A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody

A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody Yogita Rajput, Shailendra Mani, Aarti Kushwaha, Satendra Kumar, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6417069/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Regular surveillance of herd immunity and individual protection by measuring neutralizing antibody against SARS-Cov-2 would be instrumental to control future COVID-19 pandemic. The SARS-COV-2 live virus neutralization tests or other surrogate virus neutralization tests could not be used for mass scale clinical application. A robust cell based surrogate SARS-Cov-2 neutralization assay (CENA) have been designed, based on blocking of GFP-tagged RBD (RBD-GFP) to its interaction with cell surface ACE2 receptor and detected by flow cytometry. A stable and high binding of RBD-GFP on cell surface was achieved using ammonium containing buffer inhibiting receptor-ligand endocytosis. Using DMSO containing frozen cells instead of conventional fresh cultured cells in the assay, enable further reduction of endocytosis as well as simple PI stanning of the nucleus for identifying cells in ordinary flow cytometry without additional staining protocol with antibody or any washing steps.. CENA were compared with (i) commercially available surrogate neutralization kit (cPass) and (ii) live virus neutralization assay showing sensitivity 97% and 100% respectively. Specificity of CENA using pre-COVID-19 pandemic samples were 100%. CENA were highly corelated with both live virus neutralization assay (r=0.9.p=0.0016) as well as with cPass kit(r=0.9,p=0.00001 respectively). Very poor correlation with conventional binding anti-Spike protein IgG level detected by ELISA (r=0.14), suggests that the conventional ELISA tests could not be a substitute for detecting neutralizing antibody. This is first ready to use cell based neutralization assay design, to be carried out without requirement of cell culture facility at end user’s site and could be completed in two hours. Besides surveying anti-SARS-COV-2 protective antibody, the assay could also be used for low budget high throughput screening of blocking molecules. Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Clinical microbiology Biological sciences/Microbiology/Virology Health sciences/Medical research/Translational research Neutralization antibody surrogate SARS-CoV-2 cell based Microneutralization test Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The pandemic corona virus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2). Though pandemic have been controlled following vaccination, COVID-19 is still prevalent across the globe at a variable positivity rate 0.7 to 16% (WHO report Oct, 2024, 1). As per the WHO report, there is increased number of reported cases and deaths during the survey period of 28-day from 19August to 15 september comaired to earlier survey report, with over 270 000 new cases and 5700 new deaths (1). So there is always risk for future epidemic which justify the need for surveillance of protective immunity. SARS-CoV-2 is a new corona virus identified in late 2019 and belongs to a family of enveloped RNA viruses that include MERS and SARS, both of which caused serious human infections of respiratory system (2). As a first step of the viral multiplication cycle, the virus attaches to the host cell surface before entering into the host cell. The Corona virus envelope Spike protein (S protein) has been reported as a significant determinant of virus entry into host cells. The S protein receptor binding domain (RBD) recognizes and attaches to the host cell surface Angiotensin-Converting Enzyme 2 (ACE2) receptor found on the surface of type I and II pneumocytes, endothelial cells, and ciliated bronchial epithelial cells as well as oral and nasal epithelium. Following binding to the cellular receptor critical proteolytic cleavage event occurred at SARS-CoV-2 S protein, leading to membrane fusion and viral entry. After the virus enters the cells, the viral RNA genome is released into the cytoplasm and is translated into viral polyproteins and viral replication cycle is started (3). Currently no proven therapy is available for COVID-19. Though several vaccines are already available and permitted for emergency use, however its efficacy and protective immune response in different group of individuals are under investigation. Protective immune response against SARS-CoV-2 involves generation of anti spike protein antibody however only a subset antibodies against specific epitope at receptor binding domain (RBD) play the key role to neutralize the virus for binding to its receptor ACE2 and block infection to the host cells (4). Currently most of the vaccine developments against SARS-CoV-2 infection are to block the interaction of spike protein and ACE2. Immune response to natural COVID-19 infection or vaccination may not be occurred in equal efficacy in different individuals leading to different outcome of vaccination. So detection of neutralizing antibody in serum is very important to evaluate protective immunity status following natural infection or post COVID-19 vaccination. This will not only help for individualize patient management but also help in identification of acceptable donors for convalescent plasma therapy. The virus neutralization assay detects presence of the antibody which actually blocks the virus from infecting to the host cell. This antibody usually blocks the receptor binding domain of the virus protein at the interface which interacts with host cell receptor for entering into the cell. As a gold standard, neutralization assay is carried out using live virus by standard plaque reduction neutralization test (PRNT) or micro neutralization test in 96 well format. However, to carry out the test required special BSL-3 laboratory, expensive safety measure and also laborious, take at least 4-5 days for completion of the assay. While the gold standard assay could be used as reference or validation of any new assay, however it cannot be available for mass scale diagnostic and research purposes (5). Alternatively few surrogate neutralization assay have been developed recently using in vitro interaction of recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein and its receptor ACE2. Currently two class of commercially available surrogate neutralizing antibody assay kits are available for SARS CoV-2. (i) Pseudo virus based Neutralization assay kits: Cell based surrogate neutralization assays have been designed using replication-defective pseudotyped viral particles (6,7) which can be performed under BSL-2 conditions. In this assay pseudotyped virus is developed which express RBD spike protein of SARS-CoV-2 on its surface. Reporter gene (luciferase or florescent proteins) is expressed in the host cells following binding of pseudotyped virus containing SARS-CoV-2 spike protein to its cell surface ACE2 receptor and transduce genetic material into the host cells. If the pseudo typed virus stock has been prepared in advance, it will take at least 2 days to get result for the candidate samples. So it also involves multistep procedure, long time to complete the assay and high manufacturing cost limiting its clinical use. (ii) Protein based kits in ELISA format: One protein based surrogate neutralization assay for SARS-CoV-2 (8,9) is commercially available which detect blocking of soluble COVID-19 spike RBD to its corresponding receptor ACE2 bound at the plate. It detects blocking of soluble SARS-CoV-2 spike RBD to its corresponding receptor ACE2 bound at the plate or vice versa. The test could be carried out in 2-3 hrs in ordinary laboratory (10). As ACE2 protein component is membrane protein may not be fully active in cell free purified form so the invitro interaction on solid surface may not be representative of cell surface natural interaction in natural protein conformation and environment. Moreover the cost of purification of the proteins and using separate reagents for detection system are always increase manufacturing cost. So the kit is very expensive, limiting its wide spread clinical and research use. Another cell based binding assay for showing in vitro interaction of RBD of SARS-CoV-2 Spike protein with its receptor ACE2 have been reported for research purposes by transient transfection of RBD-sfGFP and myc tagged ACE2 (11) expression cassette plasmids into a suspension cell line Expi293. In this assay GFP tagged RBD of SARS-CoV-2 Spike protein is secreted into the supernatant media which is separated from culture of expi293 cells which were transiently transfected with the plasmid. Expi293 cell expressing Myc tagged ACE2 on cell surface were produced by transient transfection of pCEP4-myc-ACE2 plasmid containing the expression cassette of ACE2 taged with Myc (for its in vitro detection by anti Myc antibody). In this binding assay format The supernatant media containing the RBD-GFP was added into cell suspension expressing ACE2 cells on its surface. For staining ACE2, anti-Myc antibody was used in the reaction. DAPI was used for staining dead cells. Following incubation on ice, cells were washed with PBS and flow cytometry was carried out. DAPI positive cells were excluded and Myc positive cells were gated as representative marker of ACE2 expressing cells and the RBD-GFP binding was expressed as % of ACE2 positive cells. However this assay had several limitations to be used directly as surrogate neutralization assay in a ready to use format in clinical laboratory as well as at an affordable cost. (i) In this assay suspension cells were used to make compatible with downstream flow cytometry procedure. This could bypass trypsinization steps which may affect cell surface receptor integrity. However Transfection to the suspension cells is very difficult. To ensure high transient transfection rate in the suspension cells, specially engineered Expi293 cell were used which required special media and transfection reagent. This special media and reagent is very expensive resulting increase cost of the assay. (ii) As it was transient transfection based assay, for every assay new transfection will leads to increase the cost of the assay, less reproducibility and inconvenient to be packaged as kit for diagnostic test. In suspension cells, the dead cells following transfection or due to culture propagation, cannot be easily removed. So the dead cell products will be released in the media which could degrade the RBD-GFP reducing its activity or affecting RBD-ACE2 complex to a variable degree. Dead cell degradation product of ACE2 expressing expi293 cells may block RBD-GFP reducing its effective concentration for binding to intact ACE2 expressing cells. To exclude dead cells additional stain (DAPI) also used for which additional UV channel is required in flow cytometry machine and most of the ordinary clinical laboratory this UV channel is not present. (iii) It is not feasible to maintain cell line in ordinary clinical laboratory. For every new batch of ACE2 tranfected cells, concentration of cells have to be determined and based on transfection efficiency all the assay parameter have to be readjusted before starting a assay. The neutralization titre is very much depends on ratio of number of ACE2 positive cells and concentration of RBD-GFP in the reaction mixture which cannot be maintained in transiently transfected cell lines. So those variables will contribute to gross variation and reproducibility from batch to batch and assay to assay. (iv) As cell surface binding of viral protein causes endocytosis the reaction was needed to conduct in low temperature which blocks endocytosis. However, even in low temperature also 100% blockage is not possible. So slowly the receptor protein complex is endocytosed in presence of K + containing PBS buffer system and the signal is faded with time (12-15). So there is chance of unstable reading of binding RBD-GFP to ACE2 when more samples will be assayed. All together the reported form of the cell based RBD binding assay protocol (11) is not fit for in-vitro diagnostic assay and laboratory to laboratory comparability of the data. Importantly the protocol described was not ready to use “stand alone” to be used as kit form in the clinical laboratory. (v) Additionally though most of the cell based surrogate assay mimic close to gold standard assay than an ELISA based assay, however due to biological variables most of the cell based assay have high CV making it unsuitable for diagnostic assay. Taken together, the issues discussed involving the previous protocols are unfit to be an affordable and ideal diagnostic assay kit to be carried out in ordinary clinical laboratory by less trained health staffs. Considering the all previous limitation, in this article we describe a method of cell based assay design for detecting neutralizing antibody against SARS-CoV-2 in serum/plasma using flow cytometry. Unlike other multistep cell based assay using pseudotyped virus, The method could be carried out in two step in 3 hours and without need of any washing steps as like other serological assay for eliminating background. The assay could be packaged stand alone kit format and the end users in clinical laboratory will not be needed any cell culture prior to the assay or expertise in cell culture handling and will have lowest manufacturing or establishment cost. Methods Ethical statement Study was carried out at Multi-Disciplinary Research Unit (MRU), Pt. Jawaharlal Nehru Memorial Medical College, Raipur Chhattisgarh, India and Dr. B.R.A.M. Hospital Raipur, C.G., (under Pt. Jawaharlal Nehru Memorial Medical College, Raipur) following approval of the protocol by institutional ethical committee. All procedures were carried out in accordance with relevant guidelines and regulations. Human samples were collected following informed consent from the study subjects . Cell line and cell culture HEK 293 cell line was procured from NCCS, Pune, India. HEK 293 cell was grown in MEM media (HiMedia, India) with 10 % fetal bovine serum (USA origin, HiMedia India) and 1 X Anti-Anti (HiMedia, India) incubated at 37 o C with 5% CO 2 . Preparation of the assay components: Preparation of stable SARS-CoV-2-S-RBD-GFP-HEK293 cell line HEK 293 cell was stably transfected with a plasmid pcDNA3-SARS-CoV-2-S-RBD-sfGFP, previously described (3), containing expression cassette of SARS-CoV-2 RBD tagged with sfGFP with signal sequence for its extracellular release and having mammalian selection marker. The plasmid is distributed through addgene (https://www.addgene.org/141184/sequences/). For stable transfection the RBD-GFP expression plasmid is first transfected by lipofectamin 3000 then after 48 hrs media is replaced by fresh media containing selection antibiotic Genticin 400µg/ml (G418). After two weeks of incubation in selection media, single green flurescent colonies were identified, separated and re plated into 96 well plate. Following regrowth the well was screened for most populated green florescence cells under florescence microscope. Further screening was done by checking supernatant of the wells having green florescence cells. Florescence was measured using an appropriate filter for GFP (detailed below). The colony which gave the best green florescence in the supernatant was propagated and designated as SARS-Cov-2-S-RBD-GFP-HEK293 cell line and amplified for preparing optimized RBD-GFP containing media ( component-1 ) secreted from the stable cell line. The cell line was grown upto 70 % confluence. Standardization of RBD-GFP containing media (component-1) Following 70% confluency of the cells, fresh MEM media was added (2ml for 25cm 2 flask and 6 ml media for `75cm 2 flask). After 4 days of incubation media was collected. Media was centrifuged and supernatant was collected and fluorescence is adjusted to 60-25 arbitrary fluorescent U (FU) diluting with 1 x RB (component 3) . At this range of florescence level, assay will be more linear and will have wider linear range of detection limit. The media was further calibrated with a positive control (PC) reference serum (cocktail of post vaccinated/ convalescent serum), neutralizing antibody titre of which have been predetermined by live virus neutralization assay. Dilution of the media was adjusted so that titre of the PC by the new test closely matched with the titre of gold standard assay while the % of RBD-GFP positive cells of untreated control is kept above 80% . The diluted media was dispensed in 2ml tubes and kept in -20 o C. For calibration of new batch of RBD-GFP containing media, activity of RBD-GFP is calibrated by diluting the media, adjusting % of GFP positive cells and mean florescence of single cell of the previous standardized batch. Florescence measurement protocol by Multi mode plate reader (Tecan): Measurement of Florescence of RBD-GFP containing media: 100 ul of media directly or diluted by 1x RB was dispensed into the well of 96 well black opaque plate (Corning, Thermo fisher). Florescence was measured by multi-mode plate reader, SPARK, TECAN at a wave length 485 nm excitation and 535 nm emission and band width 10nm (gain manual 70). Preparation of ACE2-HEK293 cell line and its packaging protocol: An adherent cell line HEK 293, which don’t express ACE2 was stably transfected with the plasmid pCEP4-myc-ACE2a, containing ACE2 expression cassette and mammalian selection marker (3) and distributed through addgene. For stable transfection the ACE2 expression plasmid is first transfected by lipofectamin 3000 (Thermoscientific) then after 48 hrs media is replaced by fresh media containing selection antibiotic Hygromyocin 150 g/ml. After two weeks single colonis were separated and replated into 96 well plate. Following re-growth functional screening was carried out by observing RBD-GFP binding activity under fluorescent microscope. Colony with optimal binding of RBD-GFP is propagated for the assay. Reaction buffer: All the cell washing and resuspension and downstream experiments was carried out in reaction buffer (RB). The RB was consist of NaCl close to isotonic concentration along with an inhibitor of endocytosis and a buffer system not containing phosphate. e.g. 140 mM NaCl and 25mM NH 4 Cl pH 7.4. RB will stabilizes RBD-ACE2 complex on cell surface preventing endocytosis). Cell packaging The cells were washed with reaction buffer (RB) and dislodged from the cell culture flask by scrapping by cell scraper and single cell suspension was prepared by pipetting in RB buffer and cleared by passing through cell strainer. Cells were centrifuged and supernatant was removed and resuspended in 1 x RB. Cells were counted, centrifuged and supernatant removed. Cells were resuspended in RB with 10% DMSO at a concentration 50 Lakh cell /ml and dispensed 20µl per 2 ml tube sufficient for 10 reaction and kept at -80 o C till it is used. Assay protocol Assay components Component-1: RBD-GFP containing media Component-2: ACE2-HEK293 cell line Component-3: 10X Reaction Buffer (1.40 M NaCl, 0.25M NH 4 Cl, pH7.4) Component-4: 1000X Propidium Iodide (0.5mg/ml) (Sigma) Component-5: Positive control (PC) Component-6: Negative control (NC) reference serum: Cocktail of negative serum which have been predetermined by gold standard assay (PRNT). Assay steps Blood was collected without any anti coagulant and serum was separated and aligoté was stored at -20C if was not used within 7 days. 10X RB (component 3) to be diluted to 1 X RB by sterile distilled water. Cell re-suspension buffer (CRB) was made by adding 1ul/ml 1000X PI (component 4) into the 1X RB (component 3) Serum was serially diluted with the 1X RB µl frozen ready to used standardized media containing RBD-GFP was taken into 96 well plate. 5 µl serum samples or its dilution was added into the respective well. Following incubation for 1 hour at room temperature, plate was kept on ice for additional 15 minutes. Frozen cells were (component 2) thawed on ice and 75 times diluted with pre cooled cell dilution buffer (CDB) containing 0.5µg/ml PI (e.g for 20 µl frozen cells in 1.5 ml CDB). 150µl ice cold cell suspension was added into each well. In this way 10k cell was distributed in each reaction. The reaction was mixed properly and kept for 60-90 min into ice bath. Following incubation reaction mixture was pipetted to remove any cell clump and the whole reaction mixture is added into ice cold 500µl RB kept into flow cytometry tubes. Tubes were kept into mini cooer (-20 o C) till flow cytometry measurement was performed. Flowcytometry analysis Flowcytometry analysis was performed in Sysmax flow cytometer, Germany, using inbuilt Cyflow TM software. GFP was detected by FL1 and PI was detected by FL3 Chanel. First single intact cell population is gated form SSC-FSC plot. Then PI stained cells were gated from SSC-PI plot. In a GFP-PI quadrant plot threshold is adjusted by running cells stained with PI without addition of RBD-GFP and it is set at 0 to 2% as threshold for FL1 Chanel or for GFP. % of RBD-GFP binding to the HEK293 ACE2 cells is indicated by % of GFP positive cells. Binding of RBD-GFP to the cells without incubation with any serum samples or inhibitor is considered as 100% binding. Any reduction of binding was detected as reduced % GFP positive cells, following incubation of the RBD-GFP (component 1) with any serum samples or inhibitor was the measure of neutralization. Method of getting and analysis by flow cytometry is demonstrated in Fig 3. MNT (Microneutralization Test): Vero-E6 cells were seeded into a 96-well plate at a density of 15,000 cells per well and incubated overnight at 37°C in a humidified atmosphere with 5% CO₂ to establish a monolayer. Serum samples were heat-inactivated at 56°C for 30 minutes prior to use. To assess the neutralizing capacity of serum antibodies, 50 µL of SARS-CoV-2 Wuhan strain (containing the 100 TCID₅₀ virus/50 µL) was mixed with an equal volume of heat-inactivated serum samples serially diluted (1:20 to 1:2560) and incubated at 37°C for 1 hour. After incubation, the serum-virus mixtures were added to the wells containing the pre-seeded Vero-E6 cells and incubated for 1 hour at 37°C with gentle agitation every 15 minutes to facilitate viral adsorption. Following this step, the inoculum was aspirated, and the cells were washed with serum-free medium. Subsequently, 150 µL of maintenance medium (DMEM supplemented with 2% FBS, 2× antibiotics, and non-essential amino acids) was added to each well, and the plates were incubated at 37°C with 5% CO₂ for 72 hours. The cells were then observed under a microscope for the presence of cytopathic effects (CPE), and the data were recorded. Neutralizing antibody titers were determined based on the serum dilution that inhibited CPE. Surrogate (virus free) SARS-Cov-2 neutralization antibody test (sVNT) : The test was performed by the well validated commercial test kit “cPass s SARS-CoV-2 Neutralization Antibody Detection kit”(GenScript, USA) as per the manufacturer’s protocol (https://www.fda.gov/media/143583/download) at a serum dilution 1:20 and each samples were measured in duplicate and average was determined. Anti-Spike protein SARS-Cov-2 IgG ELISA test: The test was performed using the commercial kit “EDI™ Quantitative SARS-CoV-2 Spike Protein IgG ELISA Kit“, Epitope Diagnostic Ink(EPI),CA, USA as per the manufacturer’s protocol at a serum dilution 1:100 and each samples were measured in duplicate and average was determined. Statistics % Neutralization of CENA (by only cell count) was calculated by the formulas: % NT = [1- (X-b)/(C-b)]*100 …. Equation 1 % NT(total florescence) = [1- {(X-b)/(C-b)}*{F X /F C }]*100 …Equation 2 NT: % Neutralization, X: % GFP positive cells in test serum/ treated sample C: % GFP positive cells in untreated control sample (without serum) b: background % GFP positive cell in unstained samples (without RBD-GFP) Fx : average florescence/cell in test serum/ treated sample F C : average florescence/cell in untreated control sample (without serum) F X /F C : Represents correction factor due to difference in intensity of fluorescence of RBD-GFP bound cells following neutralization of RBD-GFP by the neutralizing antibody. Though commonly in any flow cytometry based assay analysis is carried out based on % of positive cells in particular channel, in very high concentration of RBD-GFP, Equation 2 represent more accurate and linear curve. Upon serial dilution of RBD-GFP, the neutralization curve by equation-1 and Equation-2 become very closed after a certain point of dilution. Below that point simpler Equation-1 could be used for all purposes (Figure 1D I-VI). Mean, standard deviation, median and Pearson correlation and regular graphs were performed using Microsoft excel. significance of Pearson’s correlation, and sensitivity specificity test were analysed using the following web-based software: MedCalc (https://www.medcalc.org/calc/odds_ratio.php.html). Statistical significance was considered if p ≤ 0.05. For making dot plot web based software Interactive Dotplot was used. For making 4PL graph of neutralization reading at different dilution of serum, GraphPad Prism soft wear was used. *Cohen’s kappa (k) was determined using a web based software ‘Cohen’s kappa free calculator” (https://idostatistics.com/cohen-kappa-free-calculator/#risultati) as described by Landis, JR & Koch, GG (1977) for the measurement of observer agreement for categorical data. ** Interpretation of Cohen’s kappa (k) : 0.01 – 0.20 slight agreement 0.21 – 0.40 fair agreement 0.41 – 0.60 moderate agreement 0.61 – 0.80 substantial agreement 0.81 – 1.00 almost perfect or perfect agreement Result Principle of the assay is based on binding of GFP tagged receptor binding domain of SARS-COV-2 Spike protein (RBD-GFP) which is secreted into the media from a stably transfected HEK 293 cells , to cell surface ACE2 ( stably transfected into another 293 cell and expressed constitutively) and the fluorescence of the bound cells quantitated by flow cytometry as % of green fluorescence(GFP) +ve cells. If the media is pre-incubated with any chemical blocking agents or blocking antibody against Spike protein RBD of SARS-CoV-2, residual free RBD-GFP will bind to the cells and the reduction of % of GFP +ve cells to the untreated control is proportional to concentration of inhibitor or blocking/neutralizing antibody. The principle of the assay is shown in Figure 1 A (I-IV). Cells incubated with control sample shows green ring outside the cell membrane indicating binding of RBD-GFP on cell surface (Figure 1 AI). Upon incubation with serum from anti-SARS-CoV-2 vaccinated (Covishield) subject, the green hallows disappeared (Figure 1 AII) indicating blockage of interacting interface of RBD of SARS-CoV-2 Spike protein. Same observation replicated when the corresponding samples were analysed by flow cytometry (Figure 1 AIII & AIV). Though the principle of the assay was very simple however, as like other cell based assay several known and unknown contributing factor was there making the assay result inconsistent and inconvenient to be a clinical grade diagnostic test. In the present cell-based assay design effect of those factors / limitations were minimized to produce a reproducible robust assay by several strategies. which include (i) stable transfection of the expression cassette, (ii) designing optimum buffer system for stable binding of RBD-GFP to the cell surface ACE2, minimizing receptor-ligand mediated endocytosis of the viral protein, (iii) optimum dilution of media for a consistent linear results, (iv) simple method for non-antibody counter staining of cell, (v) optimizing method for packaging the assay in a kit format so that end user does not required any cell culture system, (vi) methods of calibration,(vii) reducing variation of cell based assay from batch to batch. Ammonium Chloride buffer at pH7.4 shows highest RBD-GFP binding to ACE2 expressing 293T cells and signal stability, among the tested buffers It has been shown that binding of SARS-CoV-2 virus to the cell surface ACE2 receptor leads to endocytosis and internalization of the complex leading to the disruption of the virus protein (13,15). This mechanism may cause disruption of GFP causing decaying of GFP signal. We also observed decaying of RBD-GFP signal upon binding in room temperature (data not shown). To slow the process, we had applied multiple measures such as optimum buffer system which stabilize the complex on cell surface by inhibiting the endocytosis process along with conducting the assay at low temperature as well as cell freezing. For screening optimum reaction buffer for stable and high RBD-GFP binding to HEK293 cells expressing ACE2, seven different reaction buffers (RB1,RB2, ...RB7) were tested. The detail composition of the buffer and the rational is described in table 1. Following first reading after dilution of the reaction with the corresponding RB at 2 hour, 2 nd reading was taken after 20 minutes on ice following the first reading. RB-6 showed highest level RBD-GFP binding to the cells (Figure 1 BI) and highest stability of the binding (Figure 1 BII) at 20 minutes following the first reading when compare the same to buffer RB-1, RB2, RB3(10% vs 28%, 17% reduction of signal). So for the subsequent experiment we used RB-6 and designated as RB in the subsequent text. Effect of DMSO and cell freezing on live cell Propidium Iodide staining and RBD-GFP binding on ACE2 expressing HEK 293 cells. For identifying only nucleated cells in flow cytometry removing the debris or any other non-cellular particulate components or any precipitate which may arise from serum, a nuclear stain was used which could be detected by commonly available channels. Though PI is very common and inexpensive nuclear staining dye and detected by commonly available Chanel, only could penetrate into dead cells. As DMSO and cell freezing causes increase cell permeabilization we evaluated if both may increase nuclear stanning of PI. PI was incubated with ACE2-293 cells processed in three condition (i) kept at 4ºC without DMSO (ii) kept at 4ºC with 10% DMSO, (iii) kept at -80ºC with 10% DMSO as shown in figure 1 CI. Expectedly cells frozen at -80ºC with DMSO showed highest (16%) PI staining whereas keeping at 4ºC without DMSO showed least PI staining (8.6%). As cell freezing with DMSO also will reduce metabolic activity which may further increase stability of RBD-GFP binding to ACE2 on cell surface, % of RBD-GFP binding was verified and figure 1 CII showed expectedly highest RBD-GFP binding when cells were freezed at -80ºC with 10% DMSO compared to keeping at 4ºC with or without DMSO. Optimization of RBD-GFP containing media by appropriate dilution critical for stable assay result For standardization of the assay, we prepared a cocktail of 10 positive serum samples (PC) collected from post vaccinated subjects and a cocktail of negative serum samples (NC) from unvaccinated, uninfected subjects and confirmed by live virus ant-SARS-CoV-2 neutralizing antibody assay. The PC and NC were subsequently used as assay standard. Binding of RBD-GFP to ACE2 expressing cells depend on ratio of number of free RBD-GFP in the reaction and 293 cell number expressing ACE2. Media having relatively high concentration of RBD-GFP in a given concentration of cells will cause increase intensity of average florescence of cells due to more number of RBD-GFP binding on each cell. At high concentration of RBD-GFP, as there will be excess number of RBD-GFP than total number of cell surface ACE2 in the reaction, high number of antibody will be required to block the excess RBD-GFP before it affect the % RBD-GFP bound +ve cells. So calculating neutralization by using only the single parameter % of RBD-GFP +ve cell will not be linear and will give false negative results. Average per cell fluorescence also should be considered in calculation as 2 nd parameter. So calculating reduction of % of total Florescence (% RBD-GFP +ve cells * Florescence/cells, Equation-2, material methods) is more better representation of the extent of neutralization. So in higher concentration of RBD-GFP, neutralization curve by % of RBD-GFP +ve cell count (Equation-1 in material methods) vs % of total florescence of RBD-GFP positive cells (Equation-2, material methods) will be different. We hypothesized that if RBD-GFP is serially diluted and neutralization curves are plotted at each dilution point, a point will reach, following which neutralization curves using equation 1 and equation 2 will be very close/ equivalent and give a linear curve. Figure 1D(I-VI) shows, at a florescence unit 50 (dilution 1:8 of original media,) or lower concentration, NT curves by % of RBD-GFP positive cells and % of total florescence were very similar and merged to each other. However, if the media is too much diluted linear range of detecting Neutralizing antibody will be very small and the reading will be very unstable because only small proportion of cells will be bound by RBD-GFP. To demonstrate that, the range of RBD-GFP concentration starting from 200 FU upto 6.25 FU were used to perform CENA. Value of NT was plotted in primary Y axis while % of RBD-GFP positive cells in untreated control in each of the concentrations were plotted in secondary Y axis against concentration of RBD-GFP in X axis (figure 1 E). It is interesting to observe the inverse relation of % of RBD-GFP binding and reading of NT. Media at a dilution 1:8 at FU 50 shows optimum RBD-GFP binding in control (88%) cells. More diluting the RBD-GFP caused lower % of RBD-GFP binding which causes lower resolution to detect a small difference. So, for subsequent batch of RBD-GFP containing media was calibrated accordingly using the previous batch RBD-GFP and PC. Determination of time course for optimum binding of RBD-GFP to ACE2-293 cells. Cells were incubated with RBD-GFP containing media at 50 FU on ice and flow cytometry was performed at different time interval till 90 minutes as shown in figure 1F. A Plateau of % of RBD-GFP positive cells were reached within 60 minutes. However for maximum binding for subsequent tests incubation was for 90 minutes. Determination of coefficient of variance For determination of CV at different level of neutralization of high (>60%) moderate (60-40%) and low (40-20%), serum samples were diluted appropriately and assay was performed with a 5 replica of the samples. CV for the three range of neutralization by CENA was 3%, 10% and 25% respectively shown in Table 2. While recommended CV for a cell based immune assay is 20-25%, our results showed within the range in all level of neutralization while high and moderate level neutralization had much lower CV than the recommended. Comparative correlation of CENA and commercially available cPass SARS-CoV-2 Neutralizing Antibody Detection Kit. Serum samples were used as follows: (i) Serum samples of subjects who already completed two doses of COVID-19 vaccine (53 samples) (ii) Serum collected before (before March 2020) COVID-19 pandemic (40 samples), (iv) Cocktail of serums from 10 COVID-19 vaccinated individual (PC), (iv) Cocktail of serums from individuals having no reported RT PCR positive COVID-19 infection and no vaccination (NC). cPass SARS-CoV-2 Neutralizing Antibody Detection Kit, (Genescript) is the only FDA (Emergency Use Authorization) approved well validated and internationally widely used kit for detecting SARS-CoV-2 neutralization antibody. The test was performed as per manufacturer’s protocol in duplicate at a serum dilution of 1:20 (as per recommendation). The reading was taken in SPARK multimode plate reader (TECAN). % of Neutralization was calculated as per the formula provided by the manufacturer. The cell based Neutralization assay CENA was carried out at 1:20 dilution by flow cytometry. % NT was determined as described above using a cut off 20% (CENA20). CENA20 was determined by measuring %NT of 20 pre-COVID-19 pandemic (collected before march 2020) serum samples. Mean+3SD of NT was determined to be 18% (-6%+24%). So for practical purpose 20% was used as cut off making it towards more stringent. Another 20 pre-COVID-19 samples were used for validation. For the NT assay using commercial cPass kit cut off was 30% as per manufacturer’s protocol. Results showed out of 53 post vaccinated serum samples tested 49 had agreement with both CENA and cPass kit (92.4%) and Cohen’s kappa: 0.806 suggesting almost perfect or perfect agreement (Table 3 A and B). Those 4 samples which were not with agreement with CENA had NT reading were closer to border line of cut off values. Correlation analysis was performed and Figure 4 B showed CENA have good linear correlation with the cPass SARS-CoV-2 kit ( R 0.94, p<0.00001). Corelation of CENA and conventional live virus-based SARS-COV-2 viral neutralization assay (cVNA) Total 11 post vaccinated serum samples of different range of neutralizing antibody measured by live virus neutralization assay were used to determine corelation of antibody titre with CENA. The serum samples were serially two fold diluted and each was tested for NT by CENA and also by live virus NT (micro-neutralization in 96 well plate) assay (cVNT) as described previously. Serum titre was determined by cVNT or CENA as the highest limit of serum dilution which produce 100% neutralization (cVNT100) or 50% neutralization (CENA50) respectively. Correlation analysis of serum titre measured by both the test showed CENA50 have a good correlation with the cVNA100 ( R 0.9, p<0.0016) (figure 4 A). Determination of Sensitivity and specificity of CENA For determining sensitivity of CENA, the two standard assays (i) FDA approved commercially available SARS-CoV-2 Neutralizing Antibody Detection Kit (cPass, Genescript) and (ii) SARS-CoV-2 live virus neutralization assay (cVNA) were used for detecting positive samples and pre COVID-19 (collected before March 2020) serum as true negative samples. Sensitivity of CENA using cPass kit as standard assay was 97% (37/38). 1 sample (NMR180) which were detected false negative by CENA when we compared cPass as standard test, actually proved true negative when we compared with live virus assay. However all 20 positive samples, detected positive by Live virus neutralization assay was also detected positive by CENA resulting 100% sensitivity (20/20). As no test have absolute specificity, we used pre-COVID-19 samples collected before March 2020 as true negative samples for determining specificity. We observed out of 20 samples tested by CENA all showed negative resulting 100% specificity (20/20) (Table 4). Dot plot in figure 4 E shows comparative distribution of %NT by CENA and the commercial kit (cPass) using post vaccinated serum samples and pre- COVID-19 pandemic (before March 2020) negative serum samples. While both the method showed similar distribution of %NT, however 2 of the pre-COVID-19 pandemic samples (true negative samples) were detected as positive (false positive) by Cpass commercial kit while all the pre-COVID-19 samples were detected negative by CENA suggesting CENA have better specificity. Together our assay performance shows much superior than the commercial kit Cpass. Example of using the test CENA to compare effectiveness of multiple brand of vaccines to induce neutralizing antibody against SARS-CoV-2 For comparing vaccine response to different brands of COVID-19 vaccines, serum samples of two healthy individuals before and 1 month after COVID-19 vaccination were collected for the assay. one subject was vaccinated with Sputnic V and another subject was vaccinated with Covishield. Serum samples were serially two fold diluted starting from 1:10 and neutralization assay was carried by CENA till the NT reached to baseline. IC50 was determined from 4PL graph. The result is shown in Figure 5. Comparing the two experimental subjects, the person who received Sputnic V has been shown to be less immune response following single dose than the subject receiving Covishield ( IC50: 78 vs 134). Surveying neutralizing antibody titre response of different brand of COVID-19 vaccine within a population could be carried out for implementing larger vaccine policy for the population. Alternately the same example of the assay could be used for personal protection. If a particular brand of vaccine failed to create protective response another brand of vaccine could be tried if national protocol may permit. Conventional ELISA test to detect total binding antibody against SARS-CoV-2 Spike protein can not be substitute for Neutralization test for sero surveillance. Commonly ELISA based test is used to detect total binding IgG or IgM against proteins of an organism to evaluate sero-conversion or immune status of the individual or community against the pathogenic micro-organism. Though Neutralizing antibody level represent the first line protective immunity against the new infection , however due to higher cost and more steps of the presently available NT tests, it is not used commonly for regular application or mass scale screening. Previously it was reported that NT result weakly correlated with total anti-spike protein IgG against SARS-CoV-2 (17). To verify the observation, we tested 50 post vaccinated samples using “ Quantitative SARS-CoV-2 Spike Protein IgG ELISA Kit” as per manufacturer’s protocol at a serum dilution 1:100. As per recommendation of the kit, for positive sample detection, cut off was > 60 U/ml and for negative sample detection cut off was <60U/ml. In this condition only 10 samples were detected negative (20%) while rest of the 40 samples (80%) were detected positive. However Neutralization test by CENA , even at a high serum concentration (1:20) showed out of 40 anti-Spike-protein IgG positive serum samples 34 (85%) were NT positive while 6 (15%) sample showed NT negative results. However all the 6 NT negative samples had intermediate level of IgG between 60-200U/ml (200U/ml: maximum range of calibrator; >200 : out of detection range and considered high concentration). All serum samples having anti-Spike protein IgG >200U/ml showed NT positive (Table 5). As linearity does not maintained in extreme low and high range of value, corelation with CENA and Cpass kit were evaluated for the samples having IgG value 60-200U/ml. A poor correlation was observed with NT test using both CENA (r=0.14 ) and Cpass kit (0.09). However same samples showed highly correlated result between CENA and Cpass (r=0.93, p=0.00001) (Table 5). The results suggest ELISA test for total binding IgG can’t be substitute for neutralization. This might be the reason why there was high rate of post vaccinated break through SARS-CoV-2 infection, which remain unexplained because the serum samples were not evaluated by appropriate surrogate NT test. Discussion In this article we described an assay design for detecting neutralizing antibody/ blocking agent against SARS-CoV-2 overcoming the previous drawback of the cell based assay making it an robust, low cost, surrogate virus neutralization test.This will be the first cell based neutralizing assay kit for detecting neutralizing antibody against SARS-CoV-2 optimized for application in clinical setting as diagnostic tool. As in this assay native 3D confirmation of receptor (ACE2) will be preserved on native cell membrane, unlike using purified ACE2 protein on solid support of 96 well plate, as in surrogate neutralization test (cPass, Genescript), the assay is the true representative (surrogate) of conventional viral neutralization assay as well as cost will be very cheaper because no need of purified native protein. However in contrast to live virus assay this assay could be carried out in any ordinary BSL-2 level laboratory in ready to use form. Adherent HEK 293 cell line was used in this assay instead of suspension cell Expi293 as used in previous published report (11). It is very easy to handle and to remove any debris or dead cell by washing with PBS and/ changing media in contrast to previously used Expi293 cells. The cell line also could be maintained by low cost MEM media as well as easy to transfection with ordinary transfection reagent with high efficacy. So HEK 293 cell line was stably transfected with full length ACE2 expression cassette. As the ACE2 gene expression cassette transfected cells express ACE2 on cell surface and for its interaction with spike protein it needs intact ACE2, the cells were dislodged from the plate by scraping rather than trypsinization which is commonly used for dislodging adherent cells. This transfected cell line is very loosely attached and form very fragile cell clump. So it is very easy to make single cell suspension by few times gentle pipetting against the flask’s wall. Previously it was established that SARS-CoV-2 enters cells via pH- and receptor-dependent endocytosis (10). As K + in standard Dulbecco′s Phosphate Buffered Saline (DPBS) facilitate endocytosis of receptor and ligand complex (12,13) regular PBS buffer was replaced for final cell washing and resuspension. Instead cells were washed and resuspended by buffer (RB-6, table 1) containing ammonium chloride (pH7.4). and 140 mM NaCl. Additionally Ammonium chloride inhibit endocytosis by preventing acidification of endosome (14). To make the assay ready to use as per user’s convenience and to carry out the assay in any laboratory without cell culture facility and with little steps, the cells were processed in ready to use freezing format using 10% DMSO. As instead of fresh cells, frozen cell was used in the assay, there was further reduction of metabolic activity for endocytosis. This results more stable cell surface interaction of RBD-GFP with ACE2 with better signal stability. More over assaying with frozen cell system make it feasible to package the cell based assay in kit format which was not possible using fresh cell directly from culture for every assay set up. In this assay Propidium Iodide (PI) is added to the 1 x RB to make cell resuspension buffer for cell re-suspension as well as nuclear staining to identify cells in flow cytometry. Normally PI is used for staining dead cell nucleus. PI cannot penetrate the cell membrane of fresh live cell. Previously it was shown in yeasts that nuclear DNA staining of the live cells were possible by permeabilization of the cell using DMSO (16). We also used the same principle in staining human cells. As cells were frozen with DMSO 10%, it helps in permealization of the cells enabling penetration of PI within the cells for nuclear staining. So it help gating only nucleated cells in flow cytometry removing the debris or any other non cellular component or any precipitate which may arise from serum or drugs. Though some vital nuclear stain like Hoechst 33342 could be used for staining directly fresh cells without need of using DMSO into the buffer, however that will be needed to have a special flow cytometry with laser in UV range. Most of the diagnostic laboratory does not use flow cytometry machine having UV laser. PI and GFP could be measured using ordinary single lesser basic flow cytometry machine which is commonly available in ordinary clinical laboratory. As we used stably transfected ACE2 expressing cells which express ACE2 on its surface consistently with a same high percentage, it ensure consistent percent of RBD-GFP binding. So in this protocol we able to omit step of staining ACE2 with antibody unlike previous cell based RBD-ACE2 binding assay (11). This also reduce the possible positional hindrance by the antibody for binding of RBD-GFP to the ACE2 receptor and also reduce the cost of the assay. In this assay protocol, as we omit using any florescent tagged antibody for staining ACE2 and the concentration of PI and RBD-GFP was optimized such a way that it provided negligible background and we omit washing steps making the assay further robust cell based assay. As washing step is omitted, the assay could be carried out using very low input number of cells without chances of cell loss during the procedure which further contributes to reduce the subjective variability of the cell based assay. Unlike previous protocol, as per the current protocol the components could be packaged as standalone ready to use Kit without need of cell culture by the end user. The kit component could be grouped as follows: (i)Aliquots of frozen cells (ii)Frozen standardized media containing secreted RBD-GFP, (iii)10X RB, (iv)PI solution, (v), Positive control (PC) which have known titre value , (vi)Negative control (NC) which have known titre value. Though previously it was reported that binding antibody moderately correlate with NT (17), however we observed very low correlation of NT measured by CENA as well as commercial kit cPass with binding anti-Spike IgG measured by ELISA test. This might be due to elimination of extreme readings in our study. In very high concentration of antibody by any single point serological test, result shows outside the linear range of detection. However when we included the extreme values also in the analysis, our results also was very close to previous observations (17). When we used cPass as standard test for detecting positive samples, 37 out of 38 positive samples were diagnosed correct by CENA while 1 sample was proved to be false negative. Similarly when 20 samples of pre-COVID-19 pandemic were validated with CENA and cPass, while all samples showed negative result by CENA, 18 samples showed negative result by cPass. Two true negative samples were detected false positive by cPass in our hand. The increased tendency of false positive result by cPass could be explained by at least partly due to nonspecific antibody binding to some denatured purified proteins used in the assay or variation in washing steps. To avoid such subjective variables, in our assay protocol, requirement of washing steps were omitted and the negligible background did not affect the assay results. Additionally interaction of RBD with membrane bound true native ACE2 also improve the specificity of the interaction. All improvements were indicated by 100% specificity of CENA using true negative samples. The proposed kit could be manufactured with lowest manufacturing cost due to the following reasons: (i) As the recombinant genetic materials containing expression cassette of ACE2 genes and RBD-GFP (SARS-CoV-2 spike protein receptor binding domain tagged with sfGFP) are already chromosomally integrated within the cell lines, so no need of transfection for every batch of the assay kit preparation using expensive tranfection reagents. For scaling up the production only simple propagation of the stably transfected cell lines using ordinary cell culture media will be sufficient. This will reduce the manufacturing cost of the assay as well as reduce biological variation. (ii) As like manufacturing protein based surrogate neutralization assay kits (cPass), which need series of procedures like purification of recombinant native proteins (spike protein RBD and ACE2), the protein conjugation with detection systems, special reagents for detection of signal are not required in this assay. (iii) In this assay, signal of GFP as indicator of RBD binding to the ACE2 could be directly detected by flow cytometry without any additional reagent. So the manufacturing cost for the reagents includes only cell culture media, buffer, cell culture flasks etc which are very inexpensive. The assay have flexibility to calibrate with SARS-CoV-2 conventional virus neutralization test (cVNT) by diluting RBD-GFP to an optimum concentration using a standard serum. Qualitative report could be given as % neutralization at a fixed cut off dilution. Semi-quantitative reports could be provided as limit of serum dilution which gives result as per the reporting format for conventional virus neutralization test (cVNT). This make easier to interpret the result directly comparing with the conventional standard method (cVNT). Though in this article the assay was read by flow cytometry following transferring the reaction from 96 well to flow cytometry tube, for high through put screening of blocking agent or serum samples, the assay could be optimized for directly reading in 96 well plate using flow cytometry machine having appropriate autoloader for 96 well plate. Additionally, it would be more appropriate if the autoloader have inbuild temperature control (4 o C) system for keeping the samples at low temperature during measurement. In short unlike existing conventional neutralization assay or ELISA, performing CENA at end user site is many advantages which includes : (i) could be performed in 2 steps before final reading, (ii) could be carried out in ordinary BSL-2 facility without cell culture facility at end user’s site, (iii) no need of tedious multiple washing steps like ELISA, or any other assay resulting less chance of subjective variation of background signal and increase throughput of the assay. (iv)Could be conducted with little technical expertise, (v) As the assay reading is based on the detection of cell surface GFP and no requirement of additional reagents for detection system, per test cost will be very low. Most important thing is it will have a lowest manufacturing cost, short and simple protocol qualifying its mass scale application during the pandemic. It is the first cell based kit design which could be packaged as ready to use format without need of cell culture facility at the end user’s site in the diagnostic clinical laboratory. In a post COVID-19 pandemic period still SARS-CoV-2 is propagated as low level infection across the globe. Any new favorable mutation and/or lowering the herd immunity may cause new epidemic. Considering the fact that routine ELISA does not represent the neutralizing / blocking antibody titer in the serum, CENA technique could be implemented as routine diagnostic test for the several purposes as like (but not limited to) (i)Sero surveillance, decision for booster dosing at a verge of new epidemic, Vaccine efficacy of different brands and individual vaccine response, screening convalescents serum having protective antibody for plasma therapy to severe covid-19 patients, High through put drug screening for discovering novel inhibitor against RBD spike protein of SARS-CoV-2. Same low cost surrogate neutralization assay technique could be designed for other viral diseases. Declarations Conflict of interest Dr. Jagannath Pal and Dr. Yogita Rajput had filed for Indian patent and PCT application detailed as follows : PCT Application No. PCT/IN2024/051585, Filed on: 30 August 2024, Priority No. 202321058370 dated 31-08-2023 Acknowledgements This work was supported by the Department of Health Research (DHR), Ministry of Health and Family Welfare (MOHFW), Government of India and ICMR grant # CTU/PM-ABHIM/17/10/33/2023-ECD-II. We thanks Translational Health Science and Technology Institute, Faridabad, India for supporting SARS-CoV-2 live virus neutralization tests; DM was supported by ICMR grant # CTU/PM-ABHIM/17/10/42/2023-ECD-II. Author contributions JP conceived and designed the study, data analysis, data interpretation, drafing manuscript; YR executed experiments, data analysis, manuscript editing, AK, DM, NS assisted experiments, clinical data collection; N Sherwani, AN facilitated COVID-19 diagnostic tests, infrastructure support; S Mani, SK, SM performed live virus neutralization assay; VC, MB logistic support, arrangement and management of volunteers for sampling; MS critical views, manuscript editing; All authors manuscript revising and editing. Funding Department of Health Research (DHR), Ministry of Health and Family Welfare (MOHFW), Government of India, ICMR grant # CTU/PM-ABHIM/17/10/33/2023-ECD-II. References COVID-19 epidemiological update – 9 October 2024. Edition 172 https://www.who.int/publications/m/item/covid-19-epidemiological-update- edition-172 C. Huang, Y. Wang, X. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, X. Gu, Z. Cheng, T. Yu, J. Xia, Y. Wei, W. Wu, X. Xie, W. Yin, H. Li, M. 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Appl Microbiol Biotechnol 102, 4183–4191 (2018). https://doi.org/10.1007/s00253-018-8906-8 Matusali G, Colavita F, Lapa D, Meschi S, Bordi L, Piselli P, Gagliardini R, Corpolongo A, Nicastri E, Antinori A, Ippolito G, Capobianchi MR, Castilletti C, Inmi Covid-Laboratory Team. SARS-CoV-2 Serum Neutralization Assay: A Traditional Tool for a Brand-New Virus. Viruses. 2021 Apr 10;13(4):655. Tables Table 1. Screening of different buffer for highest RBD-GFP binding and stability of signal Reaction Buffer Composition Remarks RB1 Dulbecco's Phosphate Buffered Saline, pH7.4 (DPBS) Contain K + : Facilitate endocytosis RB2 Phosphate Buffered Saline (PBS) pH 7.4, 140 mM NaCl No K + (14) RB3 Phosphate Buffered Saline (PBS) pH 8, 140 mM NaCl No K + , High pH inhibit endocytosis making a alkaline environment (12, 14). RB4 10 mM HEPES buffer pH 7.4, 140 mM NaCl Alternate buffer without K + , neutral pH. (14) RB5 10 mM HEPES buffer pH 8, 140 mM NaCl Alternate buffer without K + , alkaline pH (12, 14). RB6** 25 mM Ammonium Chloride pH7.4, 140 mM NaCl Ammonium buffer inhibit endocytosis by increasing pH of endosome (12) RB7 25 mM Ammonium Chloride Ph8, 140 mM NaCl Ammonium buffer inhibit endocytosis by increasing pH of endosome ** RB6 showed Highest RBD-GFP binding efficiency and stability to ACE2 expressing 293T cells among the tested buffers (Figure 2 A & B ) Table 2. Intraday CV at different range of neutralization test performed by CENA. Range of NT Average %NT STDV CV High (>60%) 67% 2% 3% Moderate (60-40%) 46% 5% 10% Low (40-20%) 25% 5% 24% Table 3A. Cut-off condition for comparing CENA and cPass SARS-CoV-2 Neutralizing Antibody Detection Kit Name of the assay Sample dilution cut off Positive Negative Remark Cell based SARS-CoV-2 Neutralizing Antibody detection assay (CENA) 1:20 Cut off >20% NT* Cut off 30% NT** Cut off <30% NT** sVENT30 *Determination of Cut-off 20% NT for CENA : Neutralization test (NT) by CENA using pre Ccovid-19 serum samples (20 samples, collected before March 2020) was performed. %NT (Mean +3SD=18%) 20% was used as cut off for CENA; ** Cut-off 30% NT** for cPass kit was provided as per manufacturer’s protocol. Table 3B. Clinical agreement with CENA and cPass SARS-CoV-2 Neutralizing Antibody Detection Kit for categorizing serum of post SARS-CoV-2 vaccinated serum into NT positive or negative. Result of cPass SARS-CoV-2 Neutralizing Antibody Detection Kit Positive (38) Negative (15) Total Result of Cell based SARS-CoV-2 Neutralizing Antibody detection assay (CENA) Positive 37 3 40 Negative 1 12 13 Total 38 15 53 % of total agreement 92.4% Cohen’s kappa (k)* 0.806 (interpretation: Almost perfect agreement**) Neutralization measured at a dilution 1:20. *0.81 – 1.00 almost perfect or perfect agreement Table 4. Determining sensitivity and specificity of CENA using the standard (FDA approved) commercially available SARS-CoV-2 Neutralizing Antibody Detection Kit and Live virus neutralization assay as two Gold standard assays for detecting positive samples and pre COVID-19 (collected before March 2020) serum as negative samples. Positive*: post SARS-COV-2 vaccinated serum Negative: pre COVID-19 (collected before March 2020) serum (N) Commercial Kit* ( N) Live virus assay** (N) Cell based SARS-CoV-2 Neutralizing Antibody detection assay(CENA) Positive 37 20 0 Negative 1 0 20 Total 38 20 20 Test Performance Sensitivity: 97% (95%CI: 86.2%-99.9%) Sensitivity: 100% (95%CI: 83.2%-100.0%) Specificity: 100% (95%CI: 83.2%-100.0%) *Positive samples: Neutralization test (NT)Determined by FDA approved validated cPass SARS-CoV-2 NT kit ** Positive samples: Determined by CPE based Live Virus neutralization assay (micro neutralization) (cVNA100) Table 5 Corelation of ELISA (total anti spike protein IgG) with neutralization antibody assay CENA ELISA (total anti spike protein IgG) IgG Negative (60U/ml) % (n) 60-200 >200 All positive CENA20 Positive (>20%) 40% (4) 70%(14) 100%(20) 85% (34) Negative (<20%) 60% (6) 30% (6) 0% (0) 15% (6) Total 100% (10) 100% (20) 100(20) 100%(40) Corelation CENA (NT) vs ELISA (total IgG) R=0.14 CPass (NT) vs ELISA (total IgG) R=0.09 CENA (NT) vs cPass (NT) R=0.93 (0.00001) Additional Declarations Competing interest reported. Dr. Jagannath Pal and Dr. Yogita Rajput had filed for Indian patent and PCT application detailed as follows : PCT Application No. PCT/IN2024/051585, Filed on: 30 August 2024, Priority No. 202321058370 dated 31-08-2023 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Yogita","middleName":"","lastName":"Rajput","suffix":""},{"id":440972006,"identity":"b88fec1b-7b47-4399-8c36-ec9fe410fe40","order_by":1,"name":"Shailendra Mani","email":"","orcid":"","institution":"Translational Health Science and Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Shailendra","middleName":"","lastName":"Mani","suffix":""},{"id":440972008,"identity":"214193f3-e783-4f2b-85f9-fde63aae6771","order_by":2,"name":"Aarti Kushwaha","email":"","orcid":"","institution":"Multidisciplinary Research Units (MRU) Pt. J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Aarti","middleName":"","lastName":"Kushwaha","suffix":""},{"id":440972009,"identity":"1a647fd1-4a77-411b-ab50-cfb1cbd43ab0","order_by":3,"name":"Satendra Kumar","email":"","orcid":"","institution":"Translational Health Science and Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Satendra","middleName":"","lastName":"Kumar","suffix":""},{"id":440972010,"identity":"a59ad8f2-4668-4190-83a2-d505bba6e8e5","order_by":4,"name":"Sushma Mithina","email":"","orcid":"","institution":"Translational Health Science and Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Sushma","middleName":"","lastName":"Mithina","suffix":""},{"id":440972011,"identity":"231de3dc-dce9-4d63-9d98-edeb27edbb13","order_by":5,"name":"Neetu Sahu","email":"","orcid":"","institution":"Multidisciplinary Research Units (MRU) Pt. J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Neetu","middleName":"","lastName":"Sahu","suffix":""},{"id":440972012,"identity":"65e0b42a-1bb6-4ea9-8e1b-9858ea163679","order_by":6,"name":"Diksha Mahilang","email":"","orcid":"","institution":"Multidisciplinary Research Units (MRU) Pt. J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Diksha","middleName":"","lastName":"Mahilang","suffix":""},{"id":440972013,"identity":"66836ac1-9b47-4379-ab63-65bdcc1d1eb3","order_by":7,"name":"Fulsay Paikra","email":"","orcid":"","institution":"Multidisciplinary Research Units (MRU) Pt. J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Fulsay","middleName":"","lastName":"Paikra","suffix":""},{"id":440972014,"identity":"17da295d-a394-41b9-a7d3-86996dc44544","order_by":8,"name":"Nikita Sherwani","email":"","orcid":"","institution":"Pt.J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Nikita","middleName":"","lastName":"Sherwani","suffix":""},{"id":440972015,"identity":"b997c1ac-1bf9-4b93-92f6-8c2fcc2d782f","order_by":9,"name":"Arvind Neral","email":"","orcid":"","institution":"Pt.J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Arvind","middleName":"","lastName":"Neral","suffix":""},{"id":440972016,"identity":"500a3d68-4f15-44be-9ea8-7c73cec8cb98","order_by":10,"name":"Tripti Nagaria","email":"","orcid":"","institution":"Pt.J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Tripti","middleName":"","lastName":"Nagaria","suffix":""},{"id":440972017,"identity":"06d67930-e6d7-4269-a5cd-36555330dbc7","order_by":11,"name":"Manjula Kerketta Beck","email":"","orcid":"","institution":"Pt. J.N.M. Medical College","correspondingAuthor":false,"prefix":"","firstName":"Manjula","middleName":"Kerketta","lastName":"Beck","suffix":""},{"id":440972018,"identity":"3b6fa6d9-2af0-43f6-bdca-edad0e71d049","order_by":12,"name":"Vivek Choudhary","email":"","orcid":"","institution":"Pt.J.N.M. Medical College Raipur","correspondingAuthor":false,"prefix":"","firstName":"Vivek","middleName":"","lastName":"Choudhary","suffix":""},{"id":440972019,"identity":"fd3dc166-99b0-4084-9bc5-e80c375a060c","order_by":13,"name":"Masood A. Shammas","email":"","orcid":"","institution":"Harvard (Dana Farber) Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Masood","middleName":"A.","lastName":"Shammas","suffix":""},{"id":440972020,"identity":"9b5f5629-0c18-49ac-89cc-cc8a26a6b77c","order_by":14,"name":"Jagannath Pal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACxgYowwDIPvCgAshiZm7ArR5JiwRQC8OBhDMgLXBj8AOIlsQ2FJuxA+b24xc/F+6wqzOXbn5wIHFebTR/O1DLj4ptuB3Wk1MsPfNMsoTlnGMGBxK3Hc+dcZixgbHnzG08fslJkOZtY5YwuJEA0nIstwGohZmxDY+W/jfJv3nb6oFa0j8cSJxzLHc+QS0z0o8BbTkM1JIDtKWhJncDYS1v2Kx5245LbrhzpuBAwrEDuRuBWg7i84thf/rj27xt1fwGt9s3PvhQU5c77/zhgw9+VODR0sBjAGFJgMnDYPIATvVAIM/A/gBZSx0+xaNgFIyCUTBCAQCy+WTVvuKNlgAAAABJRU5ErkJggg==","orcid":"","institution":"Multidisciplinary Research Units (MRU) Pt. J.N.M. Medical College Raipur","correspondingAuthor":true,"prefix":"","firstName":"Jagannath","middleName":"","lastName":"Pal","suffix":""}],"badges":[],"createdAt":"2025-04-10 06:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6417069/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6417069/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80361913,"identity":"62d19896-d34d-46cd-a61e-642f1abff83c","added_by":"auto","created_at":"2025-04-11 04:12:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":659649,"visible":true,"origin":"","legend":"\u003cp\u003ePanel A: Principle of CENA assay is shown by fluorescent Microscopy (A I , A II) and flowcytometry (A III, A IV). Component -2 (HEK 293 Cells stably transfected with human ACE2 receptor gene expression cassette in plasmid) was incubated with component -1 (RBD-S-GFP) on ice for\u0026nbsp; 2 hours with human serum samples\u0026nbsp; (control\u0026nbsp; serum: collected before the COVID-19 pandemic, and test serum: Serum collected from subject already vaccinated with two dose of Covishield ) as indicated. Following incubation reaction was diluted by reaction buffer and visualized by either microscopy (AI ,AII) or analysed % of GFP positive cells by flowcytometry(AII, AIV). AI, AIII: incubated control serum; AII, AIV: incubated with test serum;\u003c/p\u003e\n\u003cp\u003ePanel B. screening of different buffer system for maximum RBD-GFP binding to ACE2 expressing HEK293 cells (component 2) and binding stability. CENA was performed using the indicated reaction buffers(RB) and analysed by flowcytometry for % of RBD-GFP +ve cells. Following first reading after dilution of the reaction with the corresponding RB at 2 hour, 2\u003csup\u003end \u003c/sup\u003ereading was taken after 20 minutes on ice following the first reading.\u0026nbsp; BI: bar diagram representing % of RBD-GFP +ev cells at 2 hr using respecting RB.\u0026nbsp; B II: stability of the binding\u0026nbsp; with time (20 minutes) represented by the bar diagram as % of RBD-GFP +ev cells in reference to first reading indicated as time ‘0’; .\u003c/p\u003e\n\u003cp\u003eC: bar diagram showing effect of DMSO and cell storage conditions on Propidium Iodide (PI) stanning of ACE2 cells (CI) and RBD-GFP binding (CII).\u0026nbsp;\u003cbr\u003e\nD: Standardization of optimum RBD-GFP concentration. D1-VI: RBD-GFP containing media was serially diluted as indicated.\u0026nbsp; At each dilution, CENA was performed with serial two fold dilution of post vaccinated serum sample starting from 1:10 dilution. % neutralization was determined by (i) % GFP +ev cell count and (ii) % total florescence (% of\u0026nbsp; GFP +ev cells *mean fluorescence of the cells). 4PL graph was plotted\u0026nbsp; keeping\u0026nbsp; % neutralization in Y axis\u0026nbsp; against LOG(serum dilution) in X axis.\u003c/p\u003e\n\u003cp\u003eE:\u0026nbsp; RBD-GFP containing media was serially diluted to indicated Florescence by RB and CENA was performed with and without PC serum sample at each dilution of the RBD-GFP. Primary Y axis: NT results were plotted at indicated fluorescence (serial dilution of RBD-GFP); Secondary Y axis: % of RBD-GFP +ev cells (component-2) were plotted to increasing florescence of\u0026nbsp; RBD-GFP (X axis).\u003c/p\u003e\n\u003cp\u003eF. Time course to determine maximum binding of RBD-GFP to the ACE2 expressing cell(component-2). RBD-GFP containing media at dilution of 50FU* incubated with Component-2 on ice without any serum sample. Aliquot of samples were taken at indicated time points, diluted with RB and % of RBD-GFP +ev cells was measured by flowcytometry.\u0026nbsp;\u0026nbsp;\u0026nbsp; %RBD-GFP +ev cells are expressed as maximum % RBD-GFP +ev cells at 90 minute.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFU: Concentration of RBD-GFP is expressed as arbitrary fluorescent unit as measured by SPERK TECAN (488/520) in 96 well black, flat bottom\u0026nbsp; plate at 100ul volume and reference sample was supernatant media (equal dilution) from non transfected cells.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/10c5a3b3803c645bc0c6c6fc.png"},{"id":80361914,"identity":"65d00405-5f51-4b4b-aad2-a6d3712e063f","added_by":"auto","created_at":"2025-04-11 04:12:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":884498,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic work flow of the kit manufacturing at manufacturer’s site (A) and performing the assay at end user site(B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/7cc7428a3b26e243fba83772.png"},{"id":80361915,"identity":"6be8d1c6-c477-4142-b5b7-1f7f3d4c7d95","added_by":"auto","created_at":"2025-04-11 04:12:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185038,"visible":true,"origin":"","legend":"\u003cp\u003eDetail method of aanalysis of Neutralization of RBD of spike protein of SARS-Cov2 by the CENA using flow cytometry\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/ade9dbb50ddea0f335e3dbb2.png"},{"id":80361917,"identity":"08b7c349-78bb-4ad1-a3e9-b1c855644465","added_by":"auto","created_at":"2025-04-11 04:12:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87803,"visible":true,"origin":"","legend":"\u003cp\u003eComparative performance of CENA with different standard neutralization assay and ELISA based total binding antibody assay. \u003cstrong\u003eA:\u003c/strong\u003e Linear correlation of CENA with live virus NT assay. Total 11 post vaccinated serum samples serially two fold diluted starting from 1: 10 and serum titer is expressed as limit of serum dilution which were determined as per the cut off convention for the respective assay.The NT titer was plotted as log10(titre) along X and Y axis respectively. CENA50: The limit of serum dilution which produce at least 50% neutralization by CENA; cVNA100: The limit of serum dilution which produce 100% neutralization by the virus neutralization assay\u003cstrong\u003e; B, C, D:\u003c/strong\u003eLinear correlation analysis of commercial kits with CENA using 20 post vaccinated serum samples which showed intermediate IgG level (60-200U/ml) by total binding anti spike protein IgG ELISA kit. \u003cstrong\u003eB:\u003c/strong\u003e %NT by CENA vs cPass SARS-CoV-2 Neutralizing antibody Detection Kit (Genescript) at a serum dilution 1:20 and plotted along X and Y axis respectively; C: %NT by CENA at 1:20 dilution vs anti-Spike protein IgG ELISA at a dilution of 1:100 and plotted along X and Y axis respectively\u003cstrong\u003e; D:\u003c/strong\u003e %NT by cPass SARS-CoV-2 Neutralizing antibody Detection Kit (Genescript) at a serum dilution 1:20 vs and anti-Spike protein IgG ELISA at a dilution of 1:100 and plotted along X and Y axis; \u003cstrong\u003eE:\u003c/strong\u003e The dot-plot showing comparative performance of CENA and cPass(Genescript) kit to detect neutralizing antibody. Total 53 post SARS-CoV-2 vaccinated sample (PVAC) and 20 pre COVID-19 samples (collected before march 2020) (pre-CoV) were simultaneously tested by CENA and cPass kit for detecting SARS-CoV-2 neutralization test at a serum dilution 1:20. Cut-off for cPass NT : \u0026gt;30% positive \u0026amp; \u0026lt;30% negative , Cut-off for CENA NT: \u0026gt;20% positive \u0026amp; \u0026lt;20% negative,\u003c/p\u003e\n\u003cp\u003eN: NT Negative ; P: NT Positive; CENA-PVAC: Distribution of %NT performed by CENA in PVAC samples; CENA-pre-CoV: Distribution of %NT performed by CENA in pre-CoV samples; cPass-PVAC: Distribution of %NT performed by cPass in PVAC samples; cPass -pre-CoV: Distribution of %NT performed by cPass in pre-CoV samples; doted lines indicate respective cut off values for the tests as indicated in the figure. Brown circles : the samples detected positive by the respective test.; Blue circle : The samples detected negative by the respective test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/322802bc4769b619baeb5cc9.png"},{"id":80361918,"identity":"3eb65e5b-883c-4e59-bb31-1fc40b53a805","added_by":"auto","created_at":"2025-04-11 04:12:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95834,"visible":true,"origin":"","legend":"\u003cp\u003eNeutralizing antibody response to vaccination by two brand of SARS-Cov-2 vaccines. A: Sputnik V and B: Covishield. Pre and post vaccinated serum samples were serially diluted starting from 1:10 and CENA assay was performed. Serum dilution was plotted as log scale in X axis and % of NT in Y axis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/187b03c4c73cc50b84c844e5.png"},{"id":83212793,"identity":"d65e9237-2494-49c1-a44d-cf6147c723c4","added_by":"auto","created_at":"2025-05-21 08:39:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3434642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6417069/v1/fd478494-8a81-4fb4-916a-9cbca29a8cfe.pdf"}],"financialInterests":"Competing interest reported. Dr. Jagannath Pal and Dr. Yogita Rajput had filed for Indian patent and PCT application detailed as follows : PCT Application No. PCT/IN2024/051585, Filed on: 30 August 2024, Priority No. 202321058370 dated 31-08-2023","formattedTitle":"A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe pandemic corona virus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2).\u0026nbsp;Though pandemic have been controlled following vaccination, COVID-19 is still prevalent across the globe at a variable positivity rate 0.7 to 16% \u0026nbsp;(WHO report Oct, 2024, 1). As per the WHO report, there is increased number of reported cases and deaths during the survey period of 28-day from 19August to 15 september comaired to earlier survey report, with over 270 000 new cases and 5700 new deaths (1). So there is always risk for future epidemic which justify the need for surveillance of protective immunity.\u0026nbsp;\u003cbr\u003e\u003cbr\u003eSARS-CoV-2 is a new corona virus identified in late 2019 and belongs to a family of enveloped RNA viruses that include MERS and SARS, both of which caused serious human infections of respiratory system (2). As a first step of the viral multiplication cycle, the virus attaches to the host cell surface before entering into the host cell. The Corona virus envelope Spike protein (S protein) has been reported as a significant determinant of virus entry into host cells. The S protein receptor binding domain (RBD) recognizes and attaches to the host cell surface Angiotensin-Converting Enzyme 2 (ACE2) receptor found on the surface of type I and II pneumocytes, endothelial cells, and ciliated bronchial epithelial cells as well as oral and nasal epithelium. Following binding to the cellular receptor critical proteolytic cleavage event occurred at SARS-CoV-2 S protein, leading to membrane fusion and viral entry. After the virus enters the cells, the viral RNA genome is released into the cytoplasm and is translated into viral polyproteins and viral replication cycle is started (3).\u003c/p\u003e\n\u003cp\u003eCurrently no proven therapy is available for COVID-19. Though several vaccines are already available and permitted for emergency use, however its efficacy and protective immune response in different group of individuals are under investigation. Protective immune response against SARS-CoV-2 involves generation of anti spike protein antibody however only a subset antibodies against specific epitope at receptor binding domain (RBD) play the key role to neutralize the virus for binding to its receptor ACE2 and block infection to the host cells (4). Currently most of the vaccine developments against SARS-CoV-2 infection are to block the interaction of spike protein and ACE2. Immune response to natural COVID-19 infection or vaccination may not be occurred in equal efficacy in different individuals leading to different outcome of vaccination. So detection of neutralizing antibody in serum is very important to evaluate protective immunity status following natural infection or post COVID-19 vaccination. This will not only help for individualize patient management but also help in identification of acceptable donors for convalescent plasma therapy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe virus neutralization assay detects presence of the antibody which actually blocks the virus from infecting to the host cell. This antibody usually blocks the receptor binding domain of the virus protein at the interface which interacts with host cell receptor for entering into the cell. As a gold standard, neutralization assay is carried out using live virus by standard plaque reduction neutralization test (PRNT) or micro neutralization test in 96 well format. However, to carry out the test required special BSL-3 laboratory, expensive safety measure and also laborious, take at least 4-5 days for completion of the assay. While the gold standard assay could be used as reference or validation of any new assay, however it cannot be available for mass scale diagnostic and research purposes (5). Alternatively few surrogate neutralization assay have been developed recently using in vitro interaction of recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein and its receptor ACE2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCurrently two class of commercially available surrogate neutralizing antibody assay kits are available for SARS CoV-2. (i) Pseudo virus based Neutralization assay kits: Cell based surrogate neutralization assays have been designed using replication-defective pseudotyped viral particles (6,7) which can be performed under BSL-2 conditions. In this assay pseudotyped virus is developed which express RBD spike protein of SARS-CoV-2 on its surface. Reporter gene (luciferase or florescent proteins) is expressed in the host cells following binding of pseudotyped virus containing SARS-CoV-2 spike protein to its cell surface ACE2 receptor and transduce genetic material into the host cells. If the pseudo typed virus stock has been prepared in advance, it will take at least 2 days to get result for the candidate samples. So it also involves multistep procedure, long time to complete the assay and high manufacturing cost limiting its clinical use. (ii) Protein based kits in ELISA format: One protein based surrogate neutralization assay for SARS-CoV-2 (8,9) is commercially available which detect blocking of soluble COVID-19 spike RBD to its corresponding receptor ACE2 bound at the plate. It detects blocking of soluble SARS-CoV-2 spike RBD to its corresponding receptor ACE2 bound at the plate or vice versa. The test could be carried out in 2-3 hrs in ordinary laboratory (10). As ACE2 protein component is membrane protein may not be fully active in cell free purified form so the invitro interaction on solid surface may not be representative of cell surface natural interaction in natural protein conformation and environment. Moreover the cost of purification of the proteins and using separate reagents for detection system are always increase manufacturing cost. So the kit is very expensive, limiting its wide spread clinical and research use. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother cell based binding assay for showing in vitro interaction of RBD of SARS-CoV-2 Spike protein with its receptor ACE2 have been reported for research purposes by transient transfection of RBD-sfGFP and myc tagged ACE2 (11) expression cassette plasmids into a suspension cell line Expi293. In this assay GFP tagged RBD of SARS-CoV-2 Spike protein is secreted into the supernatant media which is separated from culture of expi293 cells which were transiently transfected with the plasmid. Expi293 cell expressing Myc tagged ACE2 on cell surface were produced by transient transfection of pCEP4-myc-ACE2 plasmid containing the expression cassette of ACE2 taged with Myc (for its in vitro detection by anti Myc antibody). In this binding assay format The supernatant media containing the RBD-GFP was added into cell suspension expressing ACE2 cells on its surface. For staining ACE2, anti-Myc antibody was used in the reaction. DAPI was used for staining dead cells. Following incubation on ice, cells were washed with PBS and flow cytometry was carried out. DAPI positive cells were excluded and Myc positive cells were gated as representative marker of ACE2 expressing cells and the RBD-GFP binding was expressed as % of ACE2 positive cells. However this assay had several limitations to be used directly as surrogate neutralization assay in a ready to use format in clinical laboratory as well as at an affordable cost. (i) In this assay suspension cells were used to make compatible with downstream flow cytometry procedure. This could bypass trypsinization steps which may affect cell surface receptor integrity. \u0026nbsp;However Transfection to the suspension cells is very difficult. To ensure high transient transfection rate in the suspension cells, specially engineered Expi293 cell were used which required special media and transfection reagent. This special media and reagent is very expensive resulting increase cost of the assay. \u0026nbsp;(ii) As it was transient transfection based assay, for every assay new transfection will leads to increase the cost of the assay, less reproducibility and inconvenient to be packaged as kit for diagnostic test. In suspension cells, the dead cells following transfection or due to culture propagation, cannot be easily removed. So the dead cell products will be released in the media which could degrade the RBD-GFP reducing its activity or affecting RBD-ACE2 complex to a variable degree. Dead cell degradation product of ACE2 expressing expi293 cells may block RBD-GFP reducing its effective concentration for binding to intact ACE2 expressing cells. To exclude dead cells additional stain (DAPI) also used for which additional UV channel is required in flow cytometry machine and most of the ordinary clinical laboratory this UV channel is not present. (iii) It is not feasible to maintain cell line in ordinary clinical laboratory. For every new batch of ACE2 tranfected cells, concentration of cells have to be determined and based on transfection efficiency all the assay parameter have to be readjusted before starting a assay. The neutralization titre is very much depends on ratio of number of ACE2 positive cells and concentration of RBD-GFP in the reaction mixture which cannot be maintained in transiently transfected cell lines. \u0026nbsp;So those variables will contribute to gross variation and reproducibility from batch to batch and assay to assay. (iv) As cell surface binding of viral protein causes endocytosis the reaction was needed to conduct in low temperature which blocks endocytosis. However, even in low temperature also 100% blockage is not possible. So slowly the receptor protein complex is endocytosed in presence of K\u003csup\u003e+\u003c/sup\u003e containing PBS buffer system and the signal is faded with time (12-15). So there is chance of unstable reading of binding RBD-GFP to ACE2 when more samples will be assayed. All together the reported form of the cell based RBD binding assay protocol (11) is not fit for in-vitro diagnostic assay and laboratory to laboratory comparability of the data. \u0026nbsp;Importantly the protocol described was not ready to use \u0026ldquo;stand alone\u0026rdquo; to be used as kit form in the clinical laboratory. (v) Additionally though most of the cell based surrogate assay mimic close to gold standard assay than an ELISA based assay, however due to biological variables most of the cell based assay have high CV making it unsuitable for diagnostic assay.\u003c/p\u003e\n\u003cp\u003eTaken together, the issues discussed involving the previous protocols are unfit to be an affordable and ideal diagnostic assay kit to be carried out in ordinary clinical laboratory by less trained health staffs. Considering the all previous limitation, in this article we describe a method of cell based assay design for detecting neutralizing antibody against SARS-CoV-2 in serum/plasma using flow cytometry. Unlike other multistep cell based assay using pseudotyped virus, The method could be carried out in two step in 3 hours and without need of any washing steps as like other serological assay for eliminating background. The assay could be packaged stand alone \u0026nbsp;kit format and the end users in clinical laboratory will not be needed any cell culture prior to the assay or expertise in cell culture handling and will have lowest manufacturing or establishment cost.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy was carried out at Multi-Disciplinary Research Unit (MRU), Pt. Jawaharlal Nehru Memorial Medical College, Raipur Chhattisgarh, India and Dr. B.R.A.M. Hospital Raipur, C.G., (under Pt. Jawaharlal Nehru Memorial Medical College, Raipur) following approval of the protocol by institutional ethical committee. All procedures were carried out in accordance with relevant guidelines and regulations. Human samples were collected following informed consent from the study subjects\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell line and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293 cell line was procured from NCCS, Pune, India. HEK 293 cell was grown in MEM media (HiMedia, India) with 10 % fetal bovine serum (USA origin, HiMedia India) and 1 X Anti-Anti (HiMedia, India) incubated at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of the assay components:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of stable SARS-CoV-2-S-RBD-GFP-HEK293 cell line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293 cell was stably transfected with a plasmid pcDNA3-SARS-CoV-2-S-RBD-sfGFP, previously described (3), containing expression cassette of SARS-CoV-2 RBD tagged with sfGFP with signal sequence for its extracellular release and having mammalian selection marker. The plasmid is distributed through addgene (https://www.addgene.org/141184/sequences/). For stable transfection the RBD-GFP expression plasmid is first transfected by lipofectamin 3000 then after 48 hrs media is replaced by fresh media containing selection antibiotic Genticin 400µg/ml (G418). After two weeks of incubation in selection media, single green flurescent colonies were identified, separated and re plated into 96 well plate. Following regrowth the well was screened for most populated green florescence cells under florescence microscope. Further screening was done by checking supernatant of the wells having green florescence cells. Florescence was measured using an appropriate filter for GFP (detailed below). The colony which gave the best green florescence in the supernatant was propagated and designated as SARS-Cov-2-S-RBD-GFP-HEK293 cell line and amplified for preparing optimized RBD-GFP containing media (\u003cstrong\u003ecomponent-1\u003c/strong\u003e) secreted from the stable cell line. The cell line was grown upto 70 % confluence. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandardization of RBD-GFP containing media (component-1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing 70% confluency of the cells, fresh MEM media was added (2ml for 25cm\u003csup\u003e2\u003c/sup\u003e flask and 6 ml media for `75cm\u003csup\u003e2 \u003c/sup\u003eflask). After 4 days of incubation media was collected. Media was centrifuged and supernatant was collected and fluorescence is adjusted to 60-25 arbitrary fluorescent U (FU) diluting with 1 x RB \u003cstrong\u003e(component 3)\u003c/strong\u003e. At this range of florescence level, assay will be more linear and will have wider linear range of detection limit. \u003c/p\u003e\n\u003cp\u003eThe media was further calibrated with a positive control (PC) reference serum (cocktail of post vaccinated/ convalescent serum), neutralizing antibody titre of which have been predetermined by live virus neutralization assay. Dilution of the media was adjusted so that titre of the PC by the new test closely matched with the titre of gold standard assay while the % of RBD-GFP positive cells of untreated control is kept above 80% . The diluted media was dispensed in 2ml tubes and kept in -20\u003csup\u003eo\u003c/sup\u003eC. For calibration of new batch of RBD-GFP containing media, activity of RBD-GFP is calibrated by diluting the media, adjusting % of GFP positive cells and mean florescence of single cell of the previous standardized batch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlorescence measurement protocol by Multi mode plate reader (Tecan): \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeasurement of Florescence of RBD-GFP containing media: 100 ul of media directly or diluted by 1x RB was dispensed into the well of 96 well black opaque plate (Corning, Thermo fisher). Florescence was measured by multi-mode plate reader, SPARK, TECAN at a wave length 485 nm excitation and 535 nm emission and band width 10nm (gain manual 70). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of ACE2-HEK293 cell line and its packaging protocol: \u003c/strong\u003eAn adherent cell line HEK 293, which don’t express ACE2 was stably transfected with the plasmid pCEP4-myc-ACE2a, containing ACE2 expression cassette and mammalian selection marker (3) and distributed through addgene. For stable transfection the ACE2 expression plasmid is first transfected by lipofectamin 3000 (Thermoscientific) then after 48 hrs media is replaced by fresh media containing selection antibiotic Hygromyocin 150\u003cimg width=\"9\" height=\"21\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAA0AAAAgCAMAAAD+BwKmAAAAAXNSR0IArs4c6QAAAEJQTFRFAAAAAAAAAAA6AABmADpmADqQAGa2OgAAOmY6Zrb/kDoAkGY6kLbbkNv/tmYAtmZmtpA6tv//2//b2////7Zm///bZBY3nwAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAAFiUAABYlAUlSJPAAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAUElEQVQoU2NgGCpAlIuJF+hWEUY2kIvpwWPkBFrEB7OPkVVYVICDkVWIH2w7OyMLjyg3IwvQTaJcYCdBgSgXqzAyj1kQmQf2A0Ilkhx58QAAC9AEcERPMNoAAAAASUVORK5CYII=\" alt=\"image\"\u003eg/ml. After two weeks single colonis were separated and replated into 96 well plate. Following re-growth functional screening was carried out by observing RBD-GFP binding activity under fluorescent microscope. Colony with optimal binding of RBD-GFP is propagated for the assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReaction buffer:\u003c/strong\u003e All the cell washing and resuspension and downstream experiments was carried out in reaction buffer (RB). The RB was consist of NaCl close to isotonic concentration along with an inhibitor of endocytosis and a buffer system not containing phosphate. e.g. 140 mM NaCl and 25mM NH\u003csub\u003e4\u003c/sub\u003eCl pH 7.4. RB will stabilizes RBD-ACE2 complex on cell surface preventing endocytosis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell packaging \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed with reaction buffer (RB) and dislodged from the cell culture flask by scrapping by cell scraper and single cell suspension was prepared by pipetting in RB buffer and cleared by passing through cell strainer. Cells were centrifuged and supernatant was removed and resuspended in 1 x RB. Cells were counted, centrifuged and supernatant removed. \u003c/p\u003e\n\u003cp\u003eCells were resuspended in RB with 10% DMSO at a concentration 50 Lakh cell /ml and dispensed 20µl per 2 ml tube sufficient for 10 reaction and kept at -80\u003csup\u003eo\u003c/sup\u003eC till it is used. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAssay components \u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eComponent-1: RBD-GFP containing media \u003c/li\u003e\n\u003cli\u003eComponent-2: ACE2-HEK293 cell line\u003c/li\u003e\n\u003cli\u003eComponent-3: 10X Reaction Buffer (1.40 M NaCl, 0.25M NH\u003csub\u003e4\u003c/sub\u003eCl, pH7.4)\u003c/li\u003e\n\u003cli\u003eComponent-4: 1000X Propidium Iodide (0.5mg/ml) (Sigma)\u003c/li\u003e\n\u003cli\u003eComponent-5: Positive control (PC) \u003c/li\u003e\n\u003cli\u003eComponent-6: Negative control (NC) reference serum: Cocktail of negative serum which have been predetermined by gold standard assay (PRNT). \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAssay steps \u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eBlood was collected without any anti coagulant and serum was separated and aligoté was stored at -20C if was not used within 7 days. \u003c/li\u003e\n\u003cli\u003e10X RB (component 3) to be diluted to 1 X RB by sterile distilled water.\u003c/li\u003e\n\u003cli\u003eCell re-suspension buffer (CRB) was made by adding 1ul/ml 1000X PI (component 4) into the 1X RB (component 3)\u003c/li\u003e\n\u003cli\u003eSerum was serially diluted with the 1X RB \u003c/li\u003e\n\u003cli\u003eµl frozen ready to used standardized media containing RBD-GFP was taken into 96 well plate. 5 µl serum samples or its dilution was added into the respective well. \u003c/li\u003e\n\u003cli\u003eFollowing incubation for 1 hour at room temperature, plate was kept on ice for additional 15 minutes.\u003c/li\u003e\n\u003cli\u003eFrozen cells were (component 2) thawed on ice and 75 times diluted with pre cooled cell dilution buffer (CDB) containing 0.5µg/ml PI (e.g for 20 µl frozen cells in 1.5 ml CDB). 150µl ice cold cell suspension was added into each well. In this way 10k cell was distributed in each reaction. \u003c/li\u003e\n\u003cli\u003eThe reaction was mixed properly and kept for 60-90 min into ice bath. \u003c/li\u003e\n\u003cli\u003eFollowing incubation reaction mixture was pipetted to remove any cell clump and the whole reaction mixture is added into ice cold 500µl RB kept into flow cytometry tubes. Tubes were kept into mini cooer (-20\u003csup\u003eo\u003c/sup\u003eC) till flow cytometry measurement was performed.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eFlowcytometry analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlowcytometry analysis was performed in Sysmax flow cytometer, Germany, using inbuilt Cyflow\u003csup\u003eTM \u003c/sup\u003esoftware. GFP was detected by FL1 and PI was detected by FL3 Chanel. First single intact cell population is gated form SSC-FSC plot. Then PI stained cells were gated from SSC-PI plot. In a GFP-PI quadrant plot threshold is adjusted by running cells stained with PI without addition of RBD-GFP and it is set at 0 to 2% as threshold for FL1 Chanel or for GFP. % of RBD-GFP binding to the HEK293 ACE2 cells is indicated by % of GFP positive cells. Binding of RBD-GFP to the cells without incubation with any serum samples or inhibitor is considered as 100% binding. Any reduction of binding was detected as reduced % GFP positive cells, following incubation of the RBD-GFP (component 1) with any serum samples or inhibitor was the measure of neutralization. Method of getting and analysis by flow cytometry is demonstrated in Fig 3. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMNT (Microneutralization Test): \u003c/strong\u003eVero-E6 cells were seeded into a 96-well plate at a density of 15,000 cells per well and incubated overnight at 37°C in a humidified atmosphere with 5% CO₂ to establish a monolayer. Serum samples were heat-inactivated at 56°C for 30 minutes prior to use. To assess the neutralizing capacity of serum antibodies, 50 µL of SARS-CoV-2 Wuhan strain (containing the 100 TCID₅₀ virus/50 µL) was mixed with an equal volume of heat-inactivated serum samples serially diluted (1:20 to 1:2560) and incubated at 37°C for 1 hour. After incubation, the serum-virus mixtures were added to the wells containing the pre-seeded Vero-E6 cells and incubated for 1 hour at 37°C with gentle agitation every 15 minutes to facilitate viral adsorption. Following this step, the inoculum was aspirated, and the cells were washed with serum-free medium. Subsequently, 150 µL of maintenance medium (DMEM supplemented with 2% FBS, 2× antibiotics, and non-essential amino acids) was added to each well, and the plates were incubated at 37°C with 5% CO₂ for 72 hours. The cells were then observed under a microscope for the presence of cytopathic effects (CPE), and the data were recorded. Neutralizing antibody titers were determined based on the serum dilution that inhibited CPE. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurrogate (virus free) SARS-Cov-2 neutralization antibody test (sVNT)\u003c/strong\u003e: The test was performed by the well validated commercial test kit “cPass s SARS-CoV-2 Neutralization Antibody Detection kit”(GenScript, USA) as per the manufacturer’s protocol (https://www.fda.gov/media/143583/download) at a serum dilution 1:20 and each samples were measured in duplicate and average was determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-Spike protein SARS-Cov-2 IgG ELISA test: \u003c/strong\u003eThe test was performed using the commercial kit “EDI™ Quantitative SARS-CoV-2 Spike Protein IgG ELISA Kit“, Epitope Diagnostic Ink(EPI),CA, USA as per the manufacturer’s protocol at a serum dilution 1:100 and each samples were measured in duplicate and average was determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e% Neutralization of CENA (by only cell count) was calculated by the formulas:\u003c/p\u003e\n\u003cp\u003e% NT = [1- (X-b)/(C-b)]*100 …. Equation 1\u003c/p\u003e\n\u003cp\u003e% NT(total florescence) = [1- {(X-b)/(C-b)}*{F\u003csub\u003eX\u003c/sub\u003e/F\u003csub\u003eC\u003c/sub\u003e}]*100 …Equation 2\u003c/p\u003e\n\u003cp\u003eNT: % Neutralization, \u003c/p\u003e\n\u003cp\u003eX: % GFP positive cells in test serum/ treated sample\u003c/p\u003e\n\u003cp\u003eC: % GFP positive cells in untreated control sample (without serum) \u003c/p\u003e\n\u003cp\u003eb: background % GFP positive cell in unstained samples (without RBD-GFP)\u003c/p\u003e\n\u003cp\u003eFx : average florescence/cell in test serum/ treated sample\u003c/p\u003e\n\u003cp\u003eF\u003csub\u003eC \u003c/sub\u003e : average florescence/cell in untreated control sample (without serum)\u003c/p\u003e\n\u003cp\u003eF\u003csub\u003eX\u003c/sub\u003e/F\u003csub\u003eC \u003c/sub\u003e : Represents correction factor due to difference in intensity of fluorescence of RBD-GFP bound cells following neutralization of RBD-GFP by the neutralizing antibody. Though commonly in any flow cytometry based assay analysis is carried out based on % of positive cells in particular channel, in very high concentration of RBD-GFP, Equation 2 represent more accurate and linear curve. Upon serial dilution of RBD-GFP, the neutralization curve by equation-1 and Equation-2 become very closed after a certain point of dilution. Below that point simpler Equation-1 could be used for all purposes (Figure 1D I-VI).\u003c/p\u003e\n\u003cp\u003eMean, standard deviation, median and Pearson correlation and regular graphs were performed using Microsoft excel. significance of Pearson’s correlation, and sensitivity specificity test were analysed using the following web-based software: MedCalc (https://www.medcalc.org/calc/odds_ratio.php.html). Statistical significance was considered if p ≤ 0.05. For making dot plot web based software Interactive Dotplot was used. For making 4PL graph of neutralization reading at different dilution of serum, GraphPad Prism soft wear was used. \u003cstrong\u003e*Cohen’s kappa (k)\u003c/strong\u003e was determined using a web based software ‘Cohen’s kappa free calculator” (https://idostatistics.com/cohen-kappa-free-calculator/#risultati) as described by Landis, JR \u0026amp; Koch, GG (1977) for the measurement of observer agreement for categorical data.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e** \u003cstrong\u003eInterpretation of Cohen’s kappa (k) :\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e0.01 – 0.20 slight agreement\u003c/p\u003e\n\u003cp\u003e0.21 – 0.40 fair agreement\u003c/p\u003e\n\u003cp\u003e0.41 – 0.60 moderate agreement\u003c/p\u003e\n\u003cp\u003e0.61 – 0.80 substantial agreement\u003c/p\u003e\n\u003cp\u003e0.81 – 1.00 \u003cstrong\u003ealmost perfect or perfect agreement\u003c/strong\u003e\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003ePrinciple of the assay is based on binding of \u0026nbsp;GFP tagged receptor binding domain of SARS-COV-2 Spike protein (RBD-GFP) which is secreted into the media from a stably transfected \u0026nbsp; HEK 293 cells , to cell surface ACE2 ( stably transfected into another 293 cell and expressed constitutively) and the fluorescence of the bound cells quantitated by flow cytometry as % of green fluorescence(GFP) +ve cells. If the media is pre-incubated with any chemical blocking agents or blocking antibody against Spike protein RBD of SARS-CoV-2, residual free RBD-GFP will bind to the cells and the reduction of % \u0026nbsp;of GFP +ve cells to the untreated control is proportional to concentration of inhibitor or blocking/neutralizing antibody. The principle of the assay is shown in Figure 1 A (I-IV). Cells incubated with control sample shows green ring outside the cell membrane indicating binding of RBD-GFP on cell surface (Figure 1 AI). Upon incubation with serum from anti-SARS-CoV-2 vaccinated (Covishield) subject, the green hallows disappeared (Figure 1 AII) indicating blockage of interacting interface of RBD of SARS-CoV-2 Spike protein. Same observation replicated when the corresponding samples were analysed by flow cytometry (Figure 1 AIII \u0026amp; AIV). Though the principle of the assay was very simple however, as like other cell based assay several known and unknown contributing factor was there making the assay result inconsistent and inconvenient to be a clinical grade diagnostic test. In the present cell-based assay design effect of those factors / limitations were minimized to produce a reproducible robust assay by several strategies. which include (i) stable transfection of the expression cassette, \u0026nbsp;(ii) designing optimum buffer system for stable binding of RBD-GFP to the cell surface ACE2, minimizing receptor-ligand mediated endocytosis of the viral protein, (iii) optimum dilution of media for a consistent linear results, \u0026nbsp;(iv) simple method for non-antibody counter staining of cell, (v) optimizing method for packaging the assay in a kit format so that end user does not required any cell culture system, (vi) methods of calibration,(vii) reducing variation of \u0026nbsp;cell based assay from batch to batch.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmmonium Chloride buffer at pH7.4 shows highest RBD-GFP binding to ACE2 expressing 293T cells and signal stability, among the tested \u0026nbsp;buffers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been shown that binding of SARS-CoV-2 virus to the cell surface ACE2 receptor leads to endocytosis and internalization of the complex leading to the disruption of the virus protein (13,15). This mechanism may cause disruption of GFP causing decaying of GFP signal. We also observed decaying of RBD-GFP signal upon binding in room temperature (data not shown). To slow the process, we had applied multiple measures such as optimum buffer system which stabilize the complex on cell surface by inhibiting the endocytosis process along with conducting the assay at low temperature as well as cell freezing.\u003c/p\u003e\n\u003cp\u003eFor screening optimum reaction buffer for stable and high RBD-GFP binding to HEK293 cells expressing ACE2, seven different reaction buffers (RB1,RB2, ...RB7) were tested. The detail composition of the buffer and the rational is described in table 1. Following first reading after dilution of the reaction with the corresponding RB at 2 hour, 2\u003csup\u003end\u0026nbsp;\u003c/sup\u003ereading was taken after 20 minutes on ice following the first reading. \u0026nbsp;RB-6 showed highest level RBD-GFP binding to the cells (Figure 1 BI) and highest stability of the binding (Figure 1 BII) at 20 minutes following the first reading when compare the same to buffer RB-1, RB2, RB3(10% vs 28%, 17% reduction of signal). So for the subsequent experiment we used RB-6 and designated as RB in the subsequent text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of DMSO and cell freezing on live cell Propidium Iodide staining and RBD-GFP binding \u0026nbsp;on ACE2 expressing HEK 293 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor identifying only nucleated cells in flow cytometry removing the debris or any other non-cellular particulate components or any precipitate which may arise from serum, a nuclear stain was used which could be detected by commonly available channels. Though PI is very common and inexpensive nuclear staining dye and detected by commonly available Chanel, only could penetrate into dead cells. As DMSO \u0026nbsp;and cell freezing causes increase cell permeabilization we evaluated if both may increase nuclear stanning of PI. \u0026nbsp;PI was incubated with ACE2-293 cells processed in three condition (i) kept at 4\u0026ordm;C without DMSO (ii) kept at 4\u0026ordm;C with 10% DMSO, (iii) kept at -80\u0026ordm;C with 10% DMSO as shown in figure 1 CI. Expectedly cells frozen at -80\u0026ordm;C with DMSO showed highest (16%) PI staining whereas keeping at 4\u0026ordm;C without DMSO showed least PI staining (8.6%).\u003c/p\u003e\n\u003cp\u003eAs cell freezing with DMSO also will reduce metabolic activity which may further increase stability of RBD-GFP binding to ACE2 on cell surface, % of RBD-GFP binding was verified and figure 1 CII showed expectedly highest RBD-GFP binding when cells were freezed at -80\u0026ordm;C with 10% DMSO compared to keeping at 4\u0026ordm;C with or without DMSO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of RBD-GFP containing media by appropriate dilution critical for stable assay result \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor standardization of the assay, we prepared a cocktail of 10 positive serum samples (PC) \u0026nbsp;collected from post vaccinated subjects and a cocktail of negative serum samples (NC) from unvaccinated, uninfected subjects \u0026nbsp;and confirmed by live virus ant-SARS-CoV-2 neutralizing antibody assay. The PC and NC were subsequently used as assay standard.\u003c/p\u003e\n\u003cp\u003eBinding of RBD-GFP to ACE2 expressing cells depend on ratio of number of free RBD-GFP in the reaction and 293 cell number expressing ACE2. \u0026nbsp;Media having relatively high concentration of RBD-GFP in a given concentration of cells will cause increase intensity of average florescence of cells due to more number of RBD-GFP binding on each cell. At high concentration of RBD-GFP, as there will be excess number of RBD-GFP than total number of cell surface ACE2 in the reaction, high number of antibody will be required to block the excess RBD-GFP before it affect the % RBD-GFP bound +ve cells. So calculating neutralization by using only the single parameter % of RBD-GFP +ve cell will not be linear and will give false negative results. Average per cell fluorescence also should be considered in calculation as 2\u003csup\u003end\u003c/sup\u003e parameter. So calculating reduction of % of total Florescence (% RBD-GFP +ve cells * Florescence/cells, Equation-2, material methods) is more better representation of the extent of neutralization. So in higher concentration of RBD-GFP, neutralization curve by % of RBD-GFP +ve cell count (Equation-1 in material methods) vs % of total florescence of RBD-GFP positive cells (Equation-2, material methods) will be different. We hypothesized that if RBD-GFP is serially diluted and neutralization curves are plotted at each dilution point, a point will reach, following which neutralization curves using equation 1 and equation 2 will be very close/ equivalent and give a linear curve. Figure 1D(I-VI) shows, at a florescence unit 50 (dilution 1:8 of original media,) or lower concentration, NT curves by % of RBD-GFP positive cells and % of total florescence were very similar and merged to each other. However, if the media is too much diluted linear range of detecting Neutralizing antibody will be very small and the reading will be very unstable because only small proportion of cells will be bound by RBD-GFP. To demonstrate that, the range of RBD-GFP concentration starting from 200 FU upto 6.25 FU were used to perform CENA. Value of NT was plotted in primary Y axis while % of RBD-GFP positive cells in untreated control in each of the concentrations were plotted in secondary Y axis against concentration of RBD-GFP in X axis (figure 1 E). It is interesting to observe the inverse relation of % of RBD-GFP binding and reading of NT. Media at a dilution 1:8 at FU 50 shows optimum RBD-GFP binding in control (88%) cells. More diluting the RBD-GFP caused lower % of RBD-GFP binding which causes lower resolution to detect a small difference. So, for subsequent batch of RBD-GFP containing media was calibrated accordingly using the previous batch RBD-GFP and PC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of time course for optimum binding of RBD-GFP to ACE2-293 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were incubated with RBD-GFP containing media at 50 FU on ice and flow cytometry was performed at different time interval till 90 minutes as shown in figure 1F. A Plateau of \u0026nbsp; % of \u0026nbsp;RBD-GFP positive cells were \u0026nbsp;reached within 60 minutes. However for maximum binding for subsequent tests incubation was for 90 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of coefficient of variance \u0026nbsp;\u003c/strong\u003e\u003cbr\u003eFor determination of CV at different level of neutralization of high (\u0026gt;60%) moderate (60-40%) and low (40-20%), serum samples were diluted appropriately and assay was performed with a 5 replica of the samples. CV for the three range of neutralization by CENA was 3%, 10% and 25% respectively shown in Table 2. While recommended CV for a cell based immune assay is 20-25%, our results showed within the range in all level of neutralization while high and moderate level neutralization had much lower CV than the recommended.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative correlation of CENA and commercially available cPass SARS-CoV-2 Neutralizing Antibody Detection Kit.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples were used as follows: (i) Serum samples of subjects who already completed two doses of COVID-19 vaccine (53 samples) (ii) Serum collected before (before March 2020) COVID-19 pandemic (40 samples), (iv) Cocktail of serums from 10 COVID-19 vaccinated individual (PC), (iv) Cocktail of serums from individuals having no reported RT PCR positive COVID-19 infection and no vaccination (NC).\u003c/p\u003e\n\u003cp\u003ecPass SARS-CoV-2 Neutralizing Antibody Detection Kit, (Genescript) is the only FDA (Emergency Use Authorization) approved well validated and internationally widely used kit for detecting SARS-CoV-2 neutralization antibody. The test was performed as per manufacturer\u0026rsquo;s protocol in duplicate at a serum dilution of 1:20 (as per recommendation). The reading was taken in SPARK multimode plate reader (TECAN). % of Neutralization was calculated as per the formula provided by the manufacturer. The cell based Neutralization assay CENA was carried out at 1:20 dilution by flow cytometry. % NT was determined as described above using a cut off 20% (CENA20). CENA20 was determined by measuring %NT of 20 pre-COVID-19 pandemic (collected before march 2020) serum samples. Mean+3SD of NT was determined to be 18% (-6%+24%). So for practical purpose 20% was used as cut off making it towards more stringent. Another 20 pre-COVID-19 samples were used for validation. For the NT assay using commercial cPass kit cut off was 30% as per manufacturer\u0026rsquo;s protocol. Results showed out of 53 post vaccinated serum samples tested 49 had agreement with both CENA and cPass kit (92.4%) and Cohen\u0026rsquo;s kappa: 0.806 suggesting almost perfect or perfect agreement (Table 3 A and B). Those 4 samples which were not with agreement with CENA had NT reading were closer to border line of cut off values. Correlation analysis was performed and Figure 4 B showed CENA have good linear correlation with the cPass SARS-CoV-2 kit (\u003cem\u003eR\u003c/em\u003e 0.94, p\u0026lt;0.00001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorelation \u003c/strong\u003e\u003cstrong\u003eof CENA and conventional live virus-based SARS-COV-2 viral neutralization assay (cVNA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 11 post vaccinated serum samples of different range of neutralizing antibody measured by live virus neutralization assay were used to determine corelation of antibody titre with CENA. The serum samples were serially two fold diluted and each was tested for NT by CENA and also by live virus NT (micro-neutralization in 96 well plate) assay (cVNT) as described previously. Serum titre was determined by cVNT or CENA as the highest limit of serum dilution which produce 100% neutralization (cVNT100) or 50% neutralization (CENA50) respectively. \u0026nbsp;Correlation analysis of serum titre measured by both the test showed CENA50 have a good correlation with the cVNA100 (\u003cem\u003eR\u003c/em\u003e 0.9, p\u0026lt;0.0016) (figure 4 A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Sensitivity and specificity of \u0026nbsp;CENA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor determining sensitivity of CENA, the two standard assays (i) FDA approved commercially available SARS-CoV-2 Neutralizing Antibody Detection Kit (cPass, Genescript) and (ii) SARS-CoV-2 live virus neutralization assay (cVNA) were used for detecting positive samples and pre COVID-19 (collected before March 2020) serum as true negative samples. Sensitivity of CENA using cPass kit as standard assay was 97% (37/38). 1 sample (NMR180) which were detected false negative by CENA when we compared cPass as standard test, actually proved true negative when we compared with live virus assay. However all 20 positive samples, detected positive by Live virus neutralization assay was also detected positive by CENA resulting 100% sensitivity (20/20). As no test have absolute specificity, we used pre-COVID-19 samples collected before March 2020 as true negative samples for determining specificity. We observed out of 20 samples tested by CENA all showed negative resulting 100% specificity (20/20) (Table 4).\u003c/p\u003e\n\u003cp\u003eDot plot in figure 4 E shows comparative distribution of %NT by CENA and the commercial kit (cPass) using post vaccinated serum samples and pre- COVID-19 pandemic (before March 2020) negative serum samples. While both the method showed similar distribution of %NT, however 2 of the pre-COVID-19 pandemic samples (true negative samples) were detected as positive (false positive) by Cpass commercial kit while all the pre-COVID-19 samples were detected negative by CENA suggesting CENA have better specificity. Together our assay performance shows much superior than the commercial kit\u0026nbsp;Cpass.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExample of using the test CENA to compare effectiveness of multiple brand of vaccines to induce neutralizing antibody against SARS-CoV-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor comparing vaccine response to different brands of COVID-19 vaccines, serum samples of two healthy individuals before and \u0026nbsp; 1 month after COVID-19 vaccination were collected for the assay. one subject was vaccinated with Sputnic V and another subject was vaccinated with Covishield. Serum samples were serially two fold \u0026nbsp;diluted starting from 1:10 and neutralization assay was carried by CENA till the NT \u0026nbsp;reached to baseline. IC50 was determined from 4PL graph. \u0026nbsp;The result is shown in Figure 5. \u0026nbsp;Comparing the two experimental subjects, the person who received Sputnic V has been shown to be less immune response following single dose than the subject receiving Covishield ( IC50: 78 \u0026nbsp;vs \u0026nbsp; 134). Surveying neutralizing antibody titre response of different brand of COVID-19 \u0026nbsp;vaccine within a population could be carried out for implementing larger vaccine policy for the population. Alternately the same example of the assay could be used for personal protection. If a particular brand of vaccine failed to create protective response another brand of vaccine could be tried if national protocol may permit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConventional ELISA test to detect total binding \u0026nbsp;antibody against SARS-CoV-2 Spike protein can not be substitute for Neutralization test for sero surveillance. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCommonly ELISA based test is used to detect total binding IgG or IgM against proteins of an organism to evaluate sero-conversion or immune status of the individual or community against the pathogenic micro-organism. Though Neutralizing antibody level represent the first line protective immunity against \u0026nbsp;the new infection , however due to higher cost and more steps of the presently available NT tests, \u0026nbsp;it is not used commonly for regular application or mass scale screening. Previously it was reported that NT result weakly correlated with \u0026nbsp;total anti-spike protein IgG against SARS-CoV-2 (17). To verify the observation, we tested 50 post vaccinated samples using \u0026ldquo; Quantitative SARS-CoV-2 Spike Protein IgG ELISA Kit\u0026rdquo; as per manufacturer\u0026rsquo;s protocol at a serum dilution 1:100. As per recommendation of the kit, \u0026nbsp;for positive sample detection, cut off was \u0026nbsp;\u0026gt; 60 U/ml \u0026nbsp;and for negative sample detection cut off was \u0026lt;60U/ml. In this condition only 10 samples were detected negative (20%) \u0026nbsp;while rest of the 40 samples (80%) were detected positive. However Neutralization test by CENA , even at a high serum concentration (1:20) showed out of 40 anti-Spike-protein IgG positive serum samples 34 (85%) were NT positive while 6 (15%) sample showed NT negative results. However all the 6 NT negative samples had intermediate level of IgG between 60-200U/ml (200U/ml: maximum range of calibrator; \u0026gt;200 : out of detection range and considered high concentration). All serum samples having anti-Spike protein IgG \u0026gt;200U/ml showed NT positive (Table 5). As linearity does not maintained in extreme low and high range of value, corelation with CENA and Cpass kit were \u0026nbsp;evaluated for the samples having IgG value 60-200U/ml. A poor correlation was observed with NT test using both CENA (r=0.14 \u0026nbsp;) and Cpass kit (0.09). However same samples showed highly correlated result between CENA and Cpass (r=0.93, p=0.00001) (Table 5). \u0026nbsp; The results suggest ELISA test for total binding IgG can\u0026rsquo;t be substitute for neutralization. This might be the reason why there was high rate of post vaccinated break through SARS-CoV-2 infection, which remain unexplained because the serum samples were not evaluated by appropriate surrogate NT test.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eIn this article we described an assay design for detecting neutralizing antibody/ blocking agent against SARS-CoV-2 overcoming the previous drawback of the cell based assay making it an robust, low cost, surrogate virus neutralization test.This will be the first cell based neutralizing assay kit for detecting neutralizing antibody against SARS-CoV-2 optimized for application in clinical setting as diagnostic tool.\u003c/p\u003e\n\u003cp\u003eAs in this assay native 3D confirmation of receptor (ACE2) will be preserved on native cell membrane, unlike using purified ACE2 protein on solid support of 96 well plate, as in surrogate neutralization test (cPass, Genescript), the assay is the true representative (surrogate) of conventional viral neutralization assay as well as cost will be very cheaper because no need of purified native protein. However in contrast to live virus assay this assay could be carried out in any ordinary BSL-2 level laboratory in ready to use form.\u003c/p\u003e\n\u003cp\u003eAdherent HEK 293 cell line was used in this assay instead of suspension cell Expi293 as used in previous published report (11). It is very easy to handle and to remove any debris or dead cell by washing with PBS and/ changing media in contrast to previously used Expi293 cells. The cell line also could be maintained by low cost MEM media as well as easy to transfection with ordinary transfection reagent with high efficacy. So HEK 293 cell line was stably transfected with full length ACE2 expression cassette. As the ACE2 gene expression cassette transfected cells express ACE2 on cell surface and for its interaction with spike protein it needs intact ACE2, the cells were dislodged from the plate by scraping rather than trypsinization which is commonly used for dislodging adherent cells. This transfected cell line is very loosely attached and form very fragile cell clump. So it is very easy to make single cell suspension by few times gentle pipetting against the flask’s wall. \u003c/p\u003e\n\u003cp\u003ePreviously it was established that SARS-CoV-2 enters cells via pH- and receptor-dependent endocytosis (10). As K\u003csup\u003e+ \u003c/sup\u003ein standard Dulbecco′s Phosphate Buffered Saline (DPBS) facilitate endocytosis of receptor and ligand complex (12,13) regular PBS buffer was replaced for final cell washing and resuspension. Instead cells were washed and resuspended by buffer (RB-6, table 1) containing ammonium chloride (pH7.4). and 140 mM NaCl. Additionally Ammonium chloride inhibit endocytosis by preventing acidification of endosome (14). \u003c/p\u003e\n\u003cp\u003eTo make the assay ready to use as per user’s convenience and to carry out the assay in any laboratory without cell culture facility and with little steps, the cells were processed in ready to use freezing format using 10% DMSO. As instead of fresh cells, frozen cell was used in the assay, there was further reduction of metabolic activity for endocytosis. This results more stable cell surface interaction of RBD-GFP with ACE2 with better signal stability. More over assaying with frozen cell system make it feasible to package the cell based assay in kit format which was not possible using fresh cell directly from culture for every assay set up. \u003c/p\u003e\n\u003cp\u003eIn this assay Propidium Iodide (PI) is added to the 1 x RB to make cell resuspension buffer for cell re-suspension as well as nuclear staining to identify cells in flow cytometry. Normally PI is used for staining dead cell nucleus. PI cannot penetrate the cell membrane of fresh live cell. Previously it was shown in yeasts that nuclear DNA staining of the live cells were possible by permeabilization of the cell using DMSO (16). We also used the same principle in staining human cells. As cells were frozen with DMSO 10%, it helps in permealization of the cells enabling penetration of PI within the cells for nuclear staining. So it help gating only nucleated cells in flow cytometry removing the debris or any other non cellular component or any precipitate which may arise from serum or drugs. Though some vital nuclear stain like Hoechst 33342 could be used for staining directly fresh cells without need of using DMSO into the buffer, however that will be needed to have a special flow cytometry with laser in UV range. Most of the diagnostic laboratory does not use flow cytometry machine having UV laser. PI and GFP could be measured using ordinary single lesser basic flow cytometry machine which is commonly available in ordinary clinical laboratory. \u003c/p\u003e\n\u003cp\u003eAs we used stably transfected ACE2 expressing cells which express ACE2 on its surface consistently with a same high percentage, it ensure consistent percent of RBD-GFP binding. So in this protocol we able to omit step of staining ACE2 with antibody unlike previous cell based RBD-ACE2 binding assay (11). This also reduce the possible positional hindrance by the antibody for binding of RBD-GFP to the ACE2 receptor and also reduce the cost of the assay. In this assay protocol, as we omit using any florescent tagged antibody for staining ACE2 and the concentration of PI and RBD-GFP was optimized such a way that it provided negligible background and we omit washing steps making the assay further robust cell based assay. As washing step is omitted, the assay could be carried out using very low input number of cells without chances of cell loss during the procedure which further contributes to reduce the subjective variability of the cell based assay. \u003c/p\u003e\n\u003cp\u003eUnlike previous protocol, as per the current protocol the components could be packaged as standalone ready to use Kit without need of cell culture by the end user. The kit component could be grouped as follows: (i)Aliquots of frozen cells (ii)Frozen standardized media containing secreted RBD-GFP, (iii)10X RB, (iv)PI solution, (v), Positive control (PC) which have known titre value , (vi)Negative control (NC) which have known titre value.\u003c/p\u003e\n\u003cp\u003eThough previously it was reported that binding antibody moderately correlate with NT (17), however we observed very low correlation of NT measured by CENA as well as commercial kit cPass with binding anti-Spike IgG measured by ELISA test. This might be due to elimination of extreme readings in our study. In very high concentration of antibody by any single point serological test, result shows outside the linear range of detection. However when we included the extreme values also in the analysis, our results also was very close to previous observations (17).\u003c/p\u003e\n\u003cp\u003eWhen we used cPass as standard test for detecting positive samples, 37 out of 38 positive samples were diagnosed correct by CENA while 1 sample was proved to be false negative. Similarly when 20 samples of pre-COVID-19 pandemic were validated with CENA and cPass, while all samples showed negative result by CENA, 18 samples showed negative result by cPass. Two true negative samples were detected false positive by cPass in our hand. The increased tendency of false positive result by cPass could be explained by at least partly due to nonspecific antibody binding to some denatured purified proteins used in the assay or variation in washing steps. To avoid such subjective variables, in our assay protocol, requirement of washing steps were omitted and the negligible background did not affect the assay results. Additionally interaction of RBD with membrane bound true native ACE2 also improve the specificity of the interaction. All improvements were indicated by 100% specificity of CENA using true negative samples.\u003c/p\u003e\n\u003cp\u003eThe proposed kit could be manufactured with lowest manufacturing cost due to the following reasons:\u003cstrong\u003e (i) \u003c/strong\u003eAs the recombinant genetic materials containing expression cassette of ACE2 genes and RBD-GFP (SARS-CoV-2 spike protein receptor binding domain tagged with sfGFP) are already chromosomally integrated within the cell lines, so no need of transfection for every batch of the assay kit preparation using expensive tranfection reagents. For scaling up the production only simple propagation of the stably transfected cell lines using ordinary cell culture media will be sufficient. This will reduce the manufacturing cost of the assay as well as reduce biological variation. (ii) As like manufacturing protein based surrogate neutralization assay kits (cPass), which need series of procedures like purification of recombinant native proteins (spike protein RBD and ACE2), the protein conjugation with detection systems, special reagents for detection of signal are not required in this assay. (iii) In this assay, signal of GFP as indicator of RBD binding to the ACE2 could be directly detected by flow cytometry without any additional reagent. So the manufacturing cost for the reagents includes only cell culture media, buffer, cell culture flasks etc which are very inexpensive. \u003c/p\u003e\n\u003cp\u003eThe assay have flexibility to calibrate with SARS-CoV-2 conventional virus neutralization test (cVNT) by diluting RBD-GFP to an optimum concentration using a standard serum. Qualitative report could be given as % neutralization at a fixed cut off dilution. Semi-quantitative reports could be provided as limit of serum dilution which gives result as per the reporting format for conventional virus neutralization test (cVNT). This make easier to interpret the result directly comparing with the conventional standard method (cVNT). Though in this article the assay was read by flow cytometry following transferring the reaction from 96 well to flow cytometry tube, for high through put screening of blocking agent or serum samples, the assay could be optimized for directly reading in 96 well plate using flow cytometry machine having appropriate autoloader for 96 well plate. Additionally, it would be more appropriate if the autoloader have inbuild temperature control (4\u003csup\u003eo\u003c/sup\u003eC) system for keeping the samples at low temperature during measurement. \u003c/p\u003e\n\u003cp\u003eIn short unlike existing conventional neutralization assay or ELISA, performing CENA at end user site is many advantages which includes : (i) could be performed in 2 steps before final reading, (ii) could be carried out in ordinary BSL-2 facility without cell culture facility at end user’s site, (iii) no need of tedious multiple washing steps like ELISA, or any other assay resulting less chance of subjective variation of background signal and increase throughput of the assay. (iv)Could be conducted with little technical expertise, (v) As the assay reading is based on the detection of cell surface GFP and no requirement of additional reagents for detection system, per test cost will be very low. Most important thing is it will have a lowest manufacturing cost, short and simple protocol qualifying its mass scale application during the pandemic. It is the first cell based kit design which could be packaged as ready to use format without need of cell culture facility at the end user’s site in the diagnostic clinical laboratory.\u003c/p\u003e\n\u003cp\u003eIn a post COVID-19 pandemic period still SARS-CoV-2 is propagated as low level infection across the globe. Any new favorable mutation and/or lowering the herd immunity may cause new epidemic. Considering the fact that routine ELISA does not represent the neutralizing / blocking antibody titer in the serum, CENA technique could be implemented as routine diagnostic test for the several purposes as like (but not limited to) (i)Sero surveillance, decision for booster dosing at a verge of new epidemic, Vaccine efficacy of different brands and individual vaccine response, screening convalescents serum having protective antibody for plasma therapy to severe covid-19 patients, High through put drug screening for discovering novel inhibitor against RBD spike protein of SARS-CoV-2. Same low cost surrogate neutralization assay technique could be designed for other viral diseases. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Jagannath Pal and Dr. Yogita Rajput had filed for Indian patent and \u0026nbsp;PCT application detailed as follows : PCT Application No. PCT/IN2024/051585, Filed on: 30 August 2024, Priority No. 202321058370 dated 31-08-2023\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Department of Health Research (DHR), Ministry of Health and Family Welfare (MOHFW), Government of India and ICMR grant # CTU/PM-ABHIM/17/10/33/2023-ECD-II. We thanks Translational Health Science and Technology Institute, Faridabad, India for supporting SARS-CoV-2 live virus neutralization tests; DM was supported by ICMR grant # CTU/PM-ABHIM/17/10/42/2023-ECD-II.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJP conceived and designed the study, data analysis, data interpretation, drafing manuscript; YR executed experiments, data analysis, manuscript editing, AK, DM, NS assisted experiments, clinical data collection; N\u0026nbsp;Sherwani, AN facilitated COVID-19 diagnostic tests, infrastructure support; S\u0026nbsp;Mani, SK, SM performed live virus neutralization assay; VC, MB logistic support, arrangement and management of volunteers for sampling; MS critical views, manuscript editing; All authors manuscript revising and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Health Research (DHR), Ministry of Health and Family Welfare (MOHFW), Government of India, ICMR grant # CTU/PM-ABHIM/17/10/33/2023-ECD-II.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCOVID-19 epidemiological update \u0026ndash; 9 October 2024. 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Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner. PLoS Pathog. 2014 Nov 6;10(11):e1004502. doi: 10.1371/journal.ppat.1004502. Erratum in: PLoS Pathog. 2015 Feb;11(2):e1004709. PMID: 25375324; PMCID: PMC4223067. \u003c/li\u003e\n\u003cli\u003eZhang, N., Fan, Y., Li, C. \u003cem\u003eet al.\u003c/em\u003e Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts. \u003cem\u003eAppl Microbiol Biotechnol\u003c/em\u003e\u003cstrong\u003e102, \u003c/strong\u003e4183\u0026ndash;4191 (2018). https://doi.org/10.1007/s00253-018-8906-8 \u003c/li\u003e\n\u003cli\u003eMatusali G, Colavita F, Lapa D, Meschi S, Bordi L, Piselli P, Gagliardini R, Corpolongo A, Nicastri E, Antinori A, Ippolito G, Capobianchi MR, Castilletti C, Inmi Covid-Laboratory Team. SARS-CoV-2 Serum Neutralization Assay: A Traditional Tool for a Brand-New Virus. Viruses. 2021 Apr 10;13(4):655. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Screening of different buffer for highest RBD-GFP binding and stability of signal\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eReaction Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003eComposition\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eRemarks\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003eDulbecco\u0026apos;s Phosphate Buffered Saline, pH7.4 (DPBS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eContain K\u003csup\u003e+\u003c/sup\u003e :\u003csup\u003e\u0026nbsp;\u003c/sup\u003eFacilitate endocytosis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003ePhosphate Buffered Saline (PBS) pH 7.4, 140 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eNo K\u003csup\u003e+ \u0026nbsp;\u0026nbsp;\u003c/sup\u003e (14)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003ePhosphate Buffered Saline (PBS) pH 8, \u0026nbsp;140 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eNo K \u003csup\u003e+\u003c/sup\u003e\u003csub\u003e,\u0026nbsp;\u003c/sub\u003e High \u0026nbsp;pH inhibit endocytosis making a alkaline environment (12, 14).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003e10 mM HEPES buffer pH 7.4, \u0026nbsp;140 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eAlternate buffer without \u0026nbsp;K\u003csup\u003e+\u0026nbsp;\u003c/sup\u003e, neutral pH. (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003e10 mM HEPES buffer pH 8, 140 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eAlternate buffer without \u0026nbsp;K\u003csup\u003e+ \u0026nbsp;\u003c/sup\u003e, alkaline pH (12, 14).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRB6**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25 mM Ammonium Chloride pH7.4, 140 mM NaCl \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmmonium buffer inhibit endocytosis by increasing pH of endosome (12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003eRB7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 300px;\"\u003e\n \u003cp\u003e25 mM Ammonium Chloride Ph8, 140 mM NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 223px;\"\u003e\n \u003cp\u003eAmmonium buffer inhibit endocytosis by increasing pH of endosome\u003csub\u003e\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 603px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e** RB6 showed Highest RBD-GFP binding \u0026nbsp; \u0026nbsp; efficiency and stability to ACE2 expressing 293T cells among the tested \u0026nbsp;buffers (Figure 2 A \u0026amp; B )\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Intraday CV at different range of neutralization test performed by CENA.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eRange of NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eAverage %NT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eSTDV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003eCV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eHigh (\u0026gt;60%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eModerate (60-40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eLow\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(40-20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e24%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 3A. Cut-off condition for comparing CENA and cPass SARS-CoV-2 Neutralizing Antibody Detection Kit\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eName of the assay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eSample dilution cut off\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eRemark\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003eCell based SARS-CoV-2 Neutralizing Antibody detection assay (CENA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e1:20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eCut off \u0026gt;20% NT*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eCut off\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;20% NT*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eCENA20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003ecPass SARS-CoV-2 Neutralizing Antibody Detection Kit (sVNT) (Genescript)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e1:20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eCut off \u0026gt;30% \u0026nbsp;NT**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eCut off\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026lt;30% \u0026nbsp;NT**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003esVENT30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Determination of Cut-off 20% NT for CENA : Neutralization test (NT) by CENA using pre Ccovid-19 serum samples (20 samples, collected before March 2020) was performed. %NT (Mean +3SD=18%) 20% was used as cut off for CENA; ** Cut-off 30% NT** for cPass kit was provided as per manufacturer\u0026rsquo;s protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3B. Clinical agreement with CENA and cPass SARS-CoV-2 Neutralizing Antibody Detection Kit for categorizing serum of post SARS-CoV-2 vaccinated serum into NT positive or negative.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"501\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 243px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 257px;\"\u003e\n \u003cp\u003eResult of cPass SARS-CoV-2 Neutralizing Antibody Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003ePositive \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNegative\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTotal\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eResult of Cell based SARS-CoV-2 Neutralizing Antibody detection assay (CENA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eNegative\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e% of total \u0026nbsp;agreement\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 257px;\"\u003e\n \u003cp\u003e92.4%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eCohen\u0026rsquo;s kappa (k)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 257px;\"\u003e\n \u003cp\u003e0.806 \u0026nbsp;(interpretation: Almost perfect agreement**)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\n\u003cp\u003eNeutralization measured at a dilution 1:20.\u003c/p\u003e\n\u003cp\u003e*0.81 \u0026ndash; 1.00 \u003cstrong\u003ealmost perfect or perfect agreement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 4. Determining sensitivity and specificity \u0026nbsp;of CENA using the standard \u0026nbsp;(FDA approved) commercially available SARS-CoV-2 Neutralizing Antibody Detection Kit and Live virus neutralization assay as two Gold standard assays \u0026nbsp;for detecting positive samples and pre COVID-19 (collected before March 2020) serum as negative samples.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"605\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 231px;\"\u003e\n \u003cp\u003ePositive*:\u003c/p\u003e\n \u003cp\u003epost SARS-COV-2 vaccinated serum \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eNegative:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003epre \u0026nbsp;COVID-19 (collected before March 2020) serum\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(N)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eCommercial Kit* ( N)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eLive virus assay** \u0026nbsp;(N)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 117px;\"\u003e\n \u003cp\u003eCell based SARS-CoV-2 Neutralizing Antibody detection assay(CENA)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eTest Performance\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003eSensitivity: 97%\u003c/p\u003e\n \u003cp\u003e(95%CI: 86.2%-99.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eSensitivity: 100%\u003c/p\u003e\n \u003cp\u003e(95%CI: 83.2%-100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eSpecificity: 100%\u003c/p\u003e\n \u003cp\u003e(95%CI: 83.2%-100.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Positive samples: Neutralization test (NT)Determined by FDA approved validated \u0026nbsp; cPass SARS-CoV-2 NT kit\u003c/p\u003e\n\u003cp\u003e** Positive samples: Determined by CPE based Live Virus neutralization assay (micro neutralization) (cVNA100)\u003c/p\u003e\n\u003cp\u003eTable 5 Corelation of ELISA (total anti spike protein IgG) with neutralization antibody assay \u0026nbsp;CENA\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"3\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 365px;\"\u003e\n \u003cp\u003eELISA (total anti spike protein IgG)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eIgG Negative\u003c/p\u003e\n \u003cp\u003e(\u0026lt;60U/ml )\u003c/p\u003e\n \u003cp\u003e% (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 268px;\"\u003e\n \u003cp\u003eIgG Positive (\u0026gt;60U/ml)\u003c/p\u003e\n \u003cp\u003e% (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e60-200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026gt;200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eAll positive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eCENA20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003ePositive (\u0026gt;20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e40% (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e70%(14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100%(20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e85% (34)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNegative (\u0026lt;20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e60% (6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e30% (6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0% (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e15% (6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003eTotal\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e100% (10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e100% (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e100(20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e100%(40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 495px;\"\u003e\n \u003cp\u003eCorelation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003eCENA (NT) vs ELISA (total IgG)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eR=0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003eCPass (NT) vs ELISA (total IgG)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eR=0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003eCENA (NT) vs cPass (NT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eR=0.93\u003c/p\u003e\n \u003cp\u003e(0.00001)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neutralization antibody, surrogate, SARS-CoV-2, cell based, Microneutralization test","lastPublishedDoi":"10.21203/rs.3.rs-6417069/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6417069/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegular surveillance of herd immunity and individual protection by measuring neutralizing antibody against SARS-Cov-2 would be instrumental to control future COVID-19 pandemic. The SARS-COV-2 live virus neutralization tests or other surrogate virus neutralization tests could not be used for mass scale clinical application. A robust cell based surrogate SARS-Cov-2 neutralization assay (CENA) have been designed, based on blocking of GFP-tagged RBD (RBD-GFP) to its interaction with cell surface ACE2 receptor and detected by flow cytometry. A stable and high binding of RBD-GFP on cell surface was achieved using ammonium containing buffer inhibiting receptor-ligand endocytosis. Using DMSO containing frozen cells instead of conventional fresh cultured cells in the assay, enable further reduction of endocytosis as well as simple PI stanning of the nucleus for identifying cells in ordinary flow cytometry without additional staining protocol with antibody or any washing steps.. CENA were compared with (i) commercially available surrogate neutralization kit (cPass) and (ii) live virus neutralization assay showing sensitivity 97% and 100% \u0026nbsp;respectively. Specificity of CENA using pre-COVID-19 pandemic samples were 100%. CENA were highly corelated with both live virus neutralization assay (r=0.9.p=0.0016) as well as with cPass kit(r=0.9,p=0.00001 respectively). Very poor correlation with conventional binding anti-Spike protein IgG level detected by ELISA (r=0.14), suggests that the conventional ELISA tests could not be a substitute for detecting neutralizing antibody. This is first ready to use cell based neutralization assay design, to be carried out without requirement of cell culture facility at end user’s site and could be completed in two hours. Besides surveying anti-SARS-COV-2 protective antibody, the assay could also be used for low budget high throughput screening of blocking molecules.\u003c/p\u003e","manuscriptTitle":"A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 04:12:16","doi":"10.21203/rs.3.rs-6417069/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23f1546f-8a85-4c53-84c7-b3b0fe678852","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46950530,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":46950531,"name":"Biological sciences/Microbiology/Clinical microbiology"},{"id":46950532,"name":"Biological sciences/Microbiology/Virology"},{"id":46950533,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2025-05-21T08:39:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-11 04:12:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6417069","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6417069","identity":"rs-6417069","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}