding specificity. This similarity, combined with the close proximity of the chains, may have facilitated the incorrect pairing of the VH chain of anti-Rh(D) with the VL chain of anti-B within FP-2c. The accessibility of antigen binding sites is essential for triabodies to function effectively. This was clearly observed in triabodies-C1 and C2 (Fig. 3 ). In triabody-C1, the arrangement of variable chains positioned the anti-Rh(D) binding site outward, making it accessible. Conversely, the same variable chains in triabody-C2 positioned the anti-Rh(D) binding site inward, rendering it inaccessible. This issue in triabody-C2 could have been prevented by incorporating an additional GS linker of at least 10 amino acids (G 4 S) 2 between the hinge region and the anti-Rh(D) variable chain, as shown in Supplementary Figure S6. The additional linker would have allowed reorientation and facilitated the formation of an intermolecular disulfide bond in the opposite direction, thereby exposing the anti-Rh(D) binding site and orienting it outward. These findings suggest that the arrangement of variable chains during triabody design can impact the final pairing of chains and ultimately their functionality. Despite the potential of triabodies to cause hemagglutination owing to their multiple binding sites, no visible hemagglutination was observed as shown in Fig. 6 A. However, microscopic analysis revealed that the triabody from the AE3-B1 fraction caused mixed-field hemagglutination only in AB + and AB − RBCs (Figs. 6 C and 6 D), with no hemagglutination observed in A + and B + RBCs (Figs. 6 E and 6 F). This selective hemagglutination in which a dual population of agglutinated and non-agglutinated RBCs is present, could be due to the distance between the A, B and Rh(D) binding sites. The distance between the A and B binding sites is greater, whereas the distances between the A and Rh(D) binding sites, as well as between the B and Rh(D) binding sites, are comparatively shorter as depicted in Fig. 4 . The greater distance between the A and B binding sites likely facilitated the observed mixed-field hemagglutination. The location of the disulfide bonds within the triabody structure is the key determinant of whether a closed structure (intermolecular disulfide bond) or an open structure (intramolecular disulfide bond) is formed. The observation of mixed-field hemagglutination specifically in AB + and AB − blood types strongly suggests that the triabody from the AE3-B1 fraction adopts an open structure. However, in this study, it was not determined whether this open structure contains intramolecular disulfide bonds. In contrast to the triabody from the AE3-B1 fraction, the triabody from the AE3-B2 fraction did not cause hemagglutination in AB + RBCs as shown in Fig. 6 G. This finding strongly supports a compact closed structure, which could only be formed through intermolecular disulfide bonds. Given that the triabody was designed to block blood group antigens on the surface of RBCs, it was essential to evaluate whether it could bind all three antigens simultaneously or whether its small size could limit this capability. To address this, the intramolecular binding cooperativity of the triabody was first determined using free antigens and then compared with RBC-bound antigens, enabling assessment of whether initial binding to one antigen affects subsequent interactions with the others. Figure 7 shows that while the triabody is capable of binding multiple antigens, its binding sites appear to function independently when the antigens are free in solution. The lack of change in the binding parameters K D and B max ​ with free antigens suggests that the binding of one site does not influence the binding of the other sites. This finding suggests that the individual monomers of the triabody, composed of the anti-A, and anti-B ds-scFv and the dsFv of anti-Rh(D), behave as distinct and separate units. However, when the intramolecular binding cooperativity of the triabody was evaluated using RBC-bound antigens, consistent changes were observed in the binding parameters: K D increased and B max decreased during both the second and third binding events. These effects were most pronounced in the third binding step, irrespective of the antigen, indicating a cumulative impact on binding efficiency. This reduction may be due to steric hindrance caused by the dense distribution of antigens on the RBC surface, which likely restricts the accessibility of the triabody and impairs its effective engagement during later binding events. The changes were especially significant when Rh(D) was the first antigen to bind. This observation may be explained by the relatively low abundance of antigens Rh(D) on RBCs compared with carbohydrate antigens A and B [ 49 , 50 ]. Fewer Rh(D) molecules require fewer triabodies to reach saturation during the initial binding. As a result, fewer triabodies remain available for subsequent interactions with antigen A or B, leading to reduced binding affinity and capacity, as indicated by the increased K D and decreased B max values. In contrast, these effects were less prominent when antigen A or B was involved in the first binding event, or when Rh(D) was engaged during the second binding step. The results of hemagglutination assay, as shown in Fig. 9 , directly confirmed the effectiveness of the triabody as a blocking fragment. The complete absence of hemagglutination of triabody-coated RBCs validated that the triabody coating has successfully prevented the IgM antibodies from interacting with blood group antigens. Importantly, these observations are consistent with those reported by Hafeez and Zaidi (2024) [ 13 ] where a similar lack of hemagglutination was observed in antigen A blocked RBCs incubated with anti-A IgM antibodies. These findings suggest that RBCs from each blood type, once coated with triabody, effectively mimic the compatibility profile of universal donor O-negative RBCs. This indicates that triabody-coated RBCs in the form of pRBCs, could be transfused into individuals of any blood type without triggering a blood type incompatibility related hemagglutination reaction, thereby significantly expanding the availability of usable blood. This study has certain limitations: 1) the presence and precise location of inter- and intramolecular disulfide bond was not determined; and 2) the potential of the triabody was determined outside a real blood environment, which is a highly complex mixture of cells, salts, and proteins. Further research is needed to determine the location of disulfide bonds using advanced structural analysis such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Additionally, the cooperative binding of the triabody should be studied via advanced techniques such as surface plasmon resonance (SPR) and other spectroscopic approaches. To fully evaluate the clinical potential of the triabody as a blocking agent, in vivo studies in animal models are necessary to assess its efficiency and safety in a real physiological environment. 5. Conclusion This study demonstrated that the trispecific triabody can effectively block blood group antigens A, B, and Rh(D) on RBCs, thereby preventing IgM-mediated hemagglutination and enabling RBCs to mimic the compatibility of universal donor O-negative RBCs. Its compact Y-shaped structure, inspired by the small size of IgG antibodies, allows efficient multivalent binding while avoiding hemagglutination. Further studies are needed to characterize disulfide bonds and to evaluate in vivo safety and efficacy. These findings highlight the strong potential of the triabody as a blocking fragment capable of targeting multiple antigens and provide a foundation for future translational and clinical research. Declarations Ethics approval The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Review Committee of the National University of Sciences and Technology (NUST) (IRB number: 09-2023-ASAB-01/02) and was performed in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no conflicts of interest. Funding This research was supported by a PhD student's research funds from the Atta-Ur-Rahman School of Applied Biosciences (ASAB) at the National University of Sciences and Technology (NUST) in Sector H-12, Islamabad, Pakistan. Muhammad Asghar was supported by Ragnar Söderberg Foundation Sweden. Authors' contributions SH and MA conceptualized and designed the study. SH developed the methodology and performed the software analysis. Validation was carried out by SH and MA. Formal analysis and investigation were performed by SH. Resources were provided by MA. The original draft of the manuscript was prepared by SH, and both SH and MA contributed to reviewing and editing the manuscript. Visualization was performed by SH and MA. Supervision of the study was provided by MA. All authors have read and approved the final manuscript. Acknowledgements Not applicable References Obeagu EI. The vitamin C paradigm: New frontiers in blood transfusion. Ann Med Surg (Lond). 2025; doi:10.1097/ms9.0000000000003018. Huet M, Cubizolles M, Buhot A. Red blood cell agglutination for blood typing within passive microfluidic biochips. High Throughput. 2018; doi:10.3390/ht7020010. Bain BJ. Blood cells: A practical guide. 5th ed. Chichester: John Wiley & Sons; 2019. Keyt BA, Baliga R, Sinclair AM, Carroll SF, Peterson MS. Structure, function, and therapeutic use of IgM antibodies. Antibodies (Basel). 2020; doi:10.3390/antib9040053. Rout P, Harewood J, Ramsey A, et al. Hemolytic transfusion reaction. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2023 Sep 12]. https://www.ncbi.nlm.nih.gov/books/NBK448158. Accessed 13 Mar 2025. Armstrong B. Antigen-antibody reactions. ISBT Sci Ser. 2020; doi:10.1111/voxs.12590. Raman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020; doi:10.1111/voxs.12591. Theis SR, Hashmi MF. Coombs test. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2022 Sep 12]. https://www.ncbi.nlm.nih.gov/books/NBK547707. Accessed 1 Apr 2025. Dean L. Blood Groups and Red Cell Antigens. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 7. https://www.ncbi.nlm.nih.gov/books/NBK2269/. Accessed 7 Aug 2025. Romanos-Sirakis EC, Desai D. ABO Blood Group System. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2025 Apr 26]. https://www.ncbi.nlm.nih.gov/books/NBK580518/. Accessed 22 Aug 2025. Waheed U, Farooq A. Bridging blood and genes: The future of transfusion medicine and molecular biology. Ann Pak Inst Med Sci. 2024; doi:10.48036/apims.v20iSuppl.2.1282. Rahfeld P, Withers SG. Toward universal donor blood: Enzymatic conversion of A and B to O type. J Biol Chem. 2020;295:325–334. Hafeez S, Zaidi N. Prevention of blood incompatibility related hemagglutination: Blocking of antigen A on red blood cells using in silico designed recombinant Anti-A scFv. Antibodies (Basel). 2024; doi:10.3390/antib13030064. Luyten U, Peeraer S, Pirlet C, Khaouch Y, Streel C, Deneys V. O-negative blood shortage management in a university hospital: Impact of transfusing RHD-positive red blood cells to RHD-negative patients. Transfus Clin Biol. 2023;30:402–409. Kundu S, Gerk A, Glatter R, Poenaru D. Innovative transfusion strategies for blood deserts in disaster settings. BMJ Glob Health. 2024; doi:10.1136/bmjgh-2024-016854. Hafeez S, Zaidi NUSS. Red blood cell substitutes: Liposome encapsulated hemoglobin and magnetite nanoparticle conjugates as oxygen carriers. Int J Mol Sci. 2023; doi:10.3390/ijms24021618. Haldar R, Chitranshi S, Singh MK, Sachan S, Gupta D. Artificial blood: A futuristic dimension of modern-day transfusion sciences. Cardiovasc Hematol Agents Med Chem. 2019;17:11–16. Olsson ML, Hill CA, De La Vega H, Liu QP, Stroud MR, Valdinocci J. Universal red blood cells: Enzymatic conversion of blood group A and B antigens. Transfus Clin Biol. 2004;11:33–39. Zhou P, Ouchari M, Xue Y, Yin Q. In vitro generation of red blood cells from stem cells and targeted therapy. Cell Transplant. 2020; doi:10.1177/0963689720946658. Mohammadi M, Nejatollahi F, Bemani P. Production of scFvs as anti-ABO antibody antagonists for prevention of hemolytic transfusion reactions resulting from ABO blood group incompatibility between donor and recipient. Hypothesis. 2016;14(1). Thomas R, Patenaude SI, MacKenzie CR, et al. Structure of an anti-blood group A FV and improvement of its binding affinity without loss of specificity. J Biol Chem. 2002;277:2059–2064. Santos-Esteban E, Curiel-Quesada E. Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library. Vox Sang. 2001;81:194–198. Siegel DL, Chang TY, Russell SL, Bunya VY. Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: Applications in immunohematology. J Immunol Methods. 1997; doi:10.1016/s0022-1759(97)00087-2 Zhao J, Yang L, Gu Z, et al. Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity. Int J Mol Sci. 2010;12:1–11. Saphire EO, Parren PW, Pantophlet R, et al. Crystal structure of a neutralizing human IgG against HIV-1: A template for vaccine design. Science. 2001; doi:10.1126/science.1061692. Yang J, Yan R, Roy A, et al. The I-TASSER suite: Protein structure and function prediction. Nat Methods. 2015;12:7–8. Christoffer C, Chen S, Bharadwaj V, et al. LZerD webserver for pairwise and multiple protein docking. Nucleic Acids Res. 2021;49:W359–W365. Schrödinger, LLC. The PyMOL molecular graphics system, version 3.1. 2025. Available from: https://pymol.org/. Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. Proteomics Protocols Handbook. New York: Humana Press; 2005. p. 571–607. doi:10.1385/1-59259-890-0:571. Martínez-Rosell G, Giorgino T, De Fabritiis G. Playmolecule ProteinPrepare: A web application for protein preparation for molecular dynamics simulations. J Chem Inf Model. 2017;57:1511–1516. Abraham MJ, Murtola T, Schulz R, et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1:19–25. Grant OC, Wentworth D, Holmes SG, et al. Generating 3D models of carbohydrates with GLYCAM-Web. 2025; doi:10.1101/2025.05.08.652828. Conroy MJ, Bullough PA, Merrick M, Avent ND. Modelling the human rhesus proteins: Implications for structure and function. Br J Haematol. 2005; doi:10.1111/j.1365-2141.2005.05786.x. Bugnon M, Röhrig UF, Goullieux M, et al. SwissDock 2024: Major enhancements for small-molecule docking with Attracting Cavities and AutoDock Vina. Nucleic Acids Res. 2024; doi:10.1093/nar/gkae300. Case DA, Cheatham TE, Darden T, et al. The Amber biomolecular simulation programs. J Comput Chem. 2005;26:1668–1688. Kumari R, Kumar R, Lynn A. g_mmpbsa—A GROMACS tool for high-throughput MM-PBSA calculations. J Chem Inf Model. 2014; doi:10.1021/ci500020m. Pope B, Kent HM. High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res. 1996;24:536–537. Woiszwillo JE. TMB formulation for soluble and precipitable HRP-ELISA. US Patent 5,006,461. United States; 1991. Syedbasha M, Linnik J, Santer D, et al. An ELISA based binding and competition method to rapidly determine ligand-receptor interactions. J Vis Exp. 2016;109:e53575. GraphPad Software. Nonlinear regression curve fitting was performed using GraphPad Prism version 9.5.1. 2023. Available from: https://www.graphpad.com/ Sang Z, Xiang Y, Bahar I, Shi Y. Llamanade: An open-source computational pipeline for robust nanobody humanization. Structure. 2022; doi:10.1016/j.str.2021.11.006. Koganei A, Tsuchiya T, Samura K, Nishikibe M. Use of whole sheep red blood cells in ELISA to assess immunosuppression in vivo. J Immunotoxicol. 2007;4:77–82. Harris LJ, Skaletsky E, McPherson A. Crystallographic structure of an intact IgG1 monoclonal antibody. J Mol Biol. 1998; doi:10.1006/jmbi.1997.1508. Focosi D, Franchini M, Maggi F. Modified hemagglutination tests for COVID-19 serology in resource-poor settings: Ready for prime-time? Vaccines. 2022; doi:10.3390/vaccines10030406. Horx P, Geyer A. Comparing the hinge-type mobility of natural and designed intermolecular bi-disulfide domains. Front Chem. 2020; doi:10.3389/fchem.2020.00025. Rosmalen MV, Krom M, Merkx M. Tuning the flexibility of glycine-serine linkers to allow rational design of multidomain proteins. Biochemistry. 2017; doi:10.1021/acs.biochem.7b00902. Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The immune system in health and disease. 5th edition. New York: Garland Science; 2001. https://www.ncbi.nlm.nih.gov/books/NBK27144/. Accessed 2 Sep 2025. Gąciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017; doi:10.1371/journal.pone.0189964. McGann PT, Despotovic JM, Howard TA, Ware RE. A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression. Am J Hematol. 2012; doi:10.1002/ajh.22254. Dean L. Blood Groups and Red Cell Antigens. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 2. https://www.ncbi.nlm.nih.gov/books/NBK2264/. Accessed 18 Sep 2025. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTriabody.docx GraphicalAbstract.docx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted Editorial decision: Revision requested 20 Jan, 2026 Reviews received at journal 18 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviews received at journal 06 Jan, 2026 Reviewers agreed at journal 30 Dec, 2025 Reviewers agreed at journal 27 Dec, 2025 Reviewers agreed at journal 23 Dec, 2025 Reviewers agreed at journal 22 Dec, 2025 Reviewers agreed at journal 22 Dec, 2025 Reviewers invited by journal 22 Dec, 2025 Editor assigned by journal 18 Dec, 2025 Submission checks completed at journal 18 Dec, 2025 First submitted to journal 15 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8369029","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561955202,"identity":"a26f8bbe-c662-4e7e-9f18-e8ef8c84c5c3","order_by":0,"name":"Saleha Hafeez","email":"","orcid":"","institution":"National University of Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Saleha","middleName":"","lastName":"Hafeez","suffix":""},{"id":561955204,"identity":"10135708-4959-48b4-b928-1452e195ce00","order_by":1,"name":"Muhammad Asghar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYFACHjDJ2AAkmIFYzoCZgfEAQwEJWoyBWhgOMBiQoCVxAwMBLbozcg9+upljJ9vAfvzx64Kae+nb2ZkPALUcZpBvb8CqxexGXrJ07rZk4waeHDPrGceKc3c2syWAtRicOYBDS44BUAtzYgNDDpsxD1tC7obDPAYQLRIJuLQY/87dVp/YwP/8mTHPv4R0A5gW+fkPcGkxA9pyOLFBIsH4MW9bQgJcC8MN7N43O/PGzDp323HjNok3Zsy8fQmGYL8kGKTzGJzB4bDjOca3c7dVy/bzpz/+zPMtQd6c//DBBx8qrOXk27F7Hw7YgEgCzkuAxRcBwPyBGFWjYBSMglEw8gAAC1lfQZasB7IAAAAASUVORK5CYII=","orcid":"","institution":"Lund University","correspondingAuthor":true,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Asghar","suffix":""}],"badges":[],"createdAt":"2025-12-15 17:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8369029/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8369029/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13036-026-00661-w","type":"published","date":"2026-03-13T15:59:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98624349,"identity":"812dd636-72fb-4967-85d9-df4e5e8de3da","added_by":"auto","created_at":"2025-12-19 17:08:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8653003,"visible":true,"origin":"","legend":"","description":"","filename":"TriabodyManuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/ec80de3bc5c8da24bd14783c.docx"},{"id":98623909,"identity":"915147eb-88da-4927-85db-9e73f7290554","added_by":"auto","created_at":"2025-12-19 17:07:47","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5504,"visible":true,"origin":"","legend":"","description":"","filename":"6c86c54007ac48d48647ad98faaed297.json","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/0ce1bedc8797a43264f61502.json"},{"id":98482232,"identity":"4d492679-2f21-4b56-8d75-7bc3ae83895c","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2920223,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTriabody.docx","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/4498cc5b01e3b3b49f7792b8.docx"},{"id":98623541,"identity":"2fb3ded0-71c1-4963-adf0-082f4fef67d3","added_by":"auto","created_at":"2025-12-19 17:06:55","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143582,"visible":true,"origin":"","legend":"","description":"","filename":"6c86c54007ac48d48647ad98faaed2971enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/04a75734ec56bd69c9b6f0f1.xml"},{"id":98482240,"identity":"d9887dcf-8f13-4bcb-abbe-c0bdf3e04070","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1443388,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/251a458cfb326c7c857cab6e.png"},{"id":98624258,"identity":"58f0df95-ac87-4af1-afb4-6cafd9a47bf6","added_by":"auto","created_at":"2025-12-19 17:08:13","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":992731,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/01dd4a0e8ba7b9cc0f153e21.png"},{"id":98623351,"identity":"b0c0db07-9ede-4f89-a43a-2e4ac8efc995","added_by":"auto","created_at":"2025-12-19 17:05:53","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160457,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/9e925633f3f3b74e197d6ca1.png"},{"id":98623743,"identity":"a0cd2eab-bfbc-4ddc-92b0-94afd3eec8c1","added_by":"auto","created_at":"2025-12-19 17:07:28","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":638790,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/518aef4ad5f3a6e6e0e3cb2b.png"},{"id":98624262,"identity":"1c7f2c6d-58e0-4ae1-b4ca-8a540e8fddc8","added_by":"auto","created_at":"2025-12-19 17:08:14","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1101259,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/d9fe0f568edbe0a3a8466228.png"},{"id":98623429,"identity":"4f9943f0-30c8-444b-b829-9b3f8fd06e62","added_by":"auto","created_at":"2025-12-19 17:06:18","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1066761,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/46aa9fe249c42269cac7cb0e.png"},{"id":98482237,"identity":"e5fb5f9b-1c5f-440c-9a67-3aad5a8ca048","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":845675,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/66bde70c6ca5028523fca346.png"},{"id":98624410,"identity":"adafab4f-bb66-495e-b3c7-749c5a1de859","added_by":"auto","created_at":"2025-12-19 17:08:23","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1722460,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/a1846892e80613661e0df61e.png"},{"id":98482242,"identity":"2e2f3fee-3348-4223-b417-d56e85caaab9","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":339339,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/3384f727f04b6b91c917cb30.png"},{"id":98624580,"identity":"da9d6564-9c30-418d-bb39-80e9c63c3edb","added_by":"auto","created_at":"2025-12-19 17:08:31","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":364931,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/7bab9edeeee349ab51934f93.png"},{"id":98482251,"identity":"fdd3a4b4-1df0-46de-8d22-13c7dc394f78","added_by":"auto","created_at":"2025-12-18 05:29:14","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202336,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/ab5a1c448c677dbea4fe599a.png"},{"id":98623318,"identity":"70d41930-c9a3-46c8-86cf-79791619a0ef","added_by":"auto","created_at":"2025-12-19 17:05:48","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158587,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/c9673592c72403d660c67aa4.png"},{"id":98482247,"identity":"7dbfd9fc-8a3a-4f3f-9fc7-80b0cf7f7e09","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36554,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/2163d71ae098223d57b107fe.png"},{"id":98482245,"identity":"a8ae039d-2a30-43fe-9cb5-8c539c4bba1f","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101814,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/a560947491cecab3c43c6903.png"},{"id":98482246,"identity":"0c8c15be-f4a5-4c59-a5d6-6f8b515ca8d0","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":212755,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/123a873b2ee4eca3bfa53708.png"},{"id":98482243,"identity":"3f9de175-3b42-4640-b27e-93aeb6fcdc61","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":195053,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/448c60ed91d181d04652ad64.png"},{"id":98482259,"identity":"e606f529-eca4-480b-8d9f-674d13a5d948","added_by":"auto","created_at":"2025-12-18 05:29:14","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135190,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/e3b7be07691f8fe19b2b8724.png"},{"id":98482253,"identity":"57b0bce3-91a2-4043-81ba-7b0bc62e38c5","added_by":"auto","created_at":"2025-12-18 05:29:14","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211876,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/2c39cd5be91b495e2e1e1397.png"},{"id":98623393,"identity":"84515ef0-58a6-4445-8531-46e1a8a530dc","added_by":"auto","created_at":"2025-12-19 17:06:05","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61275,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/850cd6f255a5604f36d57885.png"},{"id":98624033,"identity":"f55daf98-4ab8-46df-96bd-c7148d409124","added_by":"auto","created_at":"2025-12-19 17:07:55","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":62225,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/e1bce9ce87357246d3e7b7cc.png"},{"id":98482254,"identity":"4355dfd8-c3a6-4ac6-aaff-f1a7793e6981","added_by":"auto","created_at":"2025-12-18 05:29:14","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139878,"visible":true,"origin":"","legend":"","description":"","filename":"6c86c54007ac48d48647ad98faaed2971structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/4ba10a6e0ee83e1e3e39534f.xml"},{"id":98623337,"identity":"ff63a609-58f0-44b5-9322-dce39a92c549","added_by":"auto","created_at":"2025-12-19 17:05:51","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152640,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/77fc2cbe66e99a39e03bc9b4.html"},{"id":98482222,"identity":"8cea45f8-2671-4388-b1b2-b94d9821dd9a","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160457,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of the triabody. Two combinations were used for designing a triabody from two ds-scFvs of anti-A, and anti-B and dsFv of anti-Rh(D). \u003cstrong\u003eA) \u003c/strong\u003eIn the first combination, VH and VL chains of anti-Rh(D) were placed at the C-terminal region of fusion proteins and \u003cstrong\u003eB) \u003c/strong\u003ein the second combination these chains were placed at the N-terminal region of fusion proteins. \u003cstrong\u003eC)\u003c/strong\u003e Final design of triabody required from the two possible combinations of fusion proteins.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/7d374fc0d76988e9198d90ea.png"},{"id":98624675,"identity":"1094289f-8fac-45e4-9897-6fc8466b4f44","added_by":"auto","created_at":"2025-12-19 17:08:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":638790,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagrams of recombinant fusion protein constructs. A) Amino acid sequence of the first fusion protein encoded by a gene construct inserted into the pET-28a (+) plasmid. The protein of interest includes variable chains of anti-A ds-scFv fused to the VH chain of anti-Rh(D) dsFv, followed by a 6X-His tag. B) Amino acid sequence of the second fusion protein encoded by a gene construct inserted into the pET-21 (+) plasmid. The protein of interest consists of variable chains of anti-B ds-scFv fused to the VL chain of anti-Rh(D) dsFv.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/159528688b3944e6750a6be3.png"},{"id":98482224,"identity":"91099790-5117-492c-9dc5-3f1d77526640","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1101259,"visible":true,"origin":"","legend":"\u003cp\u003eTriabodies generated from the pairing of two fusion proteins from each combination. A) Pairing of fusion proteins of first combination produced both functionally closed (C1) and open (O2) form of triabody. B) In second combination, pairing of fusion proteins FP-2a and FP-2b produced one non-functionally closed (C2) and one functionally open (O2) form of triabody.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/52fb1019684f3349b7ffd6ae.png"},{"id":98623978,"identity":"eb2691c1-ca8c-4c06-a88f-24f0605abf27","added_by":"auto","created_at":"2025-12-19 17:07:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1066761,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of molecular dimensions of antibody and triabodies. A) Dimensions of IgG1 antibody. B) Dimensions of closed form of triabody-C1. C) Dimensions of open form of triabody-O1. D) Superimposed models of IgG1 antibody and triabody-C1. E) Superimposed models of IgG1 antibody and triabody-O1.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/05013376c7323f238ac2ffda.png"},{"id":98623885,"identity":"c7327f89-cb5b-4402-b6b9-40b052070124","added_by":"auto","created_at":"2025-12-19 17:07:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":845675,"visible":true,"origin":"","legend":"\u003cp\u003eA) TMB stained western blot showing purification of trifunctional triabody in a three-step process. Lanes 5-7 show purification through Ni\u003csup\u003e2+\u003c/sup\u003e-NTA chromatography. E1 shows purified his-tagged proteins at ~45 kDa, ~83 kDa and ~87 kDa. Lanes 8 and 9 show second purification through Native PAGE gel. N1 (lane 8) and N2 (lane 9) show bands at ~87 kDa and ~83 kDa respectively. Lanes 10-15 show last purification through acid-glycine elution. AE1-A1 (lane 10), AE2-RhD1 (lane 11) and AE3-B1 (lane 12) show purification of functional triabody form N1 fraction with functional A, Rh(D) and B sites, respectively. AE1-A2 (lane 13), AE2-RhD2 (lane 14) and AE3-B2 (lane 15) show purification of functional triabody from N2 fraction with functional A, Rh(D) and B sites, respectively. B) Monospecific anti-A his-tagged triabodies purified in E1, N1 and N2 elution fractions. C) Monospecific anti-A his-tagged half triabody purified in E1 elution fraction. D) Trivalent triabodies purified in E1, N1, N2, AE3-B1 and AE3-B2 elution fractions.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/5181ef04406932bef9c596c1.png"},{"id":98624563,"identity":"39d466ca-7eff-44a7-966e-6819b5f4d790","added_by":"auto","created_at":"2025-12-19 17:08:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1722460,"visible":true,"origin":"","legend":"\u003cp\u003eA) Hemagglutination testing. Well 1 shows control (A+ RBCs in anti-A IgM antibodies). Wells 2 to 5 shows AB+, AB−, A+ and B+ RBCs in triabody from AE3-B1 fraction. Well 6 shows AB+ RBCs in triabody from AE3-B2 fraction. B-G) TMB-stained immunocytochemistry. B) (control) Giemsa-stained A+ RBCs show complete hemagglutination in the presence of anti-A IgM antibodies. C) Mixed-field hemagglutination of AB+ RBCs coated with triabody from AE3-B1 fraction. D) Mixed-field hemagglutination of AB− RBCs coated with triabody from AE3-B1 fraction. E). No hemagglutination of A+ RBCs coated with triabody from AE3-B1 fraction. F) No hemagglutination of B+ RBCs coated with triabody from AE3-B1 fraction. G) No hemagglutination of AB+ RBCs coated with triabody from AE3-B2 fraction.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/6b1f5f308d0f73471866cb06.png"},{"id":98624282,"identity":"76325b87-1be1-494f-9442-2b8799b8b646","added_by":"auto","created_at":"2025-12-19 17:08:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":339339,"visible":true,"origin":"","legend":"\u003cp\u003eBinding cooperativity of the triabody with free blood group antigens. The binding parameters K\u003csub\u003eD\u003c/sub\u003e\u003cbr\u003e\nand B\u003csub\u003emax\u003c/sub\u003e are shown for A) anti-A, B) anti-B and C) anti-Rh(D) binding sites of triabody. The x-axis represents the triabody log concentration (μM), and the y-axis represents the absorbance at 450 nm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/7bd864acc23b635aeeb86038.png"},{"id":98623315,"identity":"27706acc-3570-47ef-99c5-e5c14ba0f788","added_by":"auto","created_at":"2025-12-19 17:05:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":364931,"visible":true,"origin":"","legend":"\u003cp\u003eBinding cooperativity of the triabody with RBC-bound blood group antigens. The binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e are shown for A) anti-A ds-scFv, B) anti-B ds-scFv and C) anti-Rh(D) ds-scFv. The x-axis represents the ds-scFv log concentration (μM), and the y-axis represents the absorbance at 450 nm.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/c249e33aca0e0d375abd5af4.png"},{"id":98482241,"identity":"c9314a4e-fb25-45d7-b93d-b87d20be4248","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":992731,"visible":true,"origin":"","legend":"\u003cp\u003eBlood type incompatibility related hemagglutination assay. A) Control wells consisting of hemagglutinated RBCs in corresponding blood group IgM antibodies. B) RBCs coated with triabody from the AE3-B2 fraction in blood group IgM antibodies.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/de2757918c84837f2fc9d528.png"},{"id":104739948,"identity":"af096dd9-a635-4bfb-b86e-8018cc56ff28","added_by":"auto","created_at":"2026-03-16 16:13:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8381540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/ec586c4e-d845-45cd-9ba9-7445f4ae379e.pdf"},{"id":98482230,"identity":"007ac5d0-abe4-4c54-b3bc-bb5bbf113256","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2920223,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTriabody.docx","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/7da8812befd4241b0dce2687.docx"},{"id":98482226,"identity":"d5730362-26e1-4f87-9a50-a07f8c6dc7ea","added_by":"auto","created_at":"2025-12-18 05:29:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1058754,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8369029/v1/5a9d09bcc024143bb146f55e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTransfusion medicine has long been an integral part of modern medicine, which relies heavily on blood products received from voluntary blood donors. A blood transfusion frequently represents the difference between life and death during medical emergencies, critical surgical procedures, and for patients with long-term medical conditions. A critical factor in achieving a successful transfusion is ensuring blood type compatibility, as mismatches can elicit severe and potentially fatal immune responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This compatibility is governed by the presence of specific antigens on the surface of red blood cells (RBCs) and the corresponding antibodies found in the plasma [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. RBCs clump together in the presence of these antibodies through a process called agglutination [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe effectiveness of hemagglutination mainly depends on the type of antibody involved i.e., immunoglobulin IgG or IgM. The IgM antibody, a large pentameric antibody, is capable of binding ten antigens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Its multivalency and considerable size make it highly effective at bridging the distance between multiple RBCs. In particular, its large molecular structure enables it to overcome the natural repulsive forces that keep RBCs apart. The anti-A and anti-B antibodies belonging to the major ABO blood group system are typically IgM. Consequently, transfusions related to ABO blood type incompatibility lead to a rapid and immediate hemagglutination reaction, which can result in a life-threatening acute hemolytic transfusion reaction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, IgG antibodies are smaller and bivalent (able to bind only two antigens) in nature. Due to their compact size, IgG antibodies can sensitize the RBCs but are inefficient at bridging the gap between RBCs to overcome the negative zeta potential [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, IgG-mediated hemagglutination is not directly visible and requires specialized techniques such as antiglobulin testing, also known as the Coombs test, which utilizes anti-human antibodies (secondary antibodies) to enable visible clumping of cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Antibodies belonging to the Rh blood group system are predominantly IgG in nature. An initial exposure of an Rh(D)-negative individual to Rh(D)-positive blood can lead to the development of anti-Rh(D) antibodies. A subsequent blood transfusion of Rh(D)-positive blood can lead to a delayed hemolytic transfusion reaction [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination of the ABO and Rh blood group systems gives us various blood types. Among these, O-negative RBCs hold a special position as \"universal donors\" due to the absence of A, B and Rh(D) antigens. The lack of immunogenic antigens makes them invaluable in time-sensitive situations where patient\u0026rsquo;s blood type is unknown, or their true blood type has been masked by a recent blood transfusion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite their indispensable role, the supply of O-negative blood is limited and often faces significant depletion due to the constant high demand. This persistent need for a constant supply of blood highlights the need for readily available universally compatible blood [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This challenge is more prominent and acute in blood deserts. Blood deserts are geographical regions where 75% of blood transfusion cases are deprived of timely and affordable blood components [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe urgent requirement for universal blood has driven the research into alternative solutions, including the development of blood substitutes such as perfluorocarbons (PFCs), hemoglobin-based oxygen carriers HBOCs and RBC substitutes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], enzymatic conversion of A and B blood types to O type blood [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], masking or blocking the surface antigens to increase compatibility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and the generation of cultured RBCs from stem cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While these strategies hold promise, no such alternative solution has been approved for blood transfusion yet.\u003c/p\u003e \u003cp\u003eGiven the advantages of O-negative blood and its persistent demand, the present study introduces a uniquely designed trispecific triabody as a blocking fragment to address situations where immediate cross-matching is not available and to increase the pool of usable blood for emergency use. Building upon previous antigen blocking strategies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], our research introduces a uniquely designed trispecific triabody to block the A, B and Rh(D) blood group antigens. Besides its blocking capabilities, the triabody\u0026rsquo;s small and compact size is an important feature that allows it to effectively block antigens without causing hemagglutination, thus ensuring a safe method for making RBCs universally compatible. This could drastically improve the outcomes in underserved regions by providing readily available blood products, especially packed red blood cells (pRBCs) for transfusion when compatible blood is unavailable.\u003c/p\u003e \u003cp\u003eHere, we computationally designed and modeled all possible structures of the triabody. Molecular docking was performed between the selected structure of the triabody and blood group antigens A, B and Rh(D), followed by computational prediction of intramolecular cooperative binding affinities. Next, triabody was produced from \u003cem\u003eE.coli\u003c/em\u003e BL21(DE3) using a two-plasmid system and purified through a three-step process. The immunocytochemistry (ICC) technique was then performed to microscopically determine its hemagglutination capabilities. To determine intramolecular binding cooperativity, ELISA-based experiments were conducted using both free and RBC-bound antigens. Finally, RBC-bound antigens were blocked by the triabody, and a hemagglutination assay was performed to evaluate its effectiveness as a blocking fragment in preventing RBC hemagglutination.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Software, web servers and chemicals\u003c/h2\u003e \u003cp\u003eAll the software, web servers, chemicals and molecular biology products used in this study are mentioned in the supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Protein designing, modeling and refinement\u003c/h2\u003e \u003cp\u003eThe sequences of anti-A and anti-B fragment variables (Fvs) were taken from PDB: 1JV5 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and research article published by Santos-Esteban and Curiel‐Quesada (2001) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] respectively. The sequences of anti-Rh(D) variable light (VL) and heavy (VH) chains were taken from accession nos AAC13488.1 (anti-Rh(D) VL chain) and AAC13447.1 (anti-Rh(D) VH chain) respectively [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Both chains of anti-A and anti-B Fvs were joined from VH to VL via a (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e3\u003c/sub\u003e flexible linker (VH-Linker-VL). Interdomain disulfide bonds were added into each Fv at the site described by Hafeez and Zaidi (2024) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Zhao et al. (2010) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After the addition of disulfide bonds, the sequences were arranged into two combinations with two fusion proteins in each, as shown in detail in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The complete disulfide-stabilized single chain fragment variable (ds-scFv) in each fusion protein of both combinations was joined to the variable chain of the other Fv through the hinge region (EPKSCDKTHTCPPCPAPELLGGP) of the IgG1 antibody [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. All proteins were modeled through the I-TASSER web server [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Models with correct domain locations and orientations of VH-VL were joined together through molecular docking using LZerD web server [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] to form a triabody. Best structures were selected on the basis of the accessibility of the binding site, i.e., all structures in which the binding site was not easily accessible for interaction were discarded. PyMOL 3.1 was used to measure all the molecular dimensions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Theoretical molecular weights were predicted through the ProtParam tool [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe selected structures were protonated to pH 7.4 using the PrepareProtein tool of the PlayMolecule web server [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The protonation states of amino acid residues (histidine, aspartic acid, and glutamic acid) with pKa values close to pH 7.4 were manually assigned. Intramolecular disulfide bonds were preserved while assigning protonation states. For intermolecular disulfide bonds, hydrogen atoms were removed from sulfur group of cysteine residues and bonds were formed via pdb2gmx with (-ss) and (-merge all) flags on GROMACS 2024.2 software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Molecular dynamics (MD) simulations were conducted to refine the structures of the triabodies. Simulations were performed for 100 ns using the Amber force field ff99SB at a temperature of 298 K in an explicit water environment, using a TIP3P model. Each protein was solvated in a cubic box and neutralized by adding Na\u0026thinsp;+\u0026thinsp;and Cl\u0026thinsp;\u0026minus;\u0026thinsp;ions at concentration of 0.154 M.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Modeling of antigens and molecular docking\u003c/h2\u003e \u003cp\u003eThe structures of the trisaccharide antigens (A-trisaccharide and B-trisaccharide) were built using the carbohydrate building tool of the Glycam web server [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The protein sequence of the antigen Rh(D) was taken from a research article published by Conroy et al. (2005) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and modeled using the I-TASSER web server. Molecular docking was performed to study the interaction between blood group antigens and the triabody. The SwissDock web server [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] was used to study the interaction of trisaccharide antigens with triabody. The grid boxes were focused on the respective antigen binding sites for each trisaccharide. For protein-protein docking, the antigen Rh(D) was first protonated to pH 7.4 as mentioned above in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e and then docked using the LZerD web server.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 MD simulations\u003c/h2\u003e \u003cp\u003eFor MD simulations, best docked structures in the case of trisaccharide antigens were selected on the basis of a single criterion: the terminal monosaccharides should interact. The selection of the best protein-protein docked structure was based on the location of the complex formation. MD simulations were performed to study the stability of the interactions of each antigen with the triabody under physiological conditions (Temp: 310 K, NaCl: 0.154 M and pH 7.4). Simulations were performed for 150 ns on GROMACS 2024.2 software using the Amber force field ff99SB for proteins. For trisaccharide, the Glycam-06h force field was used to generate topology using the ACPYPE tool of the AmberTools24 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Binding free energy calculations\u003c/h2\u003e \u003cp\u003eMolecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations were performed to estimate the binding free energy (ΔG\u003csub\u003eBind\u003c/sub\u003e) of the triabody-antigen complexes using the MmPbAaStat.py script within the g_mmpbsa software tool [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The final 10 ns of MD simulations for each monomer within the triabody was considered the equilibrium phase and was used for energy calculations. To computationally predict the intramolecular cooperative binding affinities, blood group antigens A, B and Rh(D) were sequentially docked in all possible orders: A\u0026rarr;B\u0026rarr;Rh(D), A\u0026rarr;Rh(D)\u0026rarr;B, B\u0026rarr;A\u0026rarr;Rh(D), B\u0026rarr;Rh(D)\u0026rarr;A, Rh(D)\u0026rarr;A\u0026rarr;B and Rh(D)\u0026rarr;B\u0026rarr;A. MD simulations were performed after each docking step as mentioned above in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e. Binding free energies were then calculated through MMPBSA to determine a trend in cooperative binding affinities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Production and purification\u003c/h2\u003e \u003cp\u003eTriabody molecule was expressed using two expression plasmids: pET-28a (+) and pET-21 (+). These plasmids harbor kanamycin and ampicillin resistance genes respectively. The designs of both recombinant fusion protein constructs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. For expression in pET-28a (+), the first fusion protein consisting of genes encoding anti-A ds-scFv and the VH chain of anti-Rh(D) dsFv was modified to include the genes encoding the OmpA signal peptide at the N-terminus and a 6X histidine tag at the C-terminus. This gene construct was then inserted between the NcoI and XhoI restriction sites. Similarly, the gene construct of the second fusion protein of the triabody consisting of genes encoding anti-B ds-scFv and the VL chain of anti-Rh(D) dsFv, was placed between the BamHI and XhoI restriction sites of the pET-21 (+) plasmid after the genes encoding the ribosomal binding site (RBS) and OmpA signal peptide at the N-terminal region were added. Both recombinant plasmids were obtained from Twist Bioscience (USA) and subsequently cotransformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) via the heat shock method. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor large-scale production, a 100 ml overnight culture of \u003cem\u003eE.coli\u003c/em\u003e BL21 (DE3) was used to inoculate 5 L of LB media supplemented with (25 \u0026micro;g/ml) kanamycin, (50 \u0026micro;g/ml) ampicillin and (1%) glucose. Incubation was performed at 37\u0026deg;C under constant shaking at 200 rpm until an OD\u003csub\u003e600\u003c/sub\u003e of 0.6 was reached. The culture was cooled to 18\u0026deg;C before slow induction was carried out with 0.25 mM IPTG at 18\u0026deg;C for 20 hours. The cells were harvested following induction and total proteins were extracted via sonication as described by Hafeez and Zaidi (2024) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The crude extract was collected and filtered through a 0.45 \u0026micro;m syringe filter. Western blotting was performed for the analysis of protein samples.\u003c/p\u003e \u003cp\u003eA trispecific triabody was purified via a three-step process. Initially, nickel affinity chromatography (Ni\u003csup\u003e2+\u003c/sup\u003e-NTA) was used to isolate the his-tagged triabodies. This was followed by extraction of different-sized triabodies from the Native PAGE gel and finally by acid-glycine elution to separate the functional triabodies.\u003c/p\u003e \u003cp\u003eIn the second step the Ni\u003csup\u003e2+\u003c/sup\u003e-NTA purified product was loaded onto a 10% Native PAGE gel. After the run, the protein ladder was used as a reference and the gel was divided into sections corresponding to molecular weights ranging from approximately 70\u0026ndash;84 kDa, and 85\u0026ndash;100 kDa. Each gel piece was placed in a 2 ml Eppendorf tube containing 0.5 ml Tris-buffered saline (TBS) (50 mM Tris, 154 mM NaCl, pH 7.4) and crushed with a Teflon pestle. The tubes were incubated at 37\u0026deg;C with shaking for 24 hours, followed by centrifugation and resuspension every 3 hours. After 24 hours, the tubes were centrifuged at 5,000 rpm, and the supernatant was analyzed via the western blotting.\u003c/p\u003e \u003cp\u003eThe third purification step involved the sequential purification of triabodies using blood type A\u0026minus;, B\u0026thinsp;\u0026minus;\u0026thinsp;and O\u0026thinsp;+\u0026thinsp;RBCs. The previously purified product (0.5 ml) was incubated with blood type A RBCs (10 \u0026micro;l) for 30 mins to purify triabodies with functional A binding sites. After incubation, unbound triabodies were removed by washing with TBS buffer A (pH 7.4). The bound triabodies were then detached by using 0.25 ml of Glycine-HCl buffer B (50 mM Glycine, pH 3.5). The final eluate-A was neutralized by using 1 ml of TBS buffer C (pH 8.5). Eluate-A was subsequently used to purify triabodies with functional Rh(D) binding sites. The resulting eluate-Rh(D) eluate served as the starting material for the purification of triabodies with functional B binding sites. The final product was lyophilized and stored at \u0026minus;\u0026thinsp;70\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunocytochemistry (ICC)\u003c/h2\u003e \u003cp\u003eImmunocytochemistry (ICC) was performed to microscopically determine the ability of the triabody to cause hemagglutination of RBCs. Prior to ICC, RBCs from the AB+, AB\u0026minus;, A\u0026thinsp;+\u0026thinsp;and B\u0026thinsp;+\u0026thinsp;blood types were also visibly checked for hemagglutination reactions before being analyzed microscopically. Briefly, 5 \u0026micro;l of RBCs were incubated for 30 mins under gentle shaking in 0.5 ml of 250 \u0026micro;M triabody solution. To prevent hemagglutination due to primary and secondary antibodies, triabody-coated RBCs were first diluted in TBS buffer (pH 7.4) and then smeared onto glass slides. The smears were air dried and fixed with methanol for 1 min. Next, primary rabbit anti-his tag polyclonal antibodies (1:1000 dilution) were added, and the samples were incubated at room temperature for one hour. Following incubation, the slides were washed with TBS buffer (pH 7.4) before being incubated with a secondary HRP-conjugated caprine anti-rabbit IgG polyclonal antibody (1:10000 dilution). After incubation, the slides were washed with TBS buffer (pH 7.4), and staining was performed via 3,3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine (TMB) staining method as described by Woiszwillo (1991) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. To prevent hemolysis, the smears were exposed to the working TMB precipitating solution (2 mM TMB, 0.1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and precipitating polymers: 0.1% alginic acid, 0.1% methyl vinyl ether/ maleic anhydride copolymer, 0.1% dextran sulfate and 0.3% carrageenan) for just 5 secs and then washed with TBS buffer (pH 7.4). This process was repeated until sufficient color was developed for optical microscopy. Any triabody molecule sample that caused hemagglutination of RBCs was excluded from further studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of intramolecular binding cooperativity of the triabody via free blood group antigens\u003c/h2\u003e \u003cp\u003eIntramolecular binding cooperativity of the triabody was further investigated through specialized ELISA-based experiments. The aim was to understand how the binding of one antigen affects the binding of other antigens. The experimental sequence began with immobilizing anti-his tag IgG antibodies (10 \u0026micro;g/ml) on protein A/G precoated plates, followed by the addition of purified his-tagged triabodies in twofold serial dilutions ranging from 0.2 nM to 100 \u0026micro;M. In studying the binding order A\u0026rarr;B\u0026rarr;Rh(D), the first binding event involved saturating the A binding sites with the A-trisaccharide, followed by saturation of the B binding sites with the B-trisaccharide (second binding event), and finally, the Rh(D) binding site was saturated with the antigen Rh(D) (third binding event). An overview of the process is given in Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The concentrations of A-trisaccharide, B-trisaccharide and antigen Rh(D) used for determining the binding cooperativity of triabody are given in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe binding parameters, including the dissociation constant K\u003csub\u003eD\u003c/sub\u003e​ and maximal binding B\u003csub\u003emax\u003c/sub\u003e of the B and Rh(D) binding sites were determined via indirect ELISA as described by Syedbasha et al. (2016) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] using unbound B-trisaccharide and the antigen Rh(D). To determine the changes in the binding parameters of the B binding site, a neoglycoconjugation process was used in which unbound B-trisaccharide was covalently coupled to bovine serum albumin (BSA) via a homobifunctional ethylenediamine (EDA) linker. This procedure proceeded with the coating of BSA (20 \u0026micro;g/ml dissolved in 0.1 M carbonate-bicarbonate buffer pH 9.6) onto 96-well plates overnight at 4\u0026deg;C. The next day, wells were washed several times with 200 \u0026micro;l of 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6). A freshly prepared solution of EDC and Sulfo-NHS (5 mM each, 1:1 molar ratio) in 0.1 M MES buffer was then added (100 \u0026micro;l per well) and incubated for 30 mins at room temperature with mild shaking. Following incubation, the wells were washed three times with 0.1 M MES buffer (pH 6).\u003c/p\u003e \u003cp\u003eFor the coupling of EDA to BSA, an excess concentration of EDA (100 mM) was prepared in 0.1 M MES buffer (pH 6). Following the washing step, 200 \u0026micro;l of EDA solution was added to each well and incubated overnight at 4\u0026deg;C. Immediately after incubation, the wells were washed three times with a 0.5 M solution of sodium borate Na\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e (pH 8.5).\u003c/p\u003e \u003cp\u003eA reductive amination reaction was performed to facilitate the attachment of B-trisaccharide to the BSA-EDA conjugates. The plates were incubated for 96 hours at 56\u0026deg;C with 100 \u0026micro;l of the unbound B-trisaccharide solution (from second binding). Simultaneously, a 50 \u0026micro;l solution, composed of 0.5 M sodium borate Na\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e4\u003c/sub\u003eO (pH 9), 1.5 M sodium sulfate Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and 0.75 M sodium cyanoborohydride NaBH\u003csub\u003e3\u003c/sub\u003eCN was added. Following incubation, the wells were washed rigorously three times with distilled water. An indirect ELISA was performed in which 100 \u0026micro;L of 10 mg/ml his-tagged triabody was used to detect B-trisaccharide. This was followed by sequential incubation with 100 \u0026micro;l of primary anti-his tagged (1:1000 dilution) and secondary HRP-conjugated antibodies (1:10000 dilution). The TMB substrate reaction was performed for 15 mins and stopped with 0.5 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Absorbance readings were taken at 450 nm, and the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e were determined via nonlinear regression curve fitting using GraphPad Prism Version 9.5.1 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and compared with other binding events.\u003c/p\u003e \u003cp\u003eFollowing the determination of the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e of the B-trisaccharide, the antigen Rh(D) was introduced and allowed to saturate the Rh(D) binding sites (third binding event). Unbound antigen Rh(D) were collected and coated on ELISA plates. An indirect ELISA was performed as described above (using triabody), and the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e were determined and compared with those of the other binding events. Similarly, the intramolecular binding cooperativity of triabody was studied in other orders A\u0026rarr;Rh(D)\u0026rarr;B and B\u0026rarr;A\u0026rarr;Rh(D), B\u0026rarr;Rh(D)\u0026rarr;A, Rh(D)\u0026rarr;A\u0026rarr;B and Rh(D)\u0026rarr;B\u0026rarr;A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Determination of intramolecular binding cooperativity of the triabody via RBC-bound blood group antigens\u003c/h2\u003e \u003cp\u003eTo determine the intramolecular binding cooperativity of a triabody designed to block blood group antigens on RBCs, an ELISA-based experiment was performed. In this experiment the CDR-grafted humanized nanobodies (VHH) of anti-A, anti-B and anti-Rh(D) were designed and used as blocking fragments of antigens A, B and Rh(D) on RBCs.\u003c/p\u003e \u003cp\u003eTo make the CDR-grafted humanized nanobodies, the CDR sequences of the anti-A, anti-B and anti-Rh(D) scFvs were taken and grafted onto the framework sequence of the camelid nanobody (Supplementary Figure S2). After that, the whole sequence was humanized via llamanade web server [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Nanobodies were then produced from \u003cem\u003eE.coli\u003c/em\u003e BL21(DE3) and the effect of changes in pH on the interaction of complexes of both triabody with antigens and nanobodies with antigens was determined. The criteria for the selection of nanobodies as blocking fragments were as follows: 1) the first blocking nanobody must be sensitive to slight fluctuations in pH compared with the second blocking nanobody, and 2) pH-dependent dissociation must occur at a different pH from that of the triabody. The pH values required for the complete dissociation of several blood group nanobodies are given in Supplementary Table S2. The selected blocking nanobodies (Supplementary Table S3) were then resuspended in TBS buffer (pH 7.4) in a concentration of 10 mg/ml.\u003c/p\u003e \u003cp\u003eTo study the order, A\u0026rarr;B\u0026rarr;Rh(D) (an overview of the process is given in Supplementary Figure S3), AB\u0026thinsp;+\u0026thinsp;RBCs (1 \u0026micro;l) were immobilized on ELISA plates using 0.3% glutaraldehyde as previously described by Koganei (2007) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and incubated in 100 \u0026micro;l of a solution of anti-B VHH (first blocking) for one hour under slow shaking. A similar blocking step was followed for anti-Rh(D) (second blocking) with one modification: an anti-Rh(D) scFv was used instead of a nanobody in the binding orders of A\u0026rarr;B\u0026rarr;Rh(D) and B\u0026rarr;A\u0026rarr;Rh(D). In other binding orders anti-Rh(D) VHH was used. Unbound blocking fragments were removed by washing several times with TBS buffer (pH 7.4). After blocking antigens B and Rh(D), the A binding sites of triabody were allowed to interact with antigens A on the RBC by incubating 100 \u0026micro;l of triabody (100 \u0026micro;M in TBS buffer, pH 7.4) for 30 mins under slow shaking. After incubation, the wells were washed three times with TBS buffer (pH 7.4). Following washing, first blocking fragment i.e., anti-B VHH was removed by washing three times with TBS (pH 7.83) buffer. After washing, 100 \u0026micro;l of TBS buffer (pH 7.4) was added, and the triabody\u0026rsquo;s B binding sites were allowed to interact with antigens B on the RBC surface. After antigen B was saturated, 100 \u0026micro;l of his-tagged anti-B ds-scFv (the concentrations and dilutions of all three ds-scFvs were the same as those of the triabody, as provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which had the same sequence as the triabody\u0026rsquo;s anti-B ds-scFv, was added, and allowed to interact with unbound antigen B or to displace weakly bound B binding sites for 30 min under constant shaking. Unbound anti-B ds-scFv was taken and indirect ELISA was performed using primary and secondary antibodies as mentioned above in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.8\u003c/span\u003e. The binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e were determined via nonlinear regression curve fitting using GraphPad Prism Version 9.5.1 and compared with the binding parameters of same antigen B in other orders.\u003c/p\u003e \u003cp\u003eThe same procedure was followed to dissociate the second blocking fragment anti-Rh(D) scFv. The binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e were determined by measuring the unbound his-tagged anti-Rh(D) ds-scFv via indirect ELISA and compared with the binding parameters of the same antigen in other orders. A similar process was used to measure the intramolecular binding cooperativity for other binding orders, such as A\u0026rarr;Rh(D)\u0026rarr;B, B\u0026rarr;A\u0026rarr;Rh(D), B\u0026rarr;Rh(D)\u0026rarr;A, Rh(D)\u0026rarr;A\u0026rarr;B, and Rh(D)\u0026rarr;B\u0026rarr;A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Coating of RBCs with a triabody and hemagglutination assay\u003c/h2\u003e \u003cp\u003eThe trispecific triabody was reconstituted in TBS buffer (pH 7.4) and then divided into 100 \u0026micro;l aliquots at a concentration of 100 \u0026micro;M. Approximately 10 \u0026micro;l of RBCs from ABO and Rh(D) blood types (A+, B+, O+, AB+, A\u0026minus;, B\u0026thinsp;\u0026minus;\u0026thinsp;and AB\u0026minus;) were incubated in 100 \u0026micro;l aliquots of triabody at 37\u0026deg;C for 30 mins. Following incubation, the RBCs were washed with TBS buffer (pH 7.4), and this process was repeated with fresh aliquots of triabody. The remaining triabody in the supernatant was quantified via indirect ELISA by using free antigens as described in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.8\u003c/span\u003e. The incubation of RBCs with the triabody was repeated until no further changes in the triabody concentration in the supernatant was observed.\u003c/p\u003e \u003cp\u003eAn immediate hemagglutination assay was performed to evaluate both the effectiveness of the triabody coating and its ability to block all blood group antigens. This involved incubating 1 \u0026micro;l of triabody-coated RBCs with 100 \u0026micro;l of each of the following reagents: anti-A IgM, anti-B IgM, and anti-Rh(D) IgM antibodies for 10 mins at room temperature.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Structures of triabody\u003c/h2\u003e \u003cp\u003eOn the basis of the placement of variable chains of anti-Rh(D), two combinations were used to model the molecules of the triabody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the fusion proteins FP-1a and FP-1b generated from combination one showed correct pairing of VH chains of both anti-A and anti-B ds-scFv with their respective VL chains. On the other hand, three types of fusion proteins (FP-2a, FP-2b and FP-2c) were observed from combination two. Like combination one, the fusion proteins FP-2a and FP-2b in combination two exhibited correct pairing of variable chains. However, the fusion protein FP-2c exhibited the interaction of VL chain of anti-Rh(D) with the VH chain of anti-B ds-scFv.\u003c/p\u003e \u003cp\u003eFor triabodies to effectively block blood group antigens, all three of their antigen binding sites must be freely accessible. Two types of triabody structures were produced from both combinations. Depending on the location of the disulfide bond formed these structures were in closed and open forms with intramolecular and intermolecular disulfide bonds respectively. The docking of two fusion proteins from combination one produced a closed triabody-C1 form with the antigen binding sites of all three Fv facing outward and an open triabody-O1 form where all the binding sites were easily accessible. Docking of two fusion proteins from combination two with correct pairing (FP-2a and FP-2b) produced an open triabody-O2 form similar to triabody-O1 of combination one and a closed triabody-C2 form with antigen binding sites of anti-A and anti-B ds-scFv facing outward and antigen binding site of anti-Rh(D) facing inward (blocked by intermolecular disulfide bond).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the best structures of triabodies were obtained from the fusion proteins of combination one. The selected triabodies had ten disulfide bonds (eight intramolecular and two intermolecular) in the closed C1 form and eleven disulfide bonds (ten intramolecular and one intermolecular) in the open O1 form. The predicted theoretical weight of both triabodies was 83.3 kDa. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the comparison of triabodies with the IgG1 antibody structure PDB: 1IGY [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The overall distance between the binding sites of triabody-C1 is half the distance between the binding sites of the IgG1 antibody. The length of triabody-O1 (14.3 nm) is almost the same as the distance between the binding sites (14.5 nm) of an IgG1 antibody. Additional computational studies such as molecular docking and MMPBSA were conducted on closed structure i.e., triabody-C1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Functional analysis\u003c/h2\u003e \u003cp\u003eMMPBSA calculations were performed to predict the intramolecular cooperative binding affinities of the triabody. The results shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e revealed no significant changes in the binding free energies of the complexes formed between the triabody and each blood group antigen, regardless of the binding order. For instance, the binding free energy of the triabody\u0026ndash;antigen A complex remained consistently around \u0026minus;\u0026thinsp;13.20 kcal/mol, irrespective of whether antigen A was the first, second, or third to bind. Similarly, the binding free energies of the triabody\u0026ndash;antigen B and triabody\u0026ndash;antigen Rh(D) complexes were approximately \u0026minus;\u0026thinsp;12.6 kcal/mol and \u0026minus;\u0026thinsp;49 kcal/mol, respectively. These results indicate a lack of intramolecular cooperativity, meaning that the binding of one antigen is independent of the binding of the others. Supplementary Figure S4 shows snapshots from the MD trajectories of the triabody complexes with all three blood group antigens at the beginning and end of the 150 ns simulation following the binding sequence A\u0026rarr;B\u0026rarr;Rh(D).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding free energies (ΔG\u003csub\u003eBind\u003c/sub\u003e) kcal/mol computed by MMPBSA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBinding Sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFirst binding\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSecond binding\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eThird binding\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026rarr;B\u0026rarr; Rh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;13.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;12.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;48.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026rarr; Rh(D)\u0026rarr;B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;13.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;49.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;12.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u0026rarr;A\u0026rarr; Rh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;12.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;13.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;48.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u0026rarr; Rh(D)\u0026rarr;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;12.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;49.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;13.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRh(D)\u0026rarr;A\u0026rarr;B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;48.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;13.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;12.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRh(D)\u0026rarr;B\u0026rarr;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRh(D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;49.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;12.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026minus;13.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Three-step purification\u003c/h2\u003e \u003cp\u003eTriabody was produced under slow induction conditions (18\u0026deg;C and 0.25 mM IPTG) to allow proper folding of the protein. His-tagged proteins were purified via Ni\u003csup\u003e2+\u003c/sup\u003e-NTA chromatography (first purification step) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and analyzed via western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, in the Ni\u003csup\u003e2+\u003c/sup\u003e-NTA elution profile E1 (lane 7), the molecular weight of the triabody (~\u0026thinsp;83 kDa) was approximately the same as the theoretical weight predicted by the ProtParam tool. In addition to the predicted triabody, a larger triabody of ~\u0026thinsp;87 kDa and a half triabody of ~\u0026thinsp;45 kDa were also observed.\u003c/p\u003e \u003cp\u003eProtein extraction from Native PAGE gel was a second step in separating individual triabody species. The Ni\u003csup\u003e2+\u003c/sup\u003e-NTA elution profile E1 was taken, and Native PAGE was carried out. Subsequently, each individual triabody species was extracted from the gel. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, a larger triabody (lane 8) and desired-sized triabody (lane 9) were successfully isolated from a mixture of different-sized his-tagged proteins.\u003c/p\u003e \u003cp\u003ePurification through acid-glycine elution was the final crucial step in obtaining a trifunctional triabody. Following Native PAGE purification, the elution profiles for the N1 fractions were AE1-A1, AE2-RhD1, and AE3-B1, whereas the elution profiles for the N2 were AE1-A2, AE2-RhD2, and AE3-B2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the elution profiles AE1-A1 (lane 10) and AE1-A2 (lane 13) show bands at ~\u0026thinsp;87 kDa and ~\u0026thinsp;83 kDa respectively. These bands represent the purification of triabodies with functional A binding sites. Similarly, the bands observed in the elution profiles of AE2-RhD1 (lane 11) and AE2-RhD2 (lane 14) represent the purified bifunctional triabodies with functional A and Rh(D) binding sites. Finally, bands in elution profiles AE3-B1 (lane 12) and AE3-B2 (lane 15) show purified trifunctional triabodies (~\u0026thinsp;87 kDa and ~\u0026thinsp;83 kDa, respectively) with functional A, Rh(D) and B binding sites. Figures\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD depict the expected purified proteins in each step.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Immunocytochemistry (ICC)\u003c/h2\u003e \u003cp\u003eImmunocytochemistry (ICC) was performed to observe the hemagglutinating properties of the triabodies. For this purpose, RBCs with at least two blood group antigens were taken and coated with triabodies. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA no visible hemagglutination of RBCs was observed when these RBCs were incubated with triabodies from either the AE3-B1 (wells 2\u0026ndash;5) or the AE3-B2 (well 6) fraction. However, when examined microscopically, mixed-field hemagglutination was observed in AB+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and AB\u0026minus; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) RBCs that were incubated with triabody from the AE3-B1 fraction. In contrast, no hemagglutination was observed in A+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and B+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) RBCs incubated with the same triabody. Similarly, no hemagglutination was observed in AB\u0026thinsp;+\u0026thinsp;RBCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) incubated with triabody from the AE3-B2 fraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Determination of intramolecular binding cooperativity of the triabody via free and RBC-bound blood group antigens\u003c/h2\u003e \u003cp\u003eTo understand how the binding of one antigen affects the binding of others, the triabody\u0026rsquo;s intramolecular binding cooperativity was determined via ELISA-based experiments. Free antigens were used to assess whether the changes in binding patterns observed with RBC-bound antigens were a result of the triabody\u0026rsquo;s small size during initial and subsequent binding events.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, there were no significant overall changes in the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e, suggesting non-cooperative binding, i.e., subsequent binding was not influenced by prior binding events. The K\u003csub\u003eD\u003c/sub\u003e​ values for the anti-A, anti-B, and anti-Rh(D) binding sites of the triabody, when binding individually, were 0.790, 0.842, and 0.353 \u0026micro;M, respectively. For the binding orders where A-trisaccharide (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) was involved in the second binding event (B\u0026rarr;A\u0026rarr;Rh(D) and Rh(D)\u0026rarr;A\u0026rarr;B), the K\u003csub\u003eD\u003c/sub\u003e​ values were 0.808 and 0.797 \u0026micro;M respectively. Similarly, in the third binding event involving A-trisaccharide (B\u0026rarr;Rh(D)\u0026rarr;A and Rh(D)\u0026rarr;B\u0026rarr;A), the K\u003csub\u003eD\u003c/sub\u003e​ values were 0.801 and 0.815 \u0026micro;M respectively. The B\u003csub\u003emax\u003c/sub\u003e values of A-trisaccharide for both the second and third binding events remained close to the B\u003csub\u003emax\u003c/sub\u003e value of the initial binding event (1.36), with values of 1.39 and 1.35 for the second binding events (B\u0026rarr;A\u0026rarr;Rh(D) and Rh(D)\u0026rarr;A\u0026rarr;B), and 1.38 and 1.37 values for the third binding events (B\u0026rarr;Rh(D)\u0026rarr;A and Rh(D)\u0026rarr;B\u0026rarr;A).\u003c/p\u003e \u003cp\u003eIn the case where B-trisaccharide (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) was involved in the second binding event, the K\u003csub\u003eD\u003c/sub\u003e​ values were 0.832 \u0026micro;M and 0.836 \u0026micro;M for the orders A\u0026rarr;B\u0026rarr;Rh(D) and Rh(D)\u0026rarr;B\u0026rarr;A, respectively. In the third binding event, the K\u003csub\u003eD\u003c/sub\u003e​ values were 0.839 \u0026micro;M and 0.847 \u0026micro;M for the orders A\u0026rarr;Rh(D)\u0026rarr;B and Rh(D)\u0026rarr;A\u0026rarr;B, respectively. Similar to the A-trisaccharide, the B\u003csub\u003emax\u003c/sub\u003e values for the B-trisaccharide were close to the initial value of 1.30. The second binding events yielded B\u003csub\u003emax\u003c/sub\u003e values of 1.25 and 1.27 for the A\u0026rarr;B\u0026rarr;Rh(D) and Rh(D)\u0026rarr;B\u0026rarr;A binding orders, respectively. The third binding events produced B\u003csub\u003emax\u003c/sub\u003e values of 1.28 for the A\u0026rarr;Rh(D)\u0026rarr;B order and 1.31 for the Rh(D)\u0026rarr;A\u0026rarr;B order.\u003c/p\u003e \u003cp\u003eFor the second binding events in the orders A\u0026rarr;Rh(D)\u0026rarr;B and B\u0026rarr;Rh(D)\u0026rarr;A, which involve the antigen Rh(D) Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the K\u003csub\u003eD\u003c/sub\u003e values were 0.330 and 0.351 \u0026micro;M, respectively. A similar case was observed for the other binding orders, A\u0026rarr;B\u0026rarr;Rh(D) and B\u0026rarr;A\u0026rarr;Rh(D), which involve the antigen Rh(D) in the third binding position, with K\u003csub\u003eD\u003c/sub\u003e values of 0.337 and 0.348 \u0026micro;M, respectively. The changes in B\u003csub\u003emax\u003c/sub\u003e values for antigen Rh(D) were similar to those observed for A-trisaccharide and B-trisaccharide, i.e., the B\u003csub\u003emax\u003c/sub\u003e values for the second and third binding events were closer to the initial value (0.940). The B\u003csub\u003emax\u003c/sub\u003e values for the binding order A\u0026rarr;Rh(D)\u0026rarr;B and B\u0026rarr;Rh(D)\u0026rarr;A, involving Rh(D) in the second binding event, were 0.931 and 0.937, respectively. For the third binding events A\u0026rarr;B\u0026rarr;Rh(D) and B\u0026rarr;A\u0026rarr;Rh(D), the B\u003csub\u003emax\u003c/sub\u003e values were 0.935 and 0.938, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe intramolecular binding cooperativity of the triabody with RBC-bound antigens was indirectly assessed by measuring changes in the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e of the anti-A, anti-B, and anti-Rh(D) ds-scFvs. This approach was crucial for understanding the observed cooperativity and how it is influenced by the physical environment of the antigen and the small size of the triabody.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, antigen A exhibited higher K\u003csub\u003eD\u003c/sub\u003e values for both the second and third binding events compared with its initial binding (0.618 \u0026micro;M). When antigen B was bound first, the K\u003csub\u003eD\u003c/sub\u003e values for antigen A increased to 0.724 \u0026micro;M and 0.730 \u0026micro;M in the sequences B\u0026rarr;A\u0026rarr;Rh(D) and B\u0026rarr;Rh(D)\u0026rarr;A, respectively. A further increase was noted when antigen Rh(D) served as the first binding partner, with K\u003csub\u003eD\u003c/sub\u003e values reaching 0.748 \u0026micro;M and 0.755 \u0026micro;M for Rh(D)\u0026rarr;A\u0026rarr;B and Rh(D)\u0026rarr;B\u0026rarr;A, respectively. After initial binding to antigen B, the B\u003csub\u003emax\u003c/sub\u003e value for antigen A decreased from 1.33 to 1.27 and 1.24, whereas even lower values of 1.14 and 1.12 were observed when antigen Rh(D) was bound first. These results suggest that binding of antigen A is more affected when antigen Rh(D) binds first, showing negative cooperativity. Also, when antigen A was involved the third binding event, K\u003csub\u003eD\u003c/sub\u003e value was higher and B\u003csub\u003emax\u003c/sub\u003e value was lower compared to the second binding event.\u003c/p\u003e \u003cp\u003eIn contrast to antigen A, the K\u003csub\u003eD\u003c/sub\u003e values for the second and third binding events involving antigen B were found to remain comparable to those observed when antigen B was the first antigen to bind (0.517 \u0026micro;M) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB. When antigen A was bound first, the K\u003csub\u003eD\u003c/sub\u003e values for antigen B were 0.520 \u0026micro;M and 0.522 \u0026micro;M for the sequences A\u0026rarr;B\u0026rarr;Rh(D) and A\u0026rarr;Rh(D)\u0026rarr;B, respectively. In comparison, when antigen Rh(D) was involved in the initial binding event, higher K\u003csub\u003eD\u003c/sub\u003e values were observed, i.e., 0.533 \u0026micro;M and 0.540 \u0026micro;M for the sequences Rh(D)\u0026rarr;B\u0026rarr;A and Rh(D)\u0026rarr;A\u0026rarr;B, respectively. Following the initial binding of antigen A, the B\u003csub\u003emax\u003c/sub\u003e values for antigen B were observed to remain close to its initial value (0.954), at 0.941 and 0.926 for the sequences A\u0026rarr;B\u0026rarr;Rh(D) and A\u0026rarr;Rh(D)\u0026rarr;B, respectively. However, B\u003csub\u003emax\u003c/sub\u003e values were significantly reduced to 0.837 and 0.830 when antigen Rh(D) was the first antigen to bind, as observed in the sequences Rh(D)\u0026rarr;B\u0026rarr;A and Rh(D)\u0026rarr;A\u0026rarr;B (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), suggesting that negative cooperativity was introduced when antigen Rh(D) bound first. When the second and third binding events of antigen B were compared, higher K\u003csub\u003eD\u003c/sub\u003e and lower B\u003csub\u003emax\u003c/sub\u003e values were observed in the third binding event.\u003c/p\u003e \u003cp\u003eUnlike antigens A and B, the binding of antigen Rh(D) remained largely unaffected by prior binding events. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC when antigen Rh(D) was involved in the second binding event, the K\u003csub\u003eD\u003c/sub\u003e values were 0.292 \u0026micro;M and 0.291 \u0026micro;M for the sequences A\u0026rarr;Rh(D)\u0026rarr;B and B\u0026rarr;Rh(D)\u0026rarr;A, respectively, which were comparable to its initial binding value of 0.289 \u0026micro;M. The B\u003csub\u003emax\u003c/sub\u003e value was 0.751, which was consistently maintained in second binding events close to the initial binding value of 0.753. However, when Rh(D) was the third antigen to bind, the K\u003csub\u003eD\u003c/sub\u003e values increased slightly to 0.298 \u0026micro;M and 0.301 \u0026micro;M for the sequences A\u0026rarr;B\u0026rarr;Rh(D) and B\u0026rarr;A\u0026rarr;Rh(D), respectively, with corresponding B\u003csub\u003emax\u003c/sub\u003e values of 0.744. The minimal increase in K\u003csub\u003eD\u003c/sub\u003e values and slight decrease in B\u003csub\u003emax\u003c/sub\u003e values suggest that the binding of Rh(D) is only marginally influenced by prior antigen interactions, indicating largely non-cooperative binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Coating of RBCs with triabody and hemagglutination assay\u003c/h2\u003e \u003cp\u003eTo make RBCs from various blood groups mimic O-negative RBCs, the triabody must block the maximum number of blood group antigens A, B and Rh(D). To achieve this, a repeated exposure protocol was used, which involved incubating RBCs in concentrated aliquots of a triabody solution. Supplementary Figure S5 shows that for all blood types of RBCs, a significant decrease in the concentration of triabody was observed in the first aliquot after 30 mins of incubation. However, no further reduction in concentration was observed in aliquots 2 and 3.\u003c/p\u003e \u003cp\u003eThe overall results (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) of the hemagglutination assay were negative, indicating that RBCs coated with trispecific triabodies purified in fraction AE3-B2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) did not hemagglutinate in the presence of associated anti-A IgM, anti-B IgM and anti-Rh(D) IgM antibodies. The control wells in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA show positive hemagglutination of uncoated RBCs of different blood types in the presence of associated blood group antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe main aim of this research was to design a novel trispecific triabody capable of acting as a blocking fragment for blood group antigens A, B, and Rh(D). By blocking these key antigens, any blood type RBCs could mimic universal donor O-negative RBCs, allowing for safe transfusions into any individual regardless of their native blood type. The design of the triabody was inspired by the compact, Y-shaped structure of IgG antibodies. Unlike IgM antibodies, IgG molecules are well known to rarely induce direct hemagglutination of RBCs due to their smaller size [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. To further minimize the potential for hemagglutination, the triabody was intentionally designed to be even smaller than an IgG antibody. Furthermore, in addition to its trivalent binding capability, triabody was designed to be trispecific. These combined features would allow the triabody to efficiently block multiple blood group antigens without causing the hemagglutination of RBCs.\u003c/p\u003e \u003cp\u003eTo design a compact Y-shaped triabody, the hinge region of an IgG1 antibody was used. The hinge region, similar to a glycine-serine (GS) linker, provides flexibility to protein structures. However, unlike GS linker, the flexibility and movement of the hinge region can be controlled by the presence of cysteine residues. These cysteine residues are capable of forming intermolecular disulfide bonds between two hinge regions, thereby stabilizing the overall structure [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the addition of hinge regions in the triabodies offered a distinct advantage over GS linkers by producing compact Y-shaped structures (triabody-C1 and C2) that closely mimic the native IgG structure. Such structural compactness would not have been achieved if a GS linker had been used instead.\u003c/p\u003e \u003cp\u003eIn general, scFvs possess a well-conserved framework and exhibit a remarkable functional diversity. Certain residues within the framework of a scFv are highly conserved and act as essential building blocks for the basic architecture of the molecule. These residues, through various interactions, provide shape and stability to the scFv e.g., cysteines are involved in intradomain disulfide bonds. Functional diversity arises from the hypervariable regions, which provide binding specificity to scFvs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, incorrect pairing was observed in the fusion protein FP-2c. This mispairing could be due to two reasons: 1) similar frameworks of both chains and 2) the close proximity of the VH chain of anti-B to the VL chain of anti-Rh(D). The VH and VL frameworks of the Fvs used in this study share a high degree of similarity, with the only significant difference located in the sequence of complementarity-determining regions (CDRs) responsible for binding specificity. This similarity, combined with the close proximity of the chains, may have facilitated the incorrect pairing of the VH chain of anti-Rh(D) with the VL chain of anti-B within FP-2c.\u003c/p\u003e \u003cp\u003eThe accessibility of antigen binding sites is essential for triabodies to function effectively. This was clearly observed in triabodies-C1 and C2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In triabody-C1, the arrangement of variable chains positioned the anti-Rh(D) binding site outward, making it accessible. Conversely, the same variable chains in triabody-C2 positioned the anti-Rh(D) binding site inward, rendering it inaccessible. This issue in triabody-C2 could have been prevented by incorporating an additional GS linker of at least 10 amino acids (G\u003csub\u003e4\u003c/sub\u003eS)\u003csub\u003e2\u003c/sub\u003e between the hinge region and the anti-Rh(D) variable chain, as shown in Supplementary Figure S6. The additional linker would have allowed reorientation and facilitated the formation of an intermolecular disulfide bond in the opposite direction, thereby exposing the anti-Rh(D) binding site and orienting it outward. These findings suggest that the arrangement of variable chains during triabody design can impact the final pairing of chains and ultimately their functionality.\u003c/p\u003e \u003cp\u003eDespite the potential of triabodies to cause hemagglutination owing to their multiple binding sites, no visible hemagglutination was observed as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. However, microscopic analysis revealed that the triabody from the AE3-B1 fraction caused mixed-field hemagglutination only in AB\u0026thinsp;+\u0026thinsp;and AB\u0026thinsp;\u0026minus;\u0026thinsp;RBCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), with no hemagglutination observed in A\u0026thinsp;+\u0026thinsp;and B\u0026thinsp;+\u0026thinsp;RBCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). This selective hemagglutination in which a dual population of agglutinated and non-agglutinated RBCs is present, could be due to the distance between the A, B and Rh(D) binding sites. The distance between the A and B binding sites is greater, whereas the distances between the A and Rh(D) binding sites, as well as between the B and Rh(D) binding sites, are comparatively shorter as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The greater distance between the A and B binding sites likely facilitated the observed mixed-field hemagglutination.\u003c/p\u003e \u003cp\u003eThe location of the disulfide bonds within the triabody structure is the key determinant of whether a closed structure (intermolecular disulfide bond) or an open structure (intramolecular disulfide bond) is formed. The observation of mixed-field hemagglutination specifically in AB\u0026thinsp;+\u0026thinsp;and AB\u0026thinsp;\u0026minus;\u0026thinsp;blood types strongly suggests that the triabody from the AE3-B1 fraction adopts an open structure. However, in this study, it was not determined whether this open structure contains intramolecular disulfide bonds.\u003c/p\u003e \u003cp\u003eIn contrast to the triabody from the AE3-B1 fraction, the triabody from the AE3-B2 fraction did not cause hemagglutination in AB\u0026thinsp;+\u0026thinsp;RBCs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG. This finding strongly supports a compact closed structure, which could only be formed through intermolecular disulfide bonds.\u003c/p\u003e \u003cp\u003eGiven that the triabody was designed to block blood group antigens on the surface of RBCs, it was essential to evaluate whether it could bind all three antigens simultaneously or whether its small size could limit this capability. To address this, the intramolecular binding cooperativity of the triabody was first determined using free antigens and then compared with RBC-bound antigens, enabling assessment of whether initial binding to one antigen affects subsequent interactions with the others. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows that while the triabody is capable of binding multiple antigens, its binding sites appear to function independently when the antigens are free in solution. The lack of change in the binding parameters K\u003csub\u003eD\u003c/sub\u003e and B\u003csub\u003emax\u003c/sub\u003e​ with free antigens suggests that the binding of one site does not influence the binding of the other sites. This finding suggests that the individual monomers of the triabody, composed of the anti-A, and anti-B ds-scFv and the dsFv of anti-Rh(D), behave as distinct and separate units.\u003c/p\u003e \u003cp\u003eHowever, when the intramolecular binding cooperativity of the triabody was evaluated using RBC-bound antigens, consistent changes were observed in the binding parameters: K\u003csub\u003eD\u003c/sub\u003e increased and B\u003csub\u003emax\u003c/sub\u003e decreased during both the second and third binding events. These effects were most pronounced in the third binding step, irrespective of the antigen, indicating a cumulative impact on binding efficiency. This reduction may be due to steric hindrance caused by the dense distribution of antigens on the RBC surface, which likely restricts the accessibility of the triabody and impairs its effective engagement during later binding events.\u003c/p\u003e \u003cp\u003eThe changes were especially significant when Rh(D) was the first antigen to bind. This observation may be explained by the relatively low abundance of antigens Rh(D) on RBCs compared with carbohydrate antigens A and B [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Fewer Rh(D) molecules require fewer triabodies to reach saturation during the initial binding. As a result, fewer triabodies remain available for subsequent interactions with antigen A or B, leading to reduced binding affinity and capacity, as indicated by the increased K\u003csub\u003eD\u003c/sub\u003e and decreased B\u003csub\u003emax\u003c/sub\u003e values. In contrast, these effects were less prominent when antigen A or B was involved in the first binding event, or when Rh(D) was engaged during the second binding step.\u003c/p\u003e \u003cp\u003eThe results of hemagglutination assay, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, directly confirmed the effectiveness of the triabody as a blocking fragment. The complete absence of hemagglutination of triabody-coated RBCs validated that the triabody coating has successfully prevented the IgM antibodies from interacting with blood group antigens. Importantly, these observations are consistent with those reported by Hafeez and Zaidi (2024) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] where a similar lack of hemagglutination was observed in antigen A blocked RBCs incubated with anti-A IgM antibodies. These findings suggest that RBCs from each blood type, once coated with triabody, effectively mimic the compatibility profile of universal donor O-negative RBCs. This indicates that triabody-coated RBCs in the form of pRBCs, could be transfused into individuals of any blood type without triggering a blood type incompatibility related hemagglutination reaction, thereby significantly expanding the availability of usable blood.\u003c/p\u003e \u003cp\u003eThis study has certain limitations: 1) the presence and precise location of inter- and intramolecular disulfide bond was not determined; and 2) the potential of the triabody was determined outside a real blood environment, which is a highly complex mixture of cells, salts, and proteins. Further research is needed to determine the location of disulfide bonds using advanced structural analysis such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Additionally, the cooperative binding of the triabody should be studied via advanced techniques such as surface plasmon resonance (SPR) and other spectroscopic approaches. To fully evaluate the clinical potential of the triabody as a blocking agent, \u003cem\u003ein vivo\u003c/em\u003e studies in animal models are necessary to assess its efficiency and safety in a real physiological environment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that the trispecific triabody can effectively block blood group antigens A, B, and Rh(D) on RBCs, thereby preventing IgM-mediated hemagglutination and enabling RBCs to mimic the compatibility of universal donor O-negative RBCs. Its compact Y-shaped structure, inspired by the small size of IgG antibodies, allows efficient multivalent binding while avoiding hemagglutination. Further studies are needed to characterize disulfide bonds and to evaluate \u003cem\u003ein vivo\u003c/em\u003e safety and efficacy. These findings highlight the strong potential of the triabody as a blocking fragment capable of targeting multiple antigens and provide a foundation for future translational and clinical research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Review Committee of the National University of Sciences and Technology (NUST) (IRB number: 09-2023-ASAB-01/02) and was performed in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a PhD student\u0026apos;s research funds from the Atta-Ur-Rahman School of Applied Biosciences (ASAB) at the National University of Sciences and Technology (NUST) in Sector H-12, Islamabad, Pakistan.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMuhammad Asghar was supported by Ragnar S\u0026ouml;derberg Foundation Sweden.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH and MA conceptualized and designed the study. SH developed the methodology and performed the software analysis. Validation was carried out by SH and MA. Formal analysis and investigation were performed by SH. Resources were provided by MA. The original draft of the manuscript was prepared by SH, and both SH and MA contributed to reviewing and editing the manuscript. Visualization was performed by SH and MA. Supervision of the study was provided by MA. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eObeagu EI. The vitamin C paradigm: New frontiers in blood transfusion. Ann Med Surg (Lond). 2025; doi:10.1097/ms9.0000000000003018.\u003c/li\u003e\n\u003cli\u003eHuet M, Cubizolles M, Buhot A. Red blood cell agglutination for blood typing within passive microfluidic biochips. High Throughput. 2018; doi:10.3390/ht7020010.\u003c/li\u003e\n\u003cli\u003eBain BJ. Blood cells: A practical guide. 5th ed. Chichester: John Wiley \u0026amp; Sons; 2019.\u003c/li\u003e\n\u003cli\u003eKeyt BA, Baliga R, Sinclair AM, Carroll SF, Peterson MS. Structure, function, and therapeutic use of IgM antibodies. Antibodies (Basel). 2020; doi:10.3390/antib9040053.\u003c/li\u003e\n\u003cli\u003eRout P, Harewood J, Ramsey A, et al. Hemolytic transfusion reaction. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2023 Sep 12]. https://www.ncbi.nlm.nih.gov/books/NBK448158. Accessed 13 Mar 2025.\u003c/li\u003e\n\u003cli\u003eArmstrong B. Antigen-antibody reactions. ISBT Sci Ser. 2020; doi:10.1111/voxs.12590.\u003c/li\u003e\n\u003cli\u003eRaman L, Armstrong B, Smart E. Principles of laboratory techniques. ISBT Sci Ser. 2020; doi:10.1111/voxs.12591.\u003c/li\u003e\n\u003cli\u003eTheis SR, Hashmi MF. Coombs test. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2022 Sep 12]. https://www.ncbi.nlm.nih.gov/books/NBK547707. Accessed 1 Apr 2025.\u003c/li\u003e\n\u003cli\u003eDean L. Blood Groups and Red Cell Antigens. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 7. https://www.ncbi.nlm.nih.gov/books/NBK2269/. Accessed 7 Aug 2025.\u003c/li\u003e\n\u003cli\u003eRomanos-Sirakis EC, Desai D. ABO Blood Group System. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [updated 2025 Apr 26]. https://www.ncbi.nlm.nih.gov/books/NBK580518/. Accessed 22 Aug 2025.\u003c/li\u003e\n\u003cli\u003eWaheed U, Farooq A. Bridging blood and genes: The future of transfusion medicine and molecular biology. Ann Pak Inst Med Sci. 2024; doi:10.48036/apims.v20iSuppl.2.1282.\u003c/li\u003e\n\u003cli\u003eRahfeld P, Withers SG. Toward universal donor blood: Enzymatic conversion of A and B to O type. J Biol Chem. 2020;295:325\u0026ndash;334.\u003c/li\u003e\n\u003cli\u003eHafeez S, Zaidi N. Prevention of blood incompatibility related hemagglutination: Blocking of antigen A on red blood cells using in silico designed recombinant Anti-A scFv. Antibodies (Basel). 2024; doi:10.3390/antib13030064.\u003c/li\u003e\n\u003cli\u003eLuyten U, Peeraer S, Pirlet C, Khaouch Y, Streel C, Deneys V. O-negative blood shortage management in a university hospital: Impact of transfusing RHD-positive red blood cells to RHD-negative patients. Transfus Clin Biol. 2023;30:402\u0026ndash;409.\u003c/li\u003e\n\u003cli\u003eKundu S, Gerk A, Glatter R, Poenaru D. Innovative transfusion strategies for blood deserts in disaster settings. BMJ Glob Health. 2024; doi:10.1136/bmjgh-2024-016854.\u003c/li\u003e\n\u003cli\u003eHafeez S, Zaidi NUSS. Red blood cell substitutes: Liposome encapsulated hemoglobin and magnetite nanoparticle conjugates as oxygen carriers. Int J Mol Sci. 2023; doi:10.3390/ijms24021618.\u003c/li\u003e\n\u003cli\u003eHaldar R, Chitranshi S, Singh MK, Sachan S, Gupta D. Artificial blood: A futuristic dimension of modern-day transfusion sciences. Cardiovasc Hematol Agents Med Chem. 2019;17:11\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eOlsson ML, Hill CA, De La Vega H, Liu QP, Stroud MR, Valdinocci J. Universal red blood cells: Enzymatic conversion of blood group A and B antigens. Transfus Clin Biol. 2004;11:33\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eZhou P, Ouchari M, Xue Y, Yin Q. In vitro generation of red blood cells from stem cells and targeted therapy. Cell Transplant. 2020; doi:10.1177/0963689720946658.\u003c/li\u003e\n\u003cli\u003eMohammadi M, Nejatollahi F, Bemani P. Production of scFvs as anti-ABO antibody antagonists for prevention of hemolytic transfusion reactions resulting from ABO blood group incompatibility between donor and recipient. Hypothesis. 2016;14(1).\u003c/li\u003e\n\u003cli\u003eThomas R, Patenaude SI, MacKenzie CR, et al. Structure of an anti-blood group A FV and improvement of its binding affinity without loss of specificity. J Biol Chem. 2002;277:2059\u0026ndash;2064.\u003c/li\u003e\n\u003cli\u003eSantos-Esteban E, Curiel-Quesada E. Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library. Vox Sang. 2001;81:194\u0026ndash;198.\u003c/li\u003e\n\u003cli\u003eSiegel DL, Chang TY, Russell SL, Bunya VY. Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: Applications in immunohematology. J Immunol Methods. 1997; doi:10.1016/s0022-1759(97)00087-2\u003c/li\u003e\n\u003cli\u003eZhao J, Yang L, Gu Z, et al. Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity. Int J Mol Sci. 2010;12:1\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003eSaphire EO, Parren PW, Pantophlet R, et al. Crystal structure of a neutralizing human IgG against HIV-1: A template for vaccine design. Science. 2001; doi:10.1126/science.1061692.\u003c/li\u003e\n\u003cli\u003eYang J, Yan R, Roy A, et al. The I-TASSER suite: Protein structure and function prediction. Nat Methods. 2015;12:7\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eChristoffer C, Chen S, Bharadwaj V, et al. LZerD webserver for pairwise and multiple protein docking. Nucleic Acids Res. 2021;49:W359\u0026ndash;W365.\u003c/li\u003e\n\u003cli\u003eSchr\u0026ouml;dinger, LLC. The PyMOL molecular graphics system, version 3.1. 2025. Available from: https://pymol.org/. \u003c/li\u003e\n\u003cli\u003eGasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. Proteomics Protocols Handbook. New York: Humana Press; 2005. p. 571\u0026ndash;607. doi:10.1385/1-59259-890-0:571.\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Rosell G, Giorgino T, De Fabritiis G. Playmolecule ProteinPrepare: A web application for protein preparation for molecular dynamics simulations. J Chem Inf Model. 2017;57:1511\u0026ndash;1516.\u003c/li\u003e\n\u003cli\u003eAbraham MJ, Murtola T, Schulz R, et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1:19\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eGrant OC, Wentworth D, Holmes SG, et al. Generating 3D models of carbohydrates with GLYCAM-Web. 2025; doi:10.1101/2025.05.08.652828.\u003c/li\u003e\n\u003cli\u003eConroy MJ, Bullough PA, Merrick M, Avent ND. Modelling the human rhesus proteins: Implications for structure and function. Br J Haematol. 2005; doi:10.1111/j.1365-2141.2005.05786.x.\u003c/li\u003e\n\u003cli\u003eBugnon M, R\u0026ouml;hrig UF, Goullieux M, et al. SwissDock 2024: Major enhancements for small-molecule docking with Attracting Cavities and AutoDock Vina. Nucleic Acids Res. 2024; doi:10.1093/nar/gkae300.\u003c/li\u003e\n\u003cli\u003eCase DA, Cheatham TE, Darden T, et al. The Amber biomolecular simulation programs. J Comput Chem. 2005;26:1668\u0026ndash;1688.\u003c/li\u003e\n\u003cli\u003eKumari R, Kumar R, Lynn A. g_mmpbsa\u0026mdash;A GROMACS tool for high-throughput MM-PBSA calculations. J Chem Inf Model. 2014; doi:10.1021/ci500020m.\u003c/li\u003e\n\u003cli\u003ePope B, Kent HM. High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res. 1996;24:536\u0026ndash;537.\u003c/li\u003e\n\u003cli\u003eWoiszwillo JE. TMB formulation for soluble and precipitable HRP-ELISA. US Patent 5,006,461. United States; 1991.\u003c/li\u003e\n\u003cli\u003eSyedbasha M, Linnik J, Santer D, et al. An ELISA based binding and competition method to rapidly determine ligand-receptor interactions. J Vis Exp. 2016;109:e53575.\u003c/li\u003e\n\u003cli\u003eGraphPad Software. Nonlinear regression curve fitting was performed using GraphPad Prism version 9.5.1. 2023. Available from: https://www.graphpad.com/\u003c/li\u003e\n\u003cli\u003eSang Z, Xiang Y, Bahar I, Shi Y. Llamanade: An open-source computational pipeline for robust nanobody humanization. Structure. 2022; doi:10.1016/j.str.2021.11.006.\u003c/li\u003e\n\u003cli\u003eKoganei A, Tsuchiya T, Samura K, Nishikibe M. Use of whole sheep red blood cells in ELISA to assess immunosuppression in vivo. J Immunotoxicol. 2007;4:77\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eHarris LJ, Skaletsky E, McPherson A. Crystallographic structure of an intact IgG1 monoclonal antibody. J Mol Biol. 1998; doi:10.1006/jmbi.1997.1508.\u003c/li\u003e\n\u003cli\u003eFocosi D, Franchini M, Maggi F. Modified hemagglutination tests for COVID-19 serology in resource-poor settings: Ready for prime-time? Vaccines. 2022; doi:10.3390/vaccines10030406.\u003c/li\u003e\n\u003cli\u003eHorx P, Geyer A. Comparing the hinge-type mobility of natural and designed intermolecular bi-disulfide domains. Front Chem. 2020; doi:10.3389/fchem.2020.00025.\u003c/li\u003e\n\u003cli\u003eRosmalen MV, Krom M, Merkx M. Tuning the flexibility of glycine-serine linkers to allow rational design of multidomain proteins. Biochemistry. 2017; doi:10.1021/acs.biochem.7b00902.\u003c/li\u003e\n\u003cli\u003eJaneway CA Jr, Travers P, Walport M, et al. Immunobiology: The immune system in health and disease. 5th edition. New York: Garland Science; 2001. https://www.ncbi.nlm.nih.gov/books/NBK27144/. Accessed 2 Sep 2025.\u003c/li\u003e\n\u003cli\u003eGąciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017; doi:10.1371/journal.pone.0189964.\u003c/li\u003e\n\u003cli\u003eMcGann PT, Despotovic JM, Howard TA, Ware RE. A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression. Am J Hematol. 2012; doi:10.1002/ajh.22254.\u003c/li\u003e\n\u003cli\u003eDean L. Blood Groups and Red Cell Antigens. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 2. https://www.ncbi.nlm.nih.gov/books/NBK2264/. Accessed 18 Sep 2025.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Trispecific triabody, Universal red blood cells, Binding cooperativity, Antigen blocking, Hemagglutination, Transfusion compatibility, ELISA","lastPublishedDoi":"10.21203/rs.3.rs-8369029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8369029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003cbr\u003e\nAccess to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells (RBCs) are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To help overcome this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs, thereby conferring universal compatibility similar to O-negative RBCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003cbr\u003e\nIn this study, two combinations based on the placement of the anti-Rh(D) variable chain at the C- or N-terminus were generated, and fusion proteins from the first combination produced the closed (C1) and open (O1) triabodies. Intramolecular cooperative binding affinities of the selected triabody-C1 were predicted computationally using blood group antigens A, B, and Rh(D), with no significant changes observed in binding free energies. The triabody-C1 was expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3) using a two-plasmid system and purified through a three-step process, yielding two fractions, AE3-B1 and AE3-B2. The hemagglutination potential of the triabody was evaluated both visually and microscopically through immunocytochemistry. Visually, no hemagglutination was observed, while microscopically, mixed-field hemagglutination occurred when AB+ and AB\u003cstrong\u003e−\u003c/strong\u003e RBCs were incubated with the triabody in fraction AE3-B1, but not when A+ or B+ RBCs were tested. No hemagglutination of AB+ RBCs was detected with the triabody in fraction AE3-B2. ELISA-based cooperative binding assays using free antigens showed that the triabody’s monomers functioned independently, with no changes in binding parameters K\u003csub\u003eD\u003c/sub\u003e or B\u003csub\u003emax\u003c/sub\u003e. In contrast, assays with RBC-bound antigens revealed increased K\u003csub\u003eD\u003c/sub\u003e and decreased B\u003csub\u003emax\u003c/sub\u003e across successive binding events, particularly when Rh(D) antigens were engaged first. Hemagglutination assays confirmed that triabody-coated RBCs exhibited a complete absence of hemagglutination with anti-A, anti-B, and anti-Rh(D) IgM antibodies, confirming effective antigen blocking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003cbr\u003e\nThe trispecific triabody effectively blocks A, B, and Rh(D) antigens, rendering RBCs with O-negative like universal compatibility and offering a promising strategy to expand the supply of universally transfusable blood, particularly in emergency and resource-limited settings.\u003c/p\u003e","manuscriptTitle":"Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 05:29:08","doi":"10.21203/rs.3.rs-8369029/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-20T18:53:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T12:06:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T14:58:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T00:14:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290128439998976805591545551654643819171","date":"2025-12-30T14:27:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220198663264191244562421306297403296139","date":"2025-12-27T21:16:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90778060879659053986772301916234493141","date":"2025-12-23T15:25:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93328356923100255398716985531105650368","date":"2025-12-22T20:18:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54042052843139778578234288730972261928","date":"2025-12-22T17:37:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-22T17:04:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-18T06:07:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-18T06:07:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Biological Engineering","date":"2025-12-15T17:41:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d563536-5656-4709-8ade-99edccae383d","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:09:00+00:00","versionOfRecord":{"articleIdentity":"rs-8369029","link":"https://doi.org/10.1186/s13036-026-00661-w","journal":{"identity":"journal-of-biological-engineering","isVorOnly":false,"title":"Journal of Biological Engineering"},"publishedOn":"2026-03-13 15:59:17","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-12-18 05:29:08","video":"","vorDoi":"10.1186/s13036-026-00661-w","vorDoiUrl":"https://doi.org/10.1186/s13036-026-00661-w","workflowStages":[]},"version":"v1","identity":"rs-8369029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8369029","identity":"rs-8369029","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}