Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles

Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles Dikshita Panwar, Deepali Shrivastava, Arvind Kumar, Lavleen Kumar Gupta, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2885310/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Aug, 2023 Read the published version in AMB Express → Version 1 posted 5 You are reading this latest preprint version Abstract Exosomes, a subpopulation of Extracellular vesicles (EVs), are cell-secreted vesicles found in the majority of biological fluids, including breast milk, tears, sweat, blood and, urine. The density and size of these vesicles depend on a variety of factors, including age, gender and the biological condition of the individual. Researchers are now focusing on the selective extraction of exosomes from bodily fluids due to the unique biomolecule composition of exosomes, which is critical for diagnosis, disease, and regeneration. Furthermore, current approaches for exosome isolation have limitations, necessitating the development of a simpler and more effective technique to achieve this goal. In this study, we investigated a quick and effective strategy for isolating exosomes from serum using a bench-top centrifuge. This was accomplished by raising antibodies against exosome surface tetraspanins (CD9, CD63 & CD81) in Leghorn chickens due to their phylogenetic distance from humans and cost-effectiveness for commercial use. In order to separate exosomes from a complex biological fluid, the antibodies were further coupled with gold nanoparticles. The findings were validated using ELISA, spectrophotometry, and electron microscopy. Using this technique, exosome isolation from serum was achieved rapidly and these were captured by using anti CD63 antibodies bound to AuNPs. To summarize, exosomes were purified from serum using anti-CD63 antibodies conjugated to gold nanoparticles (IgY@AuNPs). Consequently, the approach for exosome isolation from biological fluid could be useful for clinically monitoring the biological state of the patients. AuNPs@IgY Extracellular vesicles Exosomes ELISA Gold nanoparticles Tetraspanins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Points Exosomes have the potential to be employed as a diagnostic biomarker for the characterisation and detection of numerous pathophysiological diseases. We developed a reliable gold nanoparticles-mediated approach for detecting exosomes in body fluids. This was accomplished by employing anti-exosomal tetraspanins antibodies targeting CD9, CD63 and CD81. Introduction With recent progress in the field of biomarkers research, exosomes have secured a great position, consisting the advance usage in drug discovery and therapeutics. Exosomes can be employed as medication delivery vesicles for several diseases, such as diabetic fibrosis and brain sickness. Additionally, these are employed in the treatment of cancer carcinoma, as a biomarker and a tool for early detection in liquid biopsies using biological fluids, RNA, DNA, and proteins, and in diagnostic and imaging procedures for illnesses like Alzheimer's disease, Parkinson's inflammation, and cancer autoimmune metastasis (Huda et al. 2021 ). Therefore, exosome-mediated detection and characterization techniques have emerged as a potential and robust tool for disease diagnosis. Exosomes, naturally derived from cells, ranged between 20-150nm, and mediate intercellular signaling (Zhu et al.2022; Zhang et al. 2019 ). These are distinguished by their bioactive components, which vary from cell to cell and include proteins, lipids, DNA, and RNA (Zhang et al. 2019 ). Exosome biogenesis, in contrast to other extracellular vesicles, originates inside multi-vesicular bodies (a subset of late endosomes), where intraluminal vesicles (ILVs) are produced. These ILVs either fuse with the lysosome for degradation or connect with the plasma membrane to release the ILVs, also known as extracellular exosomes. (Zhang et al. 2019 ; Gurunathan et al. 2021 ). Exosomes are desirable as therapeutic vectors because of their reduced immunogenicity caused by biocompatibility and a bi-layered lipid structure that shields the genetic payload from destruction (Gurung et al. 2021 ). Tetraspanins family members (CD9, CD63 & CD81), endosomal sorting complex needed for transport (ESCRT) proteins (Alix, TSG101), heat shock proteins (Hsp), integrins, actin, and flotillins are the primary membrane-bound and cytosolic proteins of exosomes (Gurung et al. 2021 ). Tetraspanins can interact with a variety of receptors and signaling molecules at the membrane. Hence these either may be involved in the exosome’s attachment and its absorption by target cells, or antigen presentation as a response of the immune system (Gurung et al. 2021 ). Exosomes, being such a valuable resource in research, it is unfortunate that exosomes isolation methods including size-exclusion chromatography, precipitation, ultrafiltration, immunoaffinity and ultracentrifuge are available with lots of limitations such as slow processing volume, time-consuming, low purity, dependency on expensive instruments (Pammi et al. 2022). However, there are also commercial exosome isolation kits available, which have the advantages of quick isolation and ease of handling but high reagent costs (Pammi et al. 2022). The present study reports a gold nanoparticle (AuNPs) based quick and effective approach for exosome detection in human serum using anti-exosomal tetraspanins antibodies. Based on the specificity of anti-tetraspanins antibodies, immunoaffinity capture offers a superior exosomal population (Ludwig et al. 2019 ). Research performed in the human cell line, HEK293, analyzed that CD9, CD63, and CD81 was the most highly enriched protein of exosomes and hence have been utilized for exosome capturing and detection (Fordjour et al. 2022 ). This distinct pattern of tetraspanins expression provides an antibody-capture and detection of a subpopulation of extracellular vesicles i.e., exosomes through AuNPs (Mizenko et al.2021; Parthasarathy et al. 2009 ). Since the Middle Ages, gold has been used in the biomedical field to aid in the treatment of various diseases such as arthritis, epilepsy, heart difficulties, venereal disorders, and the diagnosis of syphilis (Bai et al. 2020 ). As a result of the growing understanding enabled by modern nanotechnology, biomedical applications of AuNPs such as imaging agents, drug delivery, nano-enzymes, irradiation, targeting, and photothermal therapy have been significantly advanced (Bai et al. 2020 ). AuNPs have exceptional physicochemical features that enable them to make a stable chemical interaction with S- and N-containing groups, giving them an edge over other nanoparticles (Bai et al. 2020 ; Yeh et al. 2012 ). These surface modifications give AuNPs exceptional biocompatibility and the ability to bind a variety of organic ligands or polymers for various purposes (Bai et al. 2020 ; Yeh et al. 2012 ). These nanomaterial-based biosensors have been widely used in liquid biopsies for tumor-derived exosomes as a source of cancer biomarkers. Here in this study, we have utilized AuNPs as a detecting and tracking agent for targeted antibodies raised against the exosomal tetraspanins i.e., CD9, CD63, and CD81. This is made possible by the various functionalized groups present on the surface of AuNPs, which are involved in nanoparticle stability, functionality, biocompatibility, and the development of novel properties (Jazayeri et al. 2016 ). Low molecular weight ligands, peptides, proteins, polysaccharides, polyunsaturated and saturated fatty acids, DNA, plasmids, and siRNA are all examples of functionalized groups that can be attached to nanoparticles (Jazayeri et al. 2016 ). Raised antibodies were bound to AuNPs in this study via physical interactions such as ionic interaction, hydrophobic interaction, and dative binding (Jazayeri et al. 2016 ). AuNPs, on the other hand, have a tendency to coagulate due to their highly reactive nature and must be stabilized for long-term storage. However, polyethylene glycol (PEG) could be used to resolve this, as its hydrophilic properties boost the biocompatibility and stability of conjugated complexes (Jazayeri et al. 2016 ; Reznickova et al. 2017 ). In this paper, we describe a rapid and effective immunoaffinity technique based on gold nanoparticles, in which anti-exosomal antibodies have been decorated on AuNPs through physically reactive interactions. These antibody-conjugated gold nanoparticles are processed further by centrifugation after being treated with human serum. The spectrophotometry and TEM were used to analyse and characterize the work described above. Materials and Methods Materials Several chemicals have been used to carry out various experiments in the present study. Chemicals such as gold (III) chloride hydrate (HAuCl4: Cat No. 254169) and poly (ethylene glycol) 2-mercaptoethanol ether acetic acid (PEG 3500Da, Cat No. 757837) have been purchased from Sigma Aldrich where as trisodium citrate dihydrate (Cat No. 85919), sodium hydroxide (NaOH (Cat No. 68151), bovine serum albumin (Cat No. 83803). Coomassie Brilliant Blue G-250, Methanol extra pure AR 99.8, Orthophosphoric acid extra pure 85%, Whatman® cellulose filter papers, bovine serum albumin (Cat No. 05470). Sodium citrate, citric acid, Sodium periodate, Ethylene glycol, Sodium carbonate/bicarbonate, Sodium cyanoborohydride, Glycerol, and Thiomersal have been purchased from SRL Chemicals. Non-fat milk powder has been purchased from a local vendor. Primary antibodies, anti CD9, CD63 & CD81, were raised in chickens for commercial use. Secondary antibodies were conjugated with Horseradish peroxidase through sodium periodate methods. Methods The overall workflow of this study is briefly described in Fig. 1 . The three exosomal surface tetraspanins, CD9, CD63, and CD81, were selected to produce functionalized antibodies to capture exosomes. In order to immunize chicken, the extracellular portion of these tetraspanins was selected as an immunogen. Antibodies affinity was confirmed through ELISA using spectrophotometry techniques. Furthermore, freshly synthesized citrate passivated gold nanoparticles were coupled with the anti-exosomal antibodies and finally introduced into the serum. This antibody-exosome coupling was investigated using an omega spectrophotometer and transmission electron microscope. Characterisation of tetraspanins for antigenic epitope detection Tetraspanins are members of a protein family with intracellular N-and C-termini, two extracellular domains (EC1 and EC2), and four transmembrane domains (Van et al. 2017). Although there are different tetraspanin varieties in each phylum, their chemical composition which includes four or more cysteine residues in a CCG motif of the EC2 domain is remarkably consistent across species (Van et al. 2017; Huang et al. 2005 ). Furthermore, antibodies that target surface markers such as tetraspanins proteins (CD9, CD63, and CD81) are used in several isolation and characterization methods for various extracellular vesicles, including immunoaffinity purification, flow cytometry, enzyme-linked immunosorbent assay, and Western blot. (Jiang et al. 2020 ). Moreover, the extracellular regions of these tetraspanins were targeted for antibodies production in this study, and the protein sequence was retrieved from the NCBI database. Among the various populations of EVs, the extracellular region of exosomal tetraspanins has high accessibility and is easy to target for their capture. Designing and synthesizing of antigen for antibody production Since the extracellular region was selected as an antigen for antibody production, a linker protein sequence is required to connect the two extracellular loops of the tetraspanins. The foundation of linker design is analogous to linkers derived from naturally occurring multi-domain proteins. Researchers’ generally classified the linkers into three types based on their structures: rigid linkers, flexible linkers, and in vivo cleavable linkers. Among these, flexible linkers are generally rich in small or polar amino acids like Glycine and Serine to provide good flexibility and solubility, and they are considered more appropriate for movements and interactions required for fusion protein domains (Chen et al. 2013 ). Here a flexible linker sequence GGGGS has been used, due to its advantages, like ensuring correct protein orientation and not interfering with the folding of the protein domain. In addition to this, the linker sequence was also reported for providing stability of fusion protein and enhancing protein expression (Chen et al. 2013 ). The selected extracellular region of CD9, CD63, and CD81 tetraspanins were joined with the linker, and the resulting nucleotide sequences were optimized through online oligo optimization tools. The gene of interest was cloned in PET 28a plasmid by Gene Universal, Delaware USA. A codon-optimized gene sequence was designed for the selected extracellular region and synthesized it from outsourcing. The desired gene was synthesized and cloned in the PET28a vector. Furthermore, the vector was transformed into BL21 Codon + cells. The entire amino acid sequence data for CD9 were obtained from UniProtKB (Swiss-Prot) using the reference URL https://www.uniprot.org/uniprotkb/P21926/entry and accession number: P21926. Similarly, the reference URL and accession number for CD63 were https://www.uniprot.org/uniprotkb/P08962/entry and P08962, while the reference URL and accession number for CD81 were https://www.uniprot.org/uniprotkb/P60033/entry and P60033. Transformation of PET28a into BL21 Codon plus competent cells The plasmids (PET28a) with the desired genes, i.e., CD9, CD63, and CD81, were transformed through the heat shock method into BL21 codon plus competence cells, which is a strain of E. coli. In this investigation, competence cells were prepared using the Li et al. method for increasing cell competence efficiency (Li et al. 2010 ). Furthermore, the transformation procedure was customized as per the requirement with the plasmid concentration of 5 ng and an antibiotic concentration (Kanamycin) of 34 g/ml (Li et al. 2010 ). Protein Expression The protein expression was modified based on a previous study, and the steps that follow are described briefly (Feng et al. 2006 ). The transformed single colony of BL21-codon plus was subjected to an LB medium containing 34 ug/ml of Kanamycin (Kan) for overnight growth at 37°C. Furthermore, 3% of this overnight culture was inoculated in 50 ml of terrific broth prepared in PBS buffer supplemented with 0.5% glycerol and 30–34 µg/ml of kanamycin, followed by the incubation at 37°C till its mid-log phase (0.6–0.8 OD) at 600 nm. The addition of 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) induced protein expression and the culture was incubated for another 3 h at 37°C with aeration at 250 rpm of stirring. Centrifugation at 6000 g for 10 min at 4°C was used to collect the cells, and pellet wash buffer (50 mM Tris-HCl and 100 mM NaCl) was used to clean the pellet. All three tetraspanins (CD9, CD63 & CD81) extracellular proteins were expressed with the identical approach, described above. Protein purification from Inclusion body After bacterial cell lysis, it was noted that the desired protein attained in the form of an inclusion body (IB) which is extremely dynamic in nature, and its molecules can reversibly disaggregate and assemble back into their native state (Peternel et al. 2010). In contrast to mechanical processes like sonication or homogenization, cell disruption by chemical lysis is considered more effective in the case of an inclusion body (Peternel et al. 2010). The bacterial pellet was resuspended and homogenized in lysis buffer-1 (0.2 mg/ml lysozyme, 10 µg/ml DNase, 50 mM Tris pH 8.8) for chemical lysis and incubated overnight at 37 ℃ in a rotator at 100 rpm. Following that, lysis buffer-2 (1M NaCl and 1% Triton X-100) was added to the same tube at a 1:1 volume ratio. The inclusion bodies were collected after complete lysis by centrifugation at 8000 g for 10 min, followed by 4 to 5 washes with wash buffer (100 mM NaCl and 50 mM Tris, pH 7.5). In addition, the IB pellet was re-suspended in denaturing buffer (6M Gu-HCl in 50 mM Tris, pH 7.4) and incubated at 4 ℃ for 30 min to denature the inclusion body. The denatured IB was centrifuged at 9000 g for 10 min, and the supernatant was collected for further protein renaturation. Furthermore, the supernatant was dropped into renature buffer (20 mM MgCl 2, 20 mM Trehalose, 100 mM NaCl, 5% Ethylene Glycol, 0.01% Tween80, 100 mM L-Arginine, 50 mM Tris, 1 mM GSSG, 10 mM GSH) while being continuously stirred at 4 ℃. The aforementioned mixture was subsequently incubated at 4°C for 48 h, and then the refolded protein was concentrated using sucrose. Additionally, the purity of refolded proteins (CD9, CD63, & CD81) was analyzed through SDS-PAGE. Considering the previous studies, the performed experiments for protein expression and purification in this study were modified based on the requirements (Peternel et al. 2021; Palmer et al. 2012; Singh et al. 2005; Yang et al. 2011 ). Immunization of antigen into chicken To raise the anti-exosomal antibodies in a cost-effectively, chicken has been preferred due to its phylogenetical difference between avian and mammalian species which enhances the sensitivity of immunological assays. In 8-month-old white leghorn chickens, the purified protein of three exosomal tetraspanins (CD9, CD63, & CD81) was injected intramuscularly every 14th day for a total of 8 weeks. Each bird received approximately 100 µg of protein dissolved in 150 µl of PBS and 350 µl of Montanide adjuvant (Seppic Inc., US) on the day of immunization. The bird that received only PBS and adjuvant was employed as a negative control. By stimulating the B-cell response, adjuvants accelerated the immune system's response to the antigen (Schade et al. 2005 ; Sunita et al. 2020). Afterwards, the eggs were collected for IgY antibody purification and their immunoaffinity was assessed using the ELISA method. Antibodies purification Based on the evaluation of earlier studies for protein purification, the isolation and purification of antibodies from the immunized chicken were carried out using the egg yolk component (Sunita et al. 2020; Panwar et al. 2023 ). In a nutshell, egg yolk from immunized chicken eggs was diluted in deionized water at a 1:10 ratio and homogenized to make a uniform suspension. The pH of the above mixture was then adjusted to 5.0 using 0.1 N HCl before being stored at -20°C overnight. To precipitate all the lipids and phospholipids while leaving the protein in the supernatant, the frozen sample was defrosted at room temperature the following day without being shaken. NaCl at a concentration of 8.8% was then added to the supernatant after it had been filtered and collected. Furthermore, the pH of the above solution was adjusted to 4.0 with 0.1 N HCl followed by the incubation at room temperature for 1 h. The precipitated protein holding IgY antibodies was collected by centrifugation at 5000 rpm for 10 min and resuspended in the required volume of PBS. The protein purity and the concentration were determined using the SDS-PAGE and UV absorbance method respectively. The purified antibodies were stored at -20°C for further usage. SDS-PAGE SDS-PAGE, as a simple tool, has been widely used for a long time to assess the purity of desired proteins. In this study, the traditional method described by Laemmli has been followed to analyze the purity of antibodies as well as refolded proteins (Muhammad et al. 2018 ). As a molecular weight marker, a protein ladder with a range of 20–95 kDa (SRL Chemicals), was utilized in this study. Afterward, the gel was stained with Coomassie Brilliant Blue for visualization. Quantitative analysis of antibodies affinity through ELISA Antibody titration was performed through Indirect-ELISA to analyze the antibody affinity towards the antigen. Briefly, in flat bottom microtiter plates (Thermo Scientific, USA), antigen was coated (as 500 ng/well) in coating buffer (100 mM sodium carbonate, 100 mM sodium bicarbonate, pH 9.5) followed by the addition of 300 µl blocking buffer (5% skimmed milk prepared in Phosphate Buffered Saline with Tween 20). Furthermore, antibodies at various concentrations (100 µg/ml to two-fold dilution till seven wells) were prepared in diluent buffer (1/10 dilution of blocking buffer in PBS) and added to the wells, along with negative control (without antibodies). Anti-chicken antibodies, as a secondary antibody was diluted in diluent and introduced into the wells at a 1:6000 ratio. Finally, colour was developed by the addition of substrate buffer (100 µg/mL of Tetramethylbenzidine (TMB), 0.01% H 2 O 2 in 50 mM citrate buffer, pH 5.0) followed by the addition of stop solution (1N H 2 SO 4 ) after 20 min incubation in dark at room temperature and optical density was measured by FLUOstar Omega spectrophotometer (BMG LABTECH’s). Excluding the substrate step, a 1 h incubation at 37 ℃ was followed by four washes with PBST (PBS containing 0.05% Tween20) used in this assay. Gold nanoparticle synthesis Nanoparticles were synthesized using the conventional method, by dissolving gold chloride in trisodium citrate at 85–90°C and resulting in a chemical reduction of gold (Polte et al. 2010 ). Here the concentration of gold chloride and tri-sodium citrate was used at 0.5 mmol/l and 5 mmol/l respectively. All glassware must be treated with aqua Regia (1:3 ratio of HNO₃ and HCl) overnight at 37°C, then thoroughly washed with double distilled water and dried before use. Briefly, the gold was introduced into an aqueous solution (triple distilled water) and heated while stirring in a temperature-controlled stirrer. Trisodium citrate was immediately added after it reached the desired temperature. The NPs were fabricated at two distinct temperatures (99°C and 80°C) for a better understanding of the AuNPs size variation. Then it was allowed to continue stirring for another 10 min until the colour of the gold solution changes from yellowish to colourless to wine red followed by a 20 min further incubation at the same temperature. The sample was extracted carefully, after being cooled at room temperature, by using the pipette for UV analysis and pH measurement (approx. 5.6-6.0). Antibody decoration on AuNPs The citrate-mediated AuNPs were conjugated with the antibodies through physical methods including ionic interactions, interaction with the thiol group, and hydrophobic interactions. To start with the removal of citrate and activation of AuNPs using 0.25% Tween-20, followed by incubation at room temperature for 2 h while stirring. After centrifuging the gold nanoparticle suspension at 13,500 rpm for 20 min, the supernatant is collected and resuspended in the phosphate buffer (pH 7.6). Additionally, the AuNPs were washed twice with phosphate buffer (10 mM, pH 7.2) and resuspended again in the same buffer. The washed NPs were adjusted to pH 8 using 100 mM of NaOH. Furthermore, antibodies were diluted in phosphate buffer to desired concentration (0.2 mg of all three antibodies/1 ml of AuNPs used in this) and added dropwise with continuous stirring followed by incubation of 2 h at room temperature while shaking. Then the NPs were blocked with the addition of 0.2 mg of BSA followed by centrifugation at 12000 rpm for 15 min to remove the unbound antibodies. Afterward, the conjugated NPs were resuspended in phosphate buffer and 0.25% of PEG and 0.5% glycerol were added for stabilization. The above-described methodology was influenced by various previous studies (Juan et al. 2020 ; Goossens et al. 2017 ; Okyem et al. 2021 ). Isolation of exosomes from serum For this purpose, blood was collected from healthy volunteers in clot activator tubes to allow the blood to clot in an upright position for 1h at room temperature. During this period, the RBCs settled and the pure serum was collected by centrifugation at 5000 rpm for 15 min, followed by aliquoting of 1 ml serum in 1.5 ml tubes and storing at -20 ˚C. For exosome isolation, 1 ml of the 2-fold diluted serum was incubated with 1 ml of anti-exosomes antibodies conjugated with AuNPs for 1 h at room temperature. Furthermore, the nanoparticles have been washed completely twice and resuspended in phosphate buffer for further analysis through ELISA or TEM. Sample Preparation for TEM Analysis of AuNPs and IgY@AuNPs The IgY@AuNPs complex bound with exosomes and plain AuNPs has been considered as a sample for TEM analysis. On a TEM grid, 10 µl of each sample was applied sequentially and the excess volume was wiped away with filter paper. The TEM grid was washed and air-dried three times before proceeding to the next sample. Each sample has been analyzed twice for better understanding and clarity. RESULTS Designing the Antigen Sequence Tetraspanins, CD9, CD63, and CD81, encode proteins with two extracellular and two intracellular loops. The extracellular loops are rich in numerous disulfide linkages that are conserved across the tetraspanin family, and palmitoylation sites aid in protein and lipid interactions. Moreover, CD9, CD63, and CD81 all encode surface integral proteins that are, respectively, 228, 238, and 236 amino acids long. According to the National Centre for Biotechnology Information (NCBI), the extracellular region of CD9, which was used as an antigen, spanned the lipoprotein membrane from 34 to 55 amino acids and 112 to 195 amino acids. Similarly, the extracellular region of the other two tetraspanins, which span 33–51, 103–203 for CD63, and 34–63, 113–201 for CD81, has served the purpose. Each tetraspanin's extracellular domain was joined by the GGGGS linker to construct a protein sequence that is 112, 124, and 127 amino acids long for CD9, CD63, and CD81, respectively (Table.1). The nucleotide sequence has been revealed for the synthesis of these three proteins which was designed with the addition of restriction enzyme sites, namely Nco 1 and Xho -1. The constructed sequences for the extracellular regions of CD9, CD63, and CD81 have the following accession numbers: OR166431, OR166432, and OR166433, respectively. The codon-optimized sequence (Table. 2) was cloned in PET 28a plasmid by Gene Universal and expressed in a strain of E. coli i.e., BL-21 codon plus. Protein expression and purification In this investigation, an E. coli strain called BL21 was used to express protein for the three tetraspanins, CD9, CD63, and CD81. The acquired colonies grew on an LB medium and were validated using the antibiotic (Kanamycin) selection method (Fig. 2 ). A single colony has been picked for further expression purposes through the IPTG method. The bacterial pellet was chemically lysed, and the SDS-PAGE result indicated that the desired protein is expressed in the inclusion body (Fig. 2 ). For CD9, CD63, and CD81, an overexpressed protein band of approximately 12.3 kDa, 13.7 kDa, and 12.3 kDa was observed. The IB was then purified and subjected to additional denaturation and renaturation to acquire its native protein structure. Relying upon the SDS-PAGE image, the purity was estimated to be between 70 and 80% following the final refolded process (Fig. 2 ). Along with tetraspanins purification, targeted antibodies are also analyzed using SDS PAGE electrophoresis, and the purity of antibodies may slightly vary due to different laboratory conditions or extraction methods. The salt precipitation of antibodies was carried out in two key steps, namely the exclusion of lipids and the precipitation of maximum antibodies, yielding more than 80% purity. SDS-gel confirmed two distinct bands, one of which corresponded to a molecular weight of approximately 68 kDa and the other to approximately 28 kDa (Fig. 2 ). Quantitative analysis of antibodies affinity through ELISA Anti-tetraspanin antibodies were analyzed in two ways: the kinetics of the antibodies in relation to the booster dosage and the titration of the antibodies with the highest affinity and avidity. The kinetics of antibodies were evaluated using an indirect ELISA method to determine the booster dosage at which antibodies acquire their maximum affinity and avidity. This was performed with consistent antigen and antibody concentrations, 0.5 ug/well antigen, and 10ug/well antibodies. Following that, secondary antibodies (anti-chicken) were added as detecting antibodies at a 1:6000 ratio, and its conjugation is described further below. The three anti-tetraspanin antibodies, CD9, CD63, and CD81, followed the same kinetics studies (Fig. 3 A, B & C). The third booster dosage resulted in the highest antibody titer, which increased considerably along with the booster dose. The titer became steady following the third booster dose, indicating that the antibodies had attained their maximal affinity for the antigen. Hence, following the third booster, antibodies were examined for titration to determine which concentration of antibodies is most specific for antigen detection. Indirect-ELISA was used for titration, with a constant concentration of antigen and a variable concentration of primary antibody. All three antibodies acquired a remarkable titer, with the resultant antibody concentration of 100–200 ng capable of detecting around 100 ng of antigen (Fig. 3 D, E & F). The titration of all three antibodies was carried out in accordance with the Indirect-ELISA methodology described above. Characterization of AuNPs and IgY@AuNPs Gold nanoparticles were fabricated at two different temperatures, one at 99°C and the other at 80°C, to determine the size and protein binding efficiency of each nanoparticle. The sizes of AuNPs synthesized at 99°C and 80°C determined by TEM are 18 ± 1 and 25 ± 1 nm, respectively, and spherical in shape. However, there was not a major difference between the two nanoparticles indicated above. These nanoparticles were then utilized to bind anti-tetraspanin antibodies to AuNPs, forming a structure known as the IgY@AuNPs complex. Additionally, the UV-spectrophotometer was used to analyze the absorption spectra of AuNPs and IgY@AuNPs complex to determine the maximum absorption wavelength of each. A volume of 200 µl of sample diluted five times in double distilled water was added in flat bottom microtiter plates (Thermo Scientific, USA) for spectroscopic analysis. The AuNPs synthesized at 99°Cand 80°C exhibited absorbance peaks at 520 nm and 526 nm, respectively (Fig. 4 A). As a negative control, double distilled water was employed in this case. The maximum spectrum of anti-tetraspanin IgY antibodies of CD9, CD63, and CD81 conjugated on AuNPs synthesized at 99°C was observed at approximately 528 nm (Fig. 4 B). Although the same three antibodies were coupled with AuNPs synthesized at 80°C, the maximum peak obtained for them was about approximately 531 nm (Fig. 4 C). The spectrums of all six conjugates were analyzed in contrast to their corresponding nanoparticles. Furthermore, TEM was used to examine the IgY@AuNPs complex in order to test the isolation of exosomes from serum. Evaluation of the AuNPs-antibody conjugation efficiency was accomplished through the Bradford assay which is well-known assay colorimetric assay to quantify the protein concentration The assay was carried out in accordance with the standard method described in the most recent study, and absorbance was measured at 595 nm (Davatgaran et al. 2018). A concentration of 100 µg of antibodies was introduced to 1 ml of AuNPs, resulting in approximately 50% binding to the nanoparticles. For further exosome isolation from serum, roughly 50 µg of antibodies were bound to 1 ml of AuNPs. Exosomes isolation and characterization from serum A single antibody conjugation, CD63 antibody conjugated to AuNPs, was employed for the analysis of serum exosome detection. The TEM results indicate that the IgY@AuNPs complex can extract exosomes from human serum (Fig. 5 ). Both the AuNPs synthesized at 80 ˚C and 99 ˚C were analyzed for exosomes capturing from the serum and TEM image revealed effectively caught exosomes in both cases. The AuNPs fabricated at 99 ˚C were scaled in the TEM at 100 nm, whereas the AuNPs synthesized at 80 ˚C were scaled at 200 nm. DISCUSSION Exosomes have been involved in a variety of fields, including drug delivery and disease diagnosis, yet there is still a shortage of exosome detection tools (Huda et al. 2021 ; Zhu et al. 2022 ). Exosomes are naturally cell-secreted signaling molecules with a size range of 20–150 nm comprising a diverse array of biomolecules like as DNA, RNA, proteins, mRNA, microRNA, long noncoding RNA, circular RNA (Zhang et al. 2019 ; Gurunathan et al. 2021 ; Gurung et al. 2021 ). There are several technologies available for isolating exosomes from various biological fluids, including ultracentrifugation, chromatography, and ultrafiltration, as well as a few commercial kits, but each has its own limitations (Pammi et al. 2022). The current work describes a gold nanoparticle (AuNPs) based rapid and successful strategy for exosome detection in human serum utilizing anti-exosomal tetraspanin antibodies. Exosomes, have various surface tetraspanin markers, but CD9, CD63, and CD81 are reported as dominantly expressed and mostly utilized for exosome isolation and purifications (Kowal et al. 2016 ; Khushman et al. 2017 ). However, their expression varies depending on the parent cell type, with CD63 being the most addressed as a highly expressed one (Khushman et al. 2017 ). As a result, three tetraspanins, CD9, CD63, and CD81, have been chosen to raise antibodies for exosome capture. The extracellular region of all three tetraspanin surface markers was adopted for antibody production. Correspondingly, the sequence was codon-optimized (Table.2), cloned in the PET 28a plasmid by Gene Universal, and expressed in the BL21 codon plus strain of E. coli . In a study, it was described a variety of expression strains as well as how BL21-Codon Plus (DE3) produced the highest protein yield (Assadi et al. 2008; Robichon et al. 2011 ). The extracellular region of CD9, CD63, and CD81 tetraspanin has been retrieved online from NCBI, which provides a wide range of online resources for true biological information and data from 38 distinct databases, including a gene bank, a nucleic acid sequence, a PubMed database, Bio Project, BLAST databases, and so on (Wheeler et al. 2005 ). Furthermore, the purity of three tetraspanins- exosomes surface marker as well as its targeted antibodies was characterized through SDS-PAGE. The following SDS-PAGE for antibodies revealed 80–90% purity and two distinct bands of roughly 68kDa and 28kDa, whereas refolded antigenic extracellular protein revealed 70–80% purity. Moreover, IgY concentration in egg yolk elevated throughout the immunization span until week 6, when it started to rise rapidly at 2 weeks and stagnated at 4 weeks. As a result, it is possible to conclude that the chicken's immune system takes approximately 2 weeks to give a boost in raising antibodies (Sudjarwo et al. 2017 ). However, a decrease in titer has been reported in the case of chickens about after 6 weeks of immunization. Even so, a significant decline in titer has been revealed in chickens approximately 6 weeks after immunization (Sudjarwo et al. 2017 ). Additionally, it was demonstrated that chickens immunized intramuscularly with the antigen, can raise targeted antibodies for more than 200 days, and also provides a high titer and tenfold greater specificity than subcutaneous injection (Sudjarwo et al. 2017 ). In this article, we demonstrate that a stagnant phase of antibodies titration has arrived after the third immunization as measured by indirect ELISA (Fig. 3 ). For indirect-ELISA anti- chicken HRP-secondary antibody has been internally conjugated in accordance with a recent article technique and yielded a titer of 1:6000 (Pavliuchenko et al. 2019 ). Although, along with the antibody kinetics, titration of all three antibodies was optimized using Indirect-ELISA and resulted in a remarkable titer (Fig. 3 ). The antibodies were conjugated to AuNPs, which were synthesized following the previously mentioned, traditional Turkevich method for citrate-stabilised gold nanoparticles (Muhammad et al. 2018 ). Optimization of particles morphology, size, and surface distribution were conducted by Transmission electron microscope and spectrophotometer. As previously reported in several studies, TEM images demonstrate that decreasing the temperature results in a larger diameter of gold nanoparticles (Dong et al. 2020 ). The nanoparticles were determined spherical in morphology and monodispersed in nature through TEM. The UV-vis absorption spectra of both AuNPs synthesized at 99 ˚C and 80 ˚C show well-defined surface plasmon resonance peaks at 520 nm and 526 nm, respectively (Fig. 4 ). Furthermore, a shift in the maximum absorbance wavelength of the UV-Vis spectra in contrast to naked AuNPs was interpreted as evidence of antibody conjugation on the AuNPs (Fig. 4 ). Two different types of nanoparticles, each coupled with three anti-tetraspanins antibodies, were synthesized and characterized here. In comparison to naked nanoparticles, the peak shift was clearly noticed at a difference of 3–4 nm for all six conjugated complexes for each type of AuNP. Hence, this distinct difference in the absorption spectra peaks indicated a successful conjugation and was utilized as a tool for exosome capturing. In this study PEG was used to stabilize of IgY@AuNPs complex and the stability of IgY@AuNPs was analyzed using 0.1 M NaCl solution. Similarly, in a study, Gold nanoparticles introduced to 0.1 M NaCl immediately resulted in a decrease in the absorbance peak, demonstrating their inability to be used in buffer solutions (Christau et al. 2017 ). Moreover, gold nanoparticles are highly unstable, and various environmental changes, such as pH changes or excessive centrifugation, cause immediate NP aggregation, as evidenced in several studies by the visible colour change (from red to purple) of the AuNPs solution, indicating an increase in particle size (Christau et al. 2017 ; Polte et al. 2010 ). Consequently, PEG is considered one of the best polymers for increasing the stability of nanoparticles even after numerous centrifugations during the process (Dallari et al. 2021 ). Here, the non-covalent immobilization technique was used to conjugate the antibody with nanoparticles, in which antibody was spontaneously absorbed onto the surface of citrate-stabilised nanoparticles with three types of interactions i.e., ionic, hydrophobic, and dative interactions. The attraction between the hydrophobic parts of the antibody and the metal surface results in the formation of a non-covalent bond (Ljungblad et al. 2009). Ionic attraction exists between negatively charged groups on the surface of gold nanoparticles and positively charged groups in antibodies caused by positively charged amino acids and their N-terminal region (Ljungblad et al. 2009; Jazayeri et al. 2016 ). Furthermore, the dative interactions (coordinate covalent bond) are responsible to form a covalent bond between the AuNPs surface and free sulfhydryl groups of the antibody (Ljungblad et al. 2009; Jazayeri et al. 2016 ). Before treating with PEG, the antibody and AuNPs complex were stabilized and blocked using BSA due to its nature to prevent the non-specific binding of molecules and so enhance the assay sensitivity. Similarly, in a recent study, BSA was reported to be effective in preventing nanoparticle aggregation (Leopold et al. 2017 ). In this article, we provide a more efficient and successful approach for isolating exosomes from human serum, which could potentially be employed for exosome isolation or exosomal cargo exploration. Anti-CD63 antibodies were applied to better understand and characterize the exosomes in human serum. Additionally, the expression of exosomal surface protein receptors was investigated, and observed that CD63 is prevalent in serum, but CD81 appears to be the rarest population (Karimi et al. 2022 ). However, they came to the conclusion that although CD9-enriched exosomes are also present, they are less numerous than CD63 (Karimi et al. 2022 ). Additionally, various groups used anti-CD63 antibodies to isolate the population of tiny EVs (Karimi et al. 2022 ; Pammi et al. 2022). Considering this, the TEM findings demonstrated that both conjugates, which were CD63 conjugated to both the 99 ˚C and 80 ˚C AuNPs, had efficiently captured the exosomes (Fig. 5 ). In a nutshell, the primary objective of this study was to establish a rapid, cost-effective, and efficient method of isolating exosomes from biological fluids by simply adding extra weight to them. This procedure begins with the identification of extracellular sites, then moves on to the production of antigen in bacterial cells, which is then introduced into chickens to produce anti-exosomal antibodies. ELISA and UV-vis spectrophotometry were employed to characterize antibody titration and synthesis of AuNPs respectively. Furthermore, antibodies were bound to AuNPs to isolate exosomes, and this complex was used to pull down the exosomes from fluids using the antibodies' specificity. The isolated exosomes were successfully captured by TEM and could be used for a variety of purposes, including disease diagnosis, drug delivery, and miRNA extraction. We anticipate that the proposed approach will be valuable in more in-depth research on exosomal subcategories, and it can be applied to different sized small extracellular vesicles. Declarations Acknowledgments The authors are thankful to the IgY Immunologix India Private Limited, Hyderabad, India for providing financial support and Vignan’s Foundation for Science, Technology and Research, Guntur for providing other facilities. Author contribution Dikshita Panwar: Carried out the work and writing the original draft; Lavleen Kumar Gupta: Conceiving the idea and designing the work; Deepali Shrivastava: Contribute in laboratory techniques; Arvind Kumar: Contribute in designing the work; Anjani Devi Chintagunta: review and editing. Funding IgY Immunologix India Private Limited, Hyderabad, India has provided financial support to carry out the present work. Availability of data and materials The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Ethics approval and consent to participate This article does not contain any studies with human or animal subjects Competing interests The authors declare that they have no conflicts of interest with respect to the work described in this manuscript. References Assadi-Porter FM, Patry S, Markley JL (2008) Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli . Protein Expr Purif 58(2):263–268. https://doi.org/10.1016/j.pep.2007.11.009 Bai X, Wang Y, Song Z, Feng Y, Chen Y, Zhang D, Feng L (2020) The basic properties of gold nanoparticles and their applications in tumor diagnosis and treatment. 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Nanomater 24:2022. https://doi.org/10.1155/2022/2042754 Tables Tables 1 to 2 are available in the Supplementary Files section Supplementary Files Tables23.62023.docx Cite Share Download PDF Status: Published Journal Publication published 28 Aug, 2023 Read the published version in AMB Express → Version 1 posted Editorial decision: Minor Revision 29 Jul, 2023 Reviewers agreed at journal 05 Jul, 2023 Reviewers invited by journal 03 Jul, 2023 Editor assigned by journal 23 Jun, 2023 First submitted to journal 23 Jun, 2023 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-2885310","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":215164488,"identity":"8b1aa500-63f7-4ad4-af3d-822a2ba05063","order_by":0,"name":"Dikshita Panwar","email":"","orcid":"","institution":"Vignan's Foundation for Science Technology and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dikshita","middleName":"","lastName":"Panwar","suffix":""},{"id":215164489,"identity":"baec7690-a3d4-447f-bde4-3788830b5b66","order_by":1,"name":"Deepali Shrivastava","email":"","orcid":"","institution":"Vignan's Foundation for Science Technology and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Deepali","middleName":"","lastName":"Shrivastava","suffix":""},{"id":215164490,"identity":"bc5cb485-74e7-45e9-8dc0-4b4806e8c48b","order_by":2,"name":"Arvind Kumar","email":"","orcid":"","institution":"Theton Biotech India Private Limited","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Arvind","middleName":"","lastName":"Kumar","suffix":""},{"id":215164491,"identity":"d2985b27-6179-4d75-b8fd-17cc1bb5bef4","order_by":3,"name":"Lavleen Kumar Gupta","email":"","orcid":"","institution":"IgY immunologix India Pvt. 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Sampath","lastName":"Kumar","suffix":""},{"id":215164493,"identity":"c5f45956-d072-4ae9-909f-180c3a892229","order_by":5,"name":"Anjani Devi Chintagunta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYPACCRDBxvABRLKTooVxBohkJsEqNmYeEEVIi/yM7MTPhXssEvsbmJ89tvm1TZ6PmYHxw8cc3FoMbuRulp7xTCJxxgE2c+PcvtuGbcwMzJIzt+HRIpG7QZrngERiwwEeNuncntuMQC1szLx4tMjPyN38G6RlPkiLZc9te4JaGG7kbgPbsgGkheHH7USCWgzOvN1mPeOAhPHGw2xmkr0Nt5PbmBmb8fpFvj138+2CA3Wy8443P5P48ee27fz25oMfPuJzGAMkIhwbQCRjG4jP2IBfPVSLPYT5h6DiUTAKRsEoGIEAALg4TZjhvDhQAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0807-8411","institution":"Vignan's Foundation for Science Technology and Research","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anjani","middleName":"Devi","lastName":"Chintagunta","suffix":""}],"badges":[],"createdAt":"2023-05-02 10:48:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2885310/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2885310/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13568-023-01592-1","type":"published","date":"2023-08-28T15:08:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":39696222,"identity":"1c81bc1c-c020-43b9-8444-2f05f2810db4","added_by":"auto","created_at":"2023-07-07 13:38:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166928,"visible":true,"origin":"","legend":"\u003cp\u003e(a) A simplified workflow depicting the introduction of antigen and the production of antibodies in chicken, (b). Systematic diagram representing the synthesis of AuNPs and their conjugation with IgY antibodies for exosome capture.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/bd6b3907bff304ae675d3ded.png"},{"id":39698118,"identity":"70c8758f-7df1-4936-9877-9411a2b90fe0","added_by":"auto","created_at":"2023-07-07 13:46:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205860,"visible":true,"origin":"","legend":"\u003cp\u003eRepresenting the (a) cloning, (b) antibody purity, (c) identification of desired proteins and (d) purified desired protein.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/9da425f5171ea5b156821645.png"},{"id":39696217,"identity":"367d3981-7749-470d-97b8-a88da88bc580","added_by":"auto","created_at":"2023-07-07 13:38:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109338,"visible":true,"origin":"","legend":"\u003cp\u003eRepresenting the kinetics of antibodies raising and their titration towards the antigen through ELISA (A) Kinetic of anti CD9 (B) Kinetic of anti-CD63 (C) Kinetic of anti-CD81 (D) Titration of anti CD9 (E) Titration of anti-CD63 (F) Titration of anti-CD81.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/9fa9d75f5a33178de0a7135c.png"},{"id":39696218,"identity":"5b1a5c74-278e-4792-a82d-f2e58a3c44a9","added_by":"auto","created_at":"2023-07-07 13:38:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61491,"visible":true,"origin":"","legend":"\u003cp\u003eRepresenting the Wavelength peaks as: (A) UV absorbance spectra of AuNPs prepared at distinct temperature (B) AuNPs prepared at 99 ˚C and conjugated with three different antibodies represented in the graph. (C) AuNPs prepared at 80 ˚C and conjugated with three different antibodies represented in the graph.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/7fd902f973a42a1677be13fe.png"},{"id":39698669,"identity":"6c8c0dfd-6ee1-47b9-a960-d527c329f9cf","added_by":"auto","created_at":"2023-07-07 13:54:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":340264,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscopy Data (A) Naked nanoparticles (99˚C) at a scale of 100 nm results as 18±1 in size (B, C) Exosomes captured complex of IgY@AuNPs (D) Naked nanoparticles (80 ˚C) at a scale of 200 nm results as a 25±1nm in size (E, F) Exosomes captured complex of IgY@AuNPs\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/6022bb97766c5eb966bb8710.png"},{"id":42781874,"identity":"720123f6-6d90-45ed-ab41-50887daf4e8b","added_by":"auto","created_at":"2023-09-07 15:13:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1272679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/09a527ef-5ac2-421d-b886-0f13776c1a84.pdf"},{"id":39696221,"identity":"c6ac9b82-79c5-4b63-973f-f6864e035f4c","added_by":"auto","created_at":"2023-07-07 13:38:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14732,"visible":true,"origin":"","legend":"","description":"","filename":"Tables23.62023.docx","url":"https://assets-eu.researchsquare.com/files/rs-2885310/v1/690a107cd9bbf42bd2b9e5aa.docx"}],"financialInterests":"","formattedTitle":"Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles","fulltext":[{"header":"Key Points","content":"\u003cul\u003e\n \u003cli\u003eExosomes have the potential to be employed as a diagnostic biomarker for the characterisation and detection of numerous pathophysiological diseases.\u003c/li\u003e\n \u003cli\u003eWe developed a reliable gold nanoparticles-mediated approach for detecting exosomes in body fluids.\u003c/li\u003e\n \u003cli\u003eThis was accomplished by employing anti-exosomal tetraspanins antibodies targeting CD9, CD63 and CD81.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eWith recent progress in the field of biomarkers research, exosomes have secured a great position, consisting the advance usage in drug discovery and therapeutics. Exosomes can be employed as medication delivery vesicles for several diseases, such as diabetic fibrosis and brain sickness. Additionally, these are employed in the treatment of cancer carcinoma, as a biomarker and a tool for early detection in liquid biopsies using biological fluids, RNA, DNA, and proteins, and in diagnostic and imaging procedures for illnesses like Alzheimer's disease, Parkinson's inflammation, and cancer autoimmune metastasis (Huda et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, exosome-mediated detection and characterization techniques have emerged as a potential and robust tool for disease diagnosis. Exosomes, naturally derived from cells, ranged between 20-150nm, and mediate intercellular signaling (Zhu et al.2022; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These are distinguished by their bioactive components, which vary from cell to cell and include proteins, lipids, DNA, and RNA (Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExosome biogenesis, in contrast to other extracellular vesicles, originates inside multi-vesicular bodies (a subset of late endosomes), where intraluminal vesicles (ILVs) are produced. These ILVs either fuse with the lysosome for degradation or connect with the plasma membrane to release the ILVs, also known as extracellular exosomes. (Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gurunathan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Exosomes are desirable as therapeutic vectors because of their reduced immunogenicity caused by biocompatibility and a bi-layered lipid structure that shields the genetic payload from destruction (Gurung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tetraspanins family members (CD9, CD63 \u0026amp; CD81), endosomal sorting complex needed for transport (ESCRT) proteins (Alix, TSG101), heat shock proteins (Hsp), integrins, actin, and flotillins are the primary membrane-bound and cytosolic proteins of exosomes (Gurung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tetraspanins can interact with a variety of receptors and signaling molecules at the membrane. Hence these either may be involved in the exosome\u0026rsquo;s attachment and its absorption by target cells, or antigen presentation as a response of the immune system (Gurung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExosomes, being such a valuable resource in research, it is unfortunate that exosomes isolation methods including size-exclusion chromatography, precipitation, ultrafiltration, immunoaffinity and ultracentrifuge are available with lots of limitations such as slow processing volume, time-consuming, low purity, dependency on expensive instruments (Pammi et al. 2022). However, there are also commercial exosome isolation kits available, which have the advantages of quick isolation and ease of handling but high reagent costs (Pammi et al. 2022). The present study reports a gold nanoparticle (AuNPs) based quick and effective approach for exosome detection in human serum using anti-exosomal tetraspanins antibodies. Based on the specificity of anti-tetraspanins antibodies, immunoaffinity capture offers a superior exosomal population (Ludwig et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Research performed in the human cell line, HEK293, analyzed that CD9, CD63, and CD81 was the most highly enriched protein of exosomes and hence have been utilized for exosome capturing and detection (Fordjour et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This distinct pattern of tetraspanins expression provides an antibody-capture and detection of a subpopulation of extracellular vesicles i.e., exosomes through AuNPs (Mizenko et al.2021; Parthasarathy et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the Middle Ages, gold has been used in the biomedical field to aid in the treatment of various diseases such as arthritis, epilepsy, heart difficulties, venereal disorders, and the diagnosis of syphilis (Bai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a result of the growing understanding enabled by modern nanotechnology, biomedical applications of AuNPs such as imaging agents, drug delivery, nano-enzymes, irradiation, targeting, and photothermal therapy have been significantly advanced (Bai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). AuNPs have exceptional physicochemical features that enable them to make a stable chemical interaction with S- and N-containing groups, giving them an edge over other nanoparticles (Bai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yeh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These surface modifications give AuNPs exceptional biocompatibility and the ability to bind a variety of organic ligands or polymers for various purposes (Bai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yeh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These nanomaterial-based biosensors have been widely used in liquid biopsies for tumor-derived exosomes as a source of cancer biomarkers. Here in this study, we have utilized AuNPs as a detecting and tracking agent for targeted antibodies raised against the exosomal tetraspanins i.e., CD9, CD63, and CD81. This is made possible by the various functionalized groups present on the surface of AuNPs, which are involved in nanoparticle stability, functionality, biocompatibility, and the development of novel properties (Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Low molecular weight ligands, peptides, proteins, polysaccharides, polyunsaturated and saturated fatty acids, DNA, plasmids, and siRNA are all examples of functionalized groups that can be attached to nanoparticles (Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Raised antibodies were bound to AuNPs in this study via physical interactions such as ionic interaction, hydrophobic interaction, and dative binding (Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). AuNPs, on the other hand, have a tendency to coagulate due to their highly reactive nature and must be stabilized for long-term storage. However, polyethylene glycol (PEG) could be used to resolve this, as its hydrophilic properties boost the biocompatibility and stability of conjugated complexes (Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Reznickova et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this paper, we describe a rapid and effective immunoaffinity technique based on gold nanoparticles, in which anti-exosomal antibodies have been decorated on AuNPs through physically reactive interactions. These antibody-conjugated gold nanoparticles are processed further by centrifugation after being treated with human serum. The spectrophotometry and TEM were used to analyse and characterize the work described above.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSeveral chemicals have been used to carry out various experiments in the present study. Chemicals such as gold (III) chloride hydrate (HAuCl4: Cat No. 254169) and poly (ethylene glycol) 2-mercaptoethanol ether acetic acid (PEG 3500Da, Cat No. 757837) have been purchased from Sigma Aldrich where as trisodium citrate dihydrate (Cat No. 85919), sodium hydroxide (NaOH (Cat No. 68151), bovine serum albumin (Cat No. 83803). Coomassie Brilliant Blue G-250, Methanol extra pure AR 99.8, Orthophosphoric acid extra pure 85%, Whatman\u0026reg; cellulose filter papers, bovine serum albumin (Cat No. 05470). Sodium citrate, citric acid, Sodium periodate, Ethylene glycol, Sodium carbonate/bicarbonate, Sodium cyanoborohydride, Glycerol, and Thiomersal have been purchased from SRL Chemicals. Non-fat milk powder has been purchased from a local vendor. Primary antibodies, anti CD9, CD63 \u0026amp; CD81, were raised in chickens for commercial use. Secondary antibodies were conjugated with Horseradish peroxidase through sodium periodate methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe overall workflow of this study is briefly described in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The three exosomal surface tetraspanins, CD9, CD63, and CD81, were selected to produce functionalized antibodies to capture exosomes. In order to immunize chicken, the extracellular portion of these tetraspanins was selected as an immunogen. Antibodies affinity was confirmed through ELISA using spectrophotometry techniques. Furthermore, freshly synthesized citrate passivated gold nanoparticles were coupled with the anti-exosomal antibodies and finally introduced into the serum. This antibody-exosome coupling was investigated using an omega spectrophotometer and transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCharacterisation of tetraspanins for antigenic epitope detection\u003c/h2\u003e \u003cp\u003eTetraspanins are members of a protein family with intracellular N-and C-termini, two extracellular domains (EC1 and EC2), and four transmembrane domains (Van et al. 2017).\u003c/p\u003e \u003cp\u003eAlthough there are different tetraspanin varieties in each phylum, their chemical composition which includes four or more cysteine residues in a CCG motif of the EC2 domain is remarkably consistent across species (Van et al. 2017; Huang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Furthermore, antibodies that target surface markers such as tetraspanins proteins (CD9, CD63, and CD81) are used in several isolation and characterization methods for various extracellular vesicles, including immunoaffinity purification, flow cytometry, enzyme-linked immunosorbent assay, and Western blot. (Jiang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the extracellular regions of these tetraspanins were targeted for antibodies production in this study, and the protein sequence was retrieved from the NCBI database. Among the various populations of EVs, the extracellular region of exosomal tetraspanins has high accessibility and is easy to target for their capture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDesigning and synthesizing of antigen for antibody production\u003c/h2\u003e \u003cp\u003eSince the extracellular region was selected as an antigen for antibody production, a linker protein sequence is required to connect the two extracellular loops of the tetraspanins. The foundation of linker design is analogous to linkers derived from naturally occurring multi-domain proteins. Researchers\u0026rsquo; generally classified the linkers into three types based on their structures: rigid linkers, flexible linkers, and in vivo cleavable linkers. Among these, flexible linkers are generally rich in small or polar amino acids like Glycine and Serine to provide good flexibility and solubility, and they are considered more appropriate for movements and interactions required for fusion protein domains (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Here a flexible linker sequence GGGGS has been used, due to its advantages, like ensuring correct protein orientation and not interfering with the folding of the protein domain. In addition to this, the linker sequence was also reported for providing stability of fusion protein and enhancing protein expression (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The selected extracellular region of CD9, CD63, and CD81 tetraspanins were joined with the linker, and the resulting nucleotide sequences were optimized through online oligo optimization tools. The gene of interest was cloned in PET 28a plasmid by Gene Universal, Delaware USA.\u003c/p\u003e \u003cp\u003eA codon-optimized gene sequence was designed for the selected extracellular region and synthesized it from outsourcing. The desired gene was synthesized and cloned in the PET28a vector. Furthermore, the vector was transformed into BL21 Codon\u0026thinsp;+\u0026thinsp;cells.\u003c/p\u003e \u003cp\u003eThe entire amino acid sequence data for CD9 were obtained from UniProtKB (Swiss-Prot) using the reference URL \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb/P21926/entry\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb/P21926/entry\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and accession number: P21926. Similarly, the reference URL and accession number for CD63 were \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb/P08962/entry\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb/P08962/entry\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and P08962, while the reference URL and accession number for CD81 were \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/uniprotkb/P60033/entry\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/uniprotkb/P60033/entry\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and P60033.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransformation of PET28a into BL21 Codon plus competent cells\u003c/h2\u003e \u003cp\u003eThe plasmids (PET28a) with the desired genes, i.e., CD9, CD63, and CD81, were transformed through the heat shock method into BL21 codon plus competence cells, which is a strain of \u003cem\u003eE. coli.\u003c/em\u003e In this investigation, competence cells were prepared using the Li et al. method for increasing cell competence efficiency (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Furthermore, the transformation procedure was customized as per the requirement with the plasmid concentration of 5 ng and an antibiotic concentration (Kanamycin) of 34 g/ml (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein Expression\u003c/h2\u003e \u003cp\u003eThe protein expression was modified based on a previous study, and the steps that follow are described briefly (Feng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The transformed single colony of BL21-codon plus was subjected to an LB medium containing 34 ug/ml of Kanamycin (Kan) for overnight growth at 37\u0026deg;C. Furthermore, 3% of this overnight culture was inoculated in 50 ml of terrific broth prepared in PBS buffer supplemented with 0.5% glycerol and 30\u0026ndash;34 \u0026micro;g/ml of kanamycin, followed by the incubation at 37\u0026deg;C till its mid-log phase (0.6\u0026ndash;0.8 OD) at 600 nm. The addition of 0.5 mM Isopropyl \u0026szlig;-D-1-thiogalactopyranoside (IPTG) induced protein expression and the culture was incubated for another 3 h at 37\u0026deg;C with aeration at 250 rpm of stirring. Centrifugation at 6000 g for 10 min at 4\u0026deg;C was used to collect the cells, and pellet wash buffer (50 mM Tris-HCl and 100 mM NaCl) was used to clean the pellet. All three tetraspanins (CD9, CD63 \u0026amp; CD81) extracellular proteins were expressed with the identical approach, described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification from Inclusion body\u003c/h2\u003e \u003cp\u003eAfter bacterial cell lysis, it was noted that the desired protein attained in the form of an inclusion body (IB) which is extremely dynamic in nature, and its molecules can reversibly disaggregate and assemble back into their native state (Peternel et al. 2010). In contrast to mechanical processes like sonication or homogenization, cell disruption by chemical lysis is considered more effective in the case of an inclusion body (Peternel et al. 2010). The bacterial pellet was resuspended and homogenized in lysis buffer-1 (0.2 mg/ml lysozyme, 10 \u0026micro;g/ml DNase, 50 mM Tris pH 8.8) for chemical lysis and incubated overnight at 37 ℃ in a rotator at 100 rpm. Following that, lysis buffer-2 (1M NaCl and 1% Triton X-100) was added to the same tube at a 1:1 volume ratio. The inclusion bodies were collected after complete lysis by centrifugation at 8000 g for 10 min, followed by 4 to 5 washes with wash buffer (100 mM NaCl and 50 mM Tris, pH 7.5). In addition, the IB pellet was re-suspended in denaturing buffer (6M Gu-HCl in 50 mM Tris, pH 7.4) and incubated at 4 ℃ for 30 min to denature the inclusion body. The denatured IB was centrifuged at 9000 g for 10 min, and the supernatant was collected for further protein renaturation. Furthermore, the supernatant was dropped into renature buffer (20 mM MgCl\u003csub\u003e2,\u003c/sub\u003e 20 mM Trehalose, 100 mM NaCl, 5% Ethylene Glycol, 0.01% Tween80, 100 mM L-Arginine, 50 mM Tris, 1 mM GSSG, 10 mM GSH) while being continuously stirred at 4 ℃. The aforementioned mixture was subsequently incubated at 4\u0026deg;C for 48 h, and then the refolded protein was concentrated using sucrose. Additionally, the purity of refolded proteins (CD9, CD63, \u0026amp; CD81) was analyzed through SDS-PAGE. Considering the previous studies, the performed experiments for protein expression and purification in this study were modified based on the requirements (Peternel et al. 2021; Palmer et al. 2012; Singh et al. 2005; Yang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunization of antigen into chicken\u003c/h2\u003e \u003cp\u003eTo raise the anti-exosomal antibodies in a cost-effectively, chicken has been preferred due to its phylogenetical difference between avian and mammalian species which enhances the sensitivity of immunological assays. In 8-month-old white leghorn chickens, the purified protein of three exosomal tetraspanins (CD9, CD63, \u0026amp; CD81) was injected intramuscularly every 14th day for a total of 8 weeks. Each bird received approximately 100 \u0026micro;g of protein dissolved in 150 \u0026micro;l of PBS and 350 \u0026micro;l of Montanide adjuvant (Seppic Inc., US) on the day of immunization. The bird that received only PBS and adjuvant was employed as a negative control. By stimulating the B-cell response, adjuvants accelerated the immune system's response to the antigen (Schade et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sunita et al. 2020). Afterwards, the eggs were collected for IgY antibody purification and their immunoaffinity was assessed using the ELISA method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies purification\u003c/h2\u003e \u003cp\u003eBased on the evaluation of earlier studies for protein purification, the isolation and purification of antibodies from the immunized chicken were carried out using the egg yolk component (Sunita et al. 2020; Panwar et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a nutshell, egg yolk from immunized chicken eggs was diluted in deionized water at a 1:10 ratio and homogenized to make a uniform suspension. The pH of the above mixture was then adjusted to 5.0 using 0.1 N HCl before being stored at -20\u0026deg;C overnight. To precipitate all the lipids and phospholipids while leaving the protein in the supernatant, the frozen sample was defrosted at room temperature the following day without being shaken. NaCl at a concentration of 8.8% was then added to the supernatant after it had been filtered and collected. Furthermore, the pH of the above solution was adjusted to 4.0 with 0.1 N HCl followed by the incubation at room temperature for 1 h. The precipitated protein holding IgY antibodies was collected by centrifugation at 5000 rpm for 10 min and resuspended in the required volume of PBS. The protein purity and the concentration were determined using the SDS-PAGE and UV absorbance method respectively. The purified antibodies were stored at -20\u0026deg;C for further usage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE\u003c/h2\u003e \u003cp\u003eSDS-PAGE, as a simple tool, has been widely used for a long time to assess the purity of desired proteins. In this study, the traditional method described by Laemmli has been followed to analyze the purity of antibodies as well as refolded proteins (Muhammad et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a molecular weight marker, a protein ladder with a range of 20\u0026ndash;95 kDa (SRL Chemicals), was utilized in this study. Afterward, the gel was stained with Coomassie Brilliant Blue for visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative analysis of antibodies affinity through ELISA\u003c/h2\u003e \u003cp\u003eAntibody titration was performed through Indirect-ELISA to analyze the antibody affinity towards the antigen. Briefly, in flat bottom microtiter plates (Thermo Scientific, USA), antigen was coated (as 500 ng/well) in coating buffer (100 mM sodium carbonate, 100 mM sodium bicarbonate, pH 9.5) followed by the addition of 300 \u0026micro;l blocking buffer (5% skimmed milk prepared in Phosphate Buffered Saline with Tween 20). Furthermore, antibodies at various concentrations (100 \u0026micro;g/ml to two-fold dilution till seven wells) were prepared in diluent buffer (1/10 dilution of blocking buffer in PBS) and added to the wells, along with negative control (without antibodies). Anti-chicken antibodies, as a secondary antibody was diluted in diluent and introduced into the wells at a 1:6000 ratio. Finally, colour was developed by the addition of substrate buffer (100 \u0026micro;g/mL of Tetramethylbenzidine (TMB), 0.01% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 50 mM citrate buffer, pH 5.0) followed by the addition of stop solution (1N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) after 20 min incubation in dark at room temperature and optical density was measured by FLUOstar Omega spectrophotometer (BMG LABTECH\u0026rsquo;s). Excluding the substrate step, a 1 h incubation at 37 ℃ was followed by four washes with PBST (PBS containing 0.05% Tween20) used in this assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGold nanoparticle synthesis\u003c/h2\u003e \u003cp\u003eNanoparticles were synthesized using the conventional method, by dissolving gold chloride in trisodium citrate at 85\u0026ndash;90\u0026deg;C and resulting in a chemical reduction of gold (Polte et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Here the concentration of gold chloride and tri-sodium citrate was used at 0.5 mmol/l and 5 mmol/l respectively. All glassware must be treated with aqua Regia (1:3 ratio of HNO₃ and HCl) overnight at 37\u0026deg;C, then thoroughly washed with double distilled water and dried before use. Briefly, the gold was introduced into an aqueous solution (triple distilled water) and heated while stirring in a temperature-controlled stirrer. Trisodium citrate was immediately added after it reached the desired temperature. The NPs were fabricated at two distinct temperatures (99\u0026deg;C and 80\u0026deg;C) for a better understanding of the AuNPs size variation. Then it was allowed to continue stirring for another 10 min until the colour of the gold solution changes from yellowish to colourless to wine red followed by a 20 min further incubation at the same temperature. The sample was extracted carefully, after being cooled at room temperature, by using the pipette for UV analysis and pH measurement (approx. 5.6-6.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntibody decoration on AuNPs\u003c/h2\u003e \u003cp\u003eThe citrate-mediated AuNPs were conjugated with the antibodies through physical methods including ionic interactions, interaction with the thiol group, and hydrophobic interactions. To start with the removal of citrate and activation of AuNPs using 0.25% Tween-20, followed by incubation at room temperature for 2 h while stirring. After centrifuging the gold nanoparticle suspension at 13,500 rpm for 20 min, the supernatant is collected and resuspended in the phosphate buffer (pH 7.6). Additionally, the AuNPs were washed twice with phosphate buffer (10 mM, pH 7.2) and resuspended again in the same buffer. The washed NPs were adjusted to pH 8 using 100 mM of NaOH. Furthermore, antibodies were diluted in phosphate buffer to desired concentration (0.2 mg of all three antibodies/1 ml of AuNPs used in this) and added dropwise with continuous stirring followed by incubation of 2 h at room temperature while shaking. Then the NPs were blocked with the addition of 0.2 mg of BSA followed by centrifugation at 12000 rpm for 15 min to remove the unbound antibodies. Afterward, the conjugated NPs were resuspended in phosphate buffer and 0.25% of PEG and 0.5% glycerol were added for stabilization. The above-described methodology was influenced by various previous studies (Juan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Goossens et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Okyem et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of exosomes from serum\u003c/h2\u003e \u003cp\u003eFor this purpose, blood was collected from healthy volunteers in clot activator tubes to allow the blood to clot in an upright position for 1h at room temperature. During this period, the RBCs settled and the pure serum was collected by centrifugation at 5000 rpm for 15 min, followed by aliquoting of 1 ml serum in 1.5 ml tubes and storing at -20 ˚C. For exosome isolation, 1 ml of the 2-fold diluted serum was incubated with 1 ml of anti-exosomes antibodies conjugated with AuNPs for 1 h at room temperature. Furthermore, the nanoparticles have been washed completely twice and resuspended in phosphate buffer for further analysis through ELISA or TEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSample Preparation for TEM Analysis of AuNPs and IgY@AuNPs\u003c/h2\u003e \u003cp\u003eThe IgY@AuNPs complex bound with exosomes and plain AuNPs has been considered as a sample for TEM analysis. On a TEM grid, 10 \u0026micro;l of each sample was applied sequentially and the excess volume was wiped away with filter paper. The TEM grid was washed and air-dried three times before proceeding to the next sample. Each sample has been analyzed twice for better understanding and clarity.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDesigning the Antigen Sequence\u003c/h2\u003e \u003cp\u003eTetraspanins, CD9, CD63, and CD81, encode proteins with two extracellular and two intracellular loops. The extracellular loops are rich in numerous disulfide linkages that are conserved across the tetraspanin family, and palmitoylation sites aid in protein and lipid interactions. Moreover, CD9, CD63, and CD81 all encode surface integral proteins that are, respectively, 228, 238, and 236 amino acids long. According to the National Centre for Biotechnology Information (NCBI), the extracellular region of CD9, which was used as an antigen, spanned the lipoprotein membrane from 34 to 55 amino acids and 112 to 195 amino acids. Similarly, the extracellular region of the other two tetraspanins, which span 33\u0026ndash;51, 103\u0026ndash;203 for CD63, and 34\u0026ndash;63, 113\u0026ndash;201 for CD81, has served the purpose. Each tetraspanin's extracellular domain was joined by the GGGGS linker to construct a protein sequence that is 112, 124, and 127 amino acids long for CD9, CD63, and CD81, respectively (Table.1). The nucleotide sequence has been revealed for the synthesis of these three proteins which was designed with the addition of restriction enzyme sites, namely \u003cem\u003eNco\u003c/em\u003e1 and \u003cem\u003eXho\u003c/em\u003e-1. The constructed sequences for the extracellular regions of CD9, CD63, and CD81 have the following accession numbers: OR166431, OR166432, and OR166433, respectively. The codon-optimized sequence (Table. 2) was cloned in PET 28a plasmid by Gene Universal and expressed in a strain of \u003cem\u003eE. coli\u003c/em\u003e i.e., BL-21 codon plus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eIn this investigation, an \u003cem\u003eE. coli\u003c/em\u003e strain called BL21 was used to express protein for the three tetraspanins, CD9, CD63, and CD81. The acquired colonies grew on an LB medium and were validated using the antibiotic (Kanamycin) selection method (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A single colony has been picked for further expression purposes through the IPTG method. The bacterial pellet was chemically lysed, and the SDS-PAGE result indicated that the desired protein is expressed in the inclusion body (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For CD9, CD63, and CD81, an overexpressed protein band of approximately 12.3 kDa, 13.7 kDa, and 12.3 kDa was observed. The IB was then purified and subjected to additional denaturation and renaturation to acquire its native protein structure. Relying upon the SDS-PAGE image, the purity was estimated to be between 70 and 80% following the final refolded process (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlong with tetraspanins purification, targeted antibodies are also analyzed using SDS PAGE electrophoresis, and the purity of antibodies may slightly vary due to different laboratory conditions or extraction methods. The salt precipitation of antibodies was carried out in two key steps, namely the exclusion of lipids and the precipitation of maximum antibodies, yielding more than 80% purity. SDS-gel confirmed two distinct bands, one of which corresponded to a molecular weight of approximately 68 kDa and the other to approximately 28 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative analysis of antibodies affinity through ELISA\u003c/h2\u003e \u003cp\u003eAnti-tetraspanin antibodies were analyzed in two ways: the kinetics of the antibodies in relation to the booster dosage and the titration of the antibodies with the highest affinity and avidity. The kinetics of antibodies were evaluated using an indirect ELISA method to determine the booster dosage at which antibodies acquire their maximum affinity and avidity. This was performed with consistent antigen and antibody concentrations, 0.5 ug/well antigen, and 10ug/well antibodies. Following that, secondary antibodies (anti-chicken) were added as detecting antibodies at a 1:6000 ratio, and its conjugation is described further below. The three anti-tetraspanin antibodies, CD9, CD63, and CD81, followed the same kinetics studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B \u0026amp; C). The third booster dosage resulted in the highest antibody titer, which increased considerably along with the booster dose. The titer became steady following the third booster dose, indicating that the antibodies had attained their maximal affinity for the antigen. Hence, following the third booster, antibodies were examined for titration to determine which concentration of antibodies is most specific for antigen detection. Indirect-ELISA was used for titration, with a constant concentration of antigen and a variable concentration of primary antibody. All three antibodies acquired a remarkable titer, with the resultant antibody concentration of 100\u0026ndash;200 ng capable of detecting around 100 ng of antigen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E \u0026amp; F). The titration of all three antibodies was carried out in accordance with the Indirect-ELISA methodology described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of AuNPs and IgY@AuNPs\u003c/h2\u003e \u003cp\u003eGold nanoparticles were fabricated at two different temperatures, one at 99\u0026deg;C and the other at 80\u0026deg;C, to determine the size and protein binding efficiency of each nanoparticle. The sizes of AuNPs synthesized at 99\u0026deg;C and 80\u0026deg;C determined by TEM are 18\u0026thinsp;\u0026plusmn;\u0026thinsp;1 and 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm, respectively, and spherical in shape. However, there was not a major difference between the two nanoparticles indicated above. These nanoparticles were then utilized to bind anti-tetraspanin antibodies to AuNPs, forming a structure known as the IgY@AuNPs complex. Additionally, the UV-spectrophotometer was used to analyze the absorption spectra of AuNPs and IgY@AuNPs complex to determine the maximum absorption wavelength of each. A volume of 200 \u0026micro;l of sample diluted five times in double distilled water was added in flat bottom microtiter plates (Thermo Scientific, USA) for spectroscopic analysis. The AuNPs synthesized at 99\u0026deg;Cand 80\u0026deg;C exhibited absorbance peaks at 520 nm and 526 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As a negative control, double distilled water was employed in this case. The maximum spectrum of anti-tetraspanin IgY antibodies of CD9, CD63, and CD81 conjugated on AuNPs synthesized at 99\u0026deg;C was observed at approximately 528 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Although the same three antibodies were coupled with AuNPs synthesized at 80\u0026deg;C, the maximum peak obtained for them was about approximately 531 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The spectrums of all six conjugates were analyzed in contrast to their corresponding nanoparticles.\u003c/p\u003e \u003cp\u003eFurthermore, TEM was used to examine the IgY@AuNPs complex in order to test the isolation of exosomes from serum. Evaluation of the AuNPs-antibody conjugation efficiency was accomplished through the Bradford assay which is well-known assay colorimetric assay to quantify the protein concentration The assay was carried out in accordance with the standard method described in the most recent study, and absorbance was measured at 595 nm (Davatgaran et al. 2018). A concentration of 100 \u0026micro;g of antibodies was introduced to 1 ml of AuNPs, resulting in approximately 50% binding to the nanoparticles. For further exosome isolation from serum, roughly 50 \u0026micro;g of antibodies were bound to 1 ml of AuNPs.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eExosomes isolation and characterization from serum\u003c/h2\u003e \u003cp\u003eA single antibody conjugation, CD63 antibody conjugated to AuNPs, was employed for the analysis of serum exosome detection. The TEM results indicate that the IgY@AuNPs complex can extract exosomes from human serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth the AuNPs synthesized at 80 ˚C and 99 ˚C were analyzed for exosomes capturing from the serum and TEM image revealed effectively caught exosomes in both cases. The AuNPs fabricated at 99 ˚C were scaled in the TEM at 100 nm, whereas the AuNPs synthesized at 80 ˚C were scaled at 200 nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eExosomes have been involved in a variety of fields, including drug delivery and disease diagnosis, yet there is still a shortage of exosome detection tools (Huda et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Exosomes are naturally cell-secreted signaling molecules with a size range of 20\u0026ndash;150 nm comprising a diverse array of biomolecules like as DNA, RNA, proteins, mRNA, microRNA, long noncoding RNA, circular RNA (Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gurunathan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gurung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There are several technologies available for isolating exosomes from various biological fluids, including ultracentrifugation, chromatography, and ultrafiltration, as well as a few commercial kits, but each has its own limitations (Pammi et al. 2022). The current work describes a gold nanoparticle (AuNPs) based rapid and successful strategy for exosome detection in human serum utilizing anti-exosomal tetraspanin antibodies. Exosomes, have various surface tetraspanin markers, but CD9, CD63, and CD81 are reported as dominantly expressed and mostly utilized for exosome isolation and purifications (Kowal et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khushman et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, their expression varies depending on the parent cell type, with CD63 being the most addressed as a highly expressed one (Khushman et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As a result, three tetraspanins, CD9, CD63, and CD81, have been chosen to raise antibodies for exosome capture. The extracellular region of all three tetraspanin surface markers was adopted for antibody production. Correspondingly, the sequence was codon-optimized (Table.2), cloned in the PET 28a plasmid by Gene Universal, and expressed in the BL21 codon plus strain of \u003cem\u003eE. coli\u003c/em\u003e. In a study, it was described a variety of expression strains as well as how BL21-Codon Plus (DE3) produced the highest protein yield (Assadi et al. 2008; Robichon et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe extracellular region of CD9, CD63, and CD81 tetraspanin has been retrieved online from NCBI, which provides a wide range of online resources for true biological information and data from 38 distinct databases, including a gene bank, a nucleic acid sequence, a PubMed database, Bio Project, BLAST databases, and so on (Wheeler et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Furthermore, the purity of three tetraspanins- exosomes surface marker as well as its targeted antibodies was characterized through SDS-PAGE. The following SDS-PAGE for antibodies revealed 80\u0026ndash;90% purity and two distinct bands of roughly 68kDa and 28kDa, whereas refolded antigenic extracellular protein revealed 70\u0026ndash;80% purity.\u003c/p\u003e \u003cp\u003eMoreover, IgY concentration in egg yolk elevated throughout the immunization span until week 6, when it started to rise rapidly at 2 weeks and stagnated at 4 weeks. As a result, it is possible to conclude that the chicken's immune system takes approximately 2 weeks to give a boost in raising antibodies (Sudjarwo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, a decrease in titer has been reported in the case of chickens about after 6 weeks of immunization. Even so, a significant decline in titer has been revealed in chickens approximately 6 weeks after immunization (Sudjarwo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, it was demonstrated that chickens immunized intramuscularly with the antigen, can raise targeted antibodies for more than 200 days, and also provides a high titer and tenfold greater specificity than subcutaneous injection (Sudjarwo et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this article, we demonstrate that a stagnant phase of antibodies titration has arrived after the third immunization as measured by indirect ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For indirect-ELISA anti- chicken HRP-secondary antibody has been internally conjugated in accordance with a recent article technique and yielded a titer of 1:6000 (Pavliuchenko et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although, along with the antibody kinetics, titration of all three antibodies was optimized using Indirect-ELISA and resulted in a remarkable titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe antibodies were conjugated to AuNPs, which were synthesized following the previously mentioned, traditional Turkevich method for citrate-stabilised gold nanoparticles (Muhammad et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Optimization of particles morphology, size, and surface distribution were conducted by Transmission electron microscope and spectrophotometer. As previously reported in several studies, TEM images demonstrate that decreasing the temperature results in a larger diameter of gold nanoparticles (Dong et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The nanoparticles were determined spherical in morphology and monodispersed in nature through TEM. The UV-vis absorption spectra of both AuNPs synthesized at 99 ˚C and 80 ˚C show well-defined surface plasmon resonance peaks at 520 nm and 526 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, a shift in the maximum absorbance wavelength of the UV-Vis spectra in contrast to naked AuNPs was interpreted as evidence of antibody conjugation on the AuNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Two different types of nanoparticles, each coupled with three anti-tetraspanins antibodies, were synthesized and characterized here. In comparison to naked nanoparticles, the peak shift was clearly noticed at a difference of 3\u0026ndash;4 nm for all six conjugated complexes for each type of AuNP. Hence, this distinct difference in the absorption spectra peaks indicated a successful conjugation and was utilized as a tool for exosome capturing.\u003c/p\u003e \u003cp\u003eIn this study PEG was used to stabilize of IgY@AuNPs complex and the stability of IgY@AuNPs was analyzed using 0.1 M NaCl solution. Similarly, in a study, Gold nanoparticles introduced to 0.1 M NaCl immediately resulted in a decrease in the absorbance peak, demonstrating their inability to be used in buffer solutions (Christau et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, gold nanoparticles are highly unstable, and various environmental changes, such as pH changes or excessive centrifugation, cause immediate NP aggregation, as evidenced in several studies by the visible colour change (from red to purple) of the AuNPs solution, indicating an increase in particle size (Christau et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Polte et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Consequently, PEG is considered one of the best polymers for increasing the stability of nanoparticles even after numerous centrifugations during the process (Dallari et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Here, the non-covalent immobilization technique was used to conjugate the antibody with nanoparticles, in which antibody was spontaneously absorbed onto the surface of citrate-stabilised nanoparticles with three types of interactions i.e., ionic, hydrophobic, and dative interactions. The attraction between the hydrophobic parts of the antibody and the metal surface results in the formation of a non-covalent bond (Ljungblad et al. 2009). Ionic attraction exists between negatively charged groups on the surface of gold nanoparticles and positively charged groups in antibodies caused by positively charged amino acids and their N-terminal region (Ljungblad et al. 2009; Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, the dative interactions (coordinate covalent bond) are responsible to form a covalent bond between the AuNPs surface and free sulfhydryl groups of the antibody (Ljungblad et al. 2009; Jazayeri et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Before treating with PEG, the antibody and AuNPs complex were stabilized and blocked using BSA due to its nature to prevent the non-specific binding of molecules and so enhance the assay sensitivity. Similarly, in a recent study, BSA was reported to be effective in preventing nanoparticle aggregation (Leopold et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this article, we provide a more efficient and successful approach for isolating exosomes from human serum, which could potentially be employed for exosome isolation or exosomal cargo exploration. Anti-CD63 antibodies were applied to better understand and characterize the exosomes in human serum. Additionally, the expression of exosomal surface protein receptors was investigated, and observed that CD63 is prevalent in serum, but CD81 appears to be the rarest population (Karimi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, they came to the conclusion that although CD9-enriched exosomes are also present, they are less numerous than CD63 (Karimi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, various groups used anti-CD63 antibodies to isolate the population of tiny EVs (Karimi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pammi et al. 2022). Considering this, the TEM findings demonstrated that both conjugates, which were CD63 conjugated to both the 99 ˚C and 80 ˚C AuNPs, had efficiently captured the exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a nutshell, the primary objective of this study was to establish a rapid, cost-effective, and efficient method of isolating exosomes from biological fluids by simply adding extra weight to them. This procedure begins with the identification of extracellular sites, then moves on to the production of antigen in bacterial cells, which is then introduced into chickens to produce anti-exosomal antibodies. ELISA and UV-vis spectrophotometry were employed to characterize antibody titration and synthesis of AuNPs respectively. Furthermore, antibodies were bound to AuNPs to isolate exosomes, and this complex was used to pull down the exosomes from fluids using the antibodies' specificity. The isolated exosomes were successfully captured by TEM and could be used for a variety of purposes, including disease diagnosis, drug delivery, and miRNA extraction. We anticipate that the proposed approach will be valuable in more in-depth research on exosomal subcategories, and it can be applied to different sized small extracellular vesicles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the IgY Immunologix India Private Limited, Hyderabad, India for providing financial support and Vignan\u0026rsquo;s Foundation for Science, Technology and Research, Guntur for providing other facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDikshita Panwar: Carried out the work and writing the original draft; Lavleen Kumar Gupta: Conceiving the idea and designing the work; Deepali Shrivastava: Contribute in laboratory techniques; Arvind Kumar: Contribute in designing the work; Anjani Devi Chintagunta: review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIgY Immunologix India Private Limited, Hyderabad, India has provided financial support to carry out the present work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human or animal subjects\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 with respect to the work described in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAssadi-Porter FM, Patry S, Markley JL (2008) Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in \u003cem\u003eE. coli\u003c/em\u003e. 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Nanomater 24:2022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/2042754\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AuNPs@IgY, Extracellular vesicles, Exosomes, ELISA, Gold nanoparticles, Tetraspanins","lastPublishedDoi":"10.21203/rs.3.rs-2885310/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2885310/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExosomes, a subpopulation of Extracellular vesicles (EVs), are cell-secreted vesicles found in the majority of biological fluids, including breast milk, tears, sweat, blood and, urine. The density and size of these vesicles depend on a variety of factors, including age, gender and the biological condition of the individual. Researchers are now focusing on the selective extraction of exosomes from bodily fluids due to the unique biomolecule composition of exosomes, which is critical for diagnosis, disease, and regeneration. Furthermore, current approaches for exosome isolation have limitations, necessitating the development of a simpler and more effective technique to achieve this goal. In this study, we investigated a quick and effective strategy for isolating exosomes from serum using a bench-top centrifuge. This was accomplished by raising antibodies against exosome surface tetraspanins (CD9, CD63 \u0026amp; CD81) in Leghorn chickens due to their phylogenetic distance from humans and cost-effectiveness for commercial use. In order to separate exosomes from a complex biological fluid, the antibodies were further coupled with gold nanoparticles. The findings were validated using ELISA, spectrophotometry, and electron microscopy. Using this technique, exosome isolation from serum was achieved rapidly and these were captured by using anti CD63 antibodies bound to AuNPs. To summarize, exosomes were purified from serum using anti-CD63 antibodies conjugated to gold nanoparticles (IgY@AuNPs). Consequently, the approach for exosome isolation from biological fluid could be useful for clinically monitoring the biological state of the patients.\u003c/p\u003e","manuscriptTitle":"Efficient strategy to isolate exosomes using anti-CD63 antibodies conjugated to gold nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-07 13:38:41","doi":"10.21203/rs.3.rs-2885310/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2023-07-29T06:22:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-07-05T15:52:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-03T06:37:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-23T14:29:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"AMB Express","date":"2023-06-23T09:20:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00a0f2a8-854f-42ce-a1cc-58d26c42547b","owner":[],"postedDate":"July 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-09-07T15:11:16+00:00","versionOfRecord":{"articleIdentity":"rs-2885310","link":"https://doi.org/10.1186/s13568-023-01592-1","journal":{"identity":"amb-express","isVorOnly":false,"title":"AMB Express"},"publishedOn":"2023-08-28 15:08:46","publishedOnDateReadable":"August 28th, 2023"},"versionCreatedAt":"2023-07-07 13:38:41","video":"","vorDoi":"10.1186/s13568-023-01592-1","vorDoiUrl":"https://doi.org/10.1186/s13568-023-01592-1","workflowStages":[]},"version":"v1","identity":"rs-2885310","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2885310","identity":"rs-2885310","version":["v1"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}