A kit and an assay for analysing the presence of neutralizing antibody against SARS-cov-2

Abstract

The present disclosure relates to in vitro identification of neutralizing antibody against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Specifically, the present disclosure relates a kit and an in vitro assay for analyzing the presence of neutralizing antibody / inhibitors which could block or inactivate receptor binding domain of the spike protein of SARS-CoV-2.

Description

[0001]A KIT AND AN ASSAY FOR ANALYSING THE PRESENCE OF NEUTRALIZING ANTIBODY AGAINST SARS-CoV-2 FIELD OF THE INVENTION The present disclosure relates to in vitro identification of neutralizing antibody against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Specifically, the present disclosure relates a kit and an in vitro assay for analyzing the presence of neutralizing antibody / inhibitors which could block or inactivate receptor binding domain of the spike protein of SARS-CoV-2. BACKGROUND OF THE INVENTION The outbreak of the novel coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has posed a global health crisis. Effects to control the spread of the virus and develop effective therapeutics and vaccines have been paramount in combating the pandemic. In context of COVID-19, protective immune response against SARS-CoV-2 causative agent of COVID-19 involves generation of anti-spike protein antibody however only a subset functional antibodies against specific site at receptor binding domain (RBD) of the spike protein play the key role to neutralize the virus for binding to its receptor ACE2 and block the virus to enter into the human cells and thus prevent infection (1).Typically, the neutralizing antibodies in patients or vaccinated people monitor disease outcomes and determine vaccine efficacy and critical to evaluate herd immunity. Many approaches were developed to identify neutralizing antibodies against SARS-CoV-2, such as micro neutralization assay, SARS-CoV-2 pseudotype virus assay, enzyme-linked immunosorbent assay (ELISA), and rapid lateral flow assay. Given the many types of serological assays for quantifying the neutralizing antibody titre, the comparison of different assay results has been a challenge. Also, in 2020, the World Health Organization proposed the first international standard as a common unit to define neutralizing antibody titre and antibody responses against SARS-CoV-2. These standards were useful for comparing the results of different assays and laboratories. However, these assays have several limitations to be used directly as neutralization assay in a ready to use format in clinical laboratory as well as at an affordable cost. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring neutralization antibodies against SARS-CoV-2, is time-consuming and limited to 6 or 24-well plate format. Thus, it is not suitable for emergency responses, as well as large-scale serology studies and has safety issues, as it requires exposure to live viruses in the biosafety level 3 laboratory. ELISA based serological assay is protein based surrogate neutralization assay for SARS-CoV-2 (2,3) is commercially available, it is based on the ELISA technology, but the kit is very expensive, limiting its wide spread clinical and research use, many components of the kit are needed to purchase from 3rdparty resulting increase cost of the product. The Pseudovirus based SARS- CoV-2 Neutralization Assay is a time consuming assay and at least 2 days to get result for the candidate samples if pseudotype virus stock has been prepared in advance. Thus, Neutralizing antibodies using surrogate assays are cell based surrogate assay mimic close to gold standard assay but due to biological variables most of the cell based assay have high CV making it unsuitable for diagnostic assay. The Cell based RBD-ACE2 binding assay, which is based on the interaction between ACE2 and receptor binding domain (RBD) employs the transient transfection of pcDNA3-SARS-CoV-2-S-RBD-sfGFP (4,5) and pCEP4-myc-ACE2 (4,6) plasmids into a suspension Expi293 cells. This leads to increase in 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. Also, it is not feasible to maintain cell line in ordinary clinical laboratory. For every new batch of ACE2 transfected cells, concentration of cells has to be determined and based on transfection efficiency all the assay parameter have to be read just before starting an assay. Making these assays unfit to be an affordable and ideal diagnostic assay kit to be carried out in ordinary clinical laboratory by less trained health staffs. Thus, taking into consideration all the above-stated points and keeping in view of the current situation around the world it seems that antibody neutralization assay needs to be updated as per the current requirements. Therefore, there exists a need for robust, stable and simple assay to analyze the neutralizing antibodies or inhibitors, to be carried out in any laboratory without any further standardization. OBJECTS OF THE INVENTION It is therefore an object of the present disclosure to propose a kit and assay for analyzing the presence of neutralizing antibody in vitro against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). An object of the present disclosure is to provide a low cost, cell based, virus free, ready to use kit and assay for semi-quantitative analysis of Neutralizing / blocking functional antibody which may neutralize / block receptor binding domain (RBD) of Spike protein of SARS-CoV-2 for its binding to the corresponding human receptor ACE2. Another object of the present disclosure is to provide a kit for identification of neutralizing antibody that is ready to use without the need of complex cell culture facility. Yet another object of the present disclosure is to provide an in vitro assay for identification of neutralizing antibody against SARS-CoV-2 that is more reproducible and comparable and can be conducted in short amount of time. These and other objects and advantages of the invention will be apparent from the ensuing description. SUMMARY OF THE INVENTION Accordingly, the present disclosure provides a kit for identifying neutralizing antibody in vitro against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in a sample comprising a frozen media comprising secreted SARS- CoV-2 spike protein receptor binding domain (RBD) for binding to human ACE2 protein tagged with a detection entity; a ready to use frozen transfected cells expressing human ACE2 protein; a reaction buffer; a DNA florescent binding dye; a Positive control; and a Negative control. In one aspect of the present disclosure, a method for preparing frozen cell line expressing human ACE2 protein is provided. In another aspect of the present disclosure, a method for preparing a frozen media comprising secreted SARS-CoV-2 spike protein receptor binding domain (RBD) of a human ACE2 protein is provided. In yet another aspect of the present disclosure, an in vitro assay for analysing a sample for presence of neutralizing antibody against SARS-CoV-2 is provided. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 provides microscopic view of surrogate cell based neutralizing assay of the present disclosure (CENA). Figure 2 depicts effect of different reaction buffers on stability of receptor binding domain – green fluorescent protein (RBD-GFP) binding over time. Figure 3 illustrates effect of DMSO and cell freezing on live cell Propidium Iodide staining and RBD-GFP binding with ACE2 expressing HEK 293 cells. Figure 4 provides analysis of neutralisation of RBD of spike protein of SARS-CoV-2 by the CENA method using flow cytometry. Figure 5 illustrates linear correlation of CENA 50 with conventional CPE based SARS-CoV- 2 live virus neutralization assay (cVNA 100). Figure 6 demonstrates comparative correlation of CENA and commercially available cPass SARS-CoV-2 neutralizing antibody detection kit. Figure 7 is a dot plot graph showing comparative performance of CENA and cPass (Genescript) kit for detecting neutralizing antibody in serum samples. Figure 8 demonstrates neutralising antibody titre against RBD-Spike protein of SARS-CoV-2 analysed in serum post vaccination against SARS-CoV-2 by the assay (CENA) of the present disclosure. Figure 9 illustrates production of component 1 and component 2 of the kit at manufacturer site Figure 10 depicts the procedure of the assay by interacting different components of the kit at end user site. DETAILED DESCRIPTION OF THE INVENTION In the present document, the word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment or implementation of the present subject matter described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments. While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure. The terminology used herein is to describe particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms “comprise”, “comprising”, “includes” and / or “including” when used herein, specify the presence of stated features, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and / or groups thereof. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The neutralizing antibody against SARS-CoV-2 is the functional antibody which directly blocks binding of the virus to its ACE2 receptor by blocking the interacting epitopes of the receptor binding domain (RBD) of the viral spike protein. Presence of this antibody following natural infection or vaccination gives protection against re-infection. So detection of this antibody by neutralizing antibody assay in biological fluid is important for evaluation of vaccine efficacy. This neutralizing antibody assay is different from the regular serological antibody assay for total detecting binding antibody. Accordingly, the present disclosure provides a cell based in vitro assay for analysing in a sample for presence of neutralizing antibody against SARS-CoV-2 using flow cytometry. Unlike other multistep cell based assay using pseudotyped virus, the assay could be carried out in two step in 3 hours without the need of any washing steps as like other serological assay for elimination background. Further, the assay could be packaged standalone kit and the kit eliminates the need for any cell culture prior to the assay. In an embodiment, the present disclosure provides a kit for detecting neutralizing antibody in vitro against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in a sample comprising: (i) a frozen media comprising secreted SARS-CoV-2 spike protein receptor binding domain (RBD) for binding to human ACE2 protein tagged with a detection entity; (ii) a ready to use frozen transfected cells expressing human ACE2 protein; (iii) a reaction buffer; (iv) a DNA binding florescent dye; (v) a Positive control; and (vi) a Negative control In an embodiment, the samples are biological samples selected from the group consisting of human serum, blood, cerebrospinal fluid, sweat, semen, saliva, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, otitis fluid, gastric fluid, and breast milk. Preferably, the sample is human serum or plasma. More preferably, the serum is from group of persons having previous exposure of SARS-CoV-2 or post COVID-19 vaccinated or both. In an embodiment, the receptor binding domain (RBD) is represented by amino acid sequence 333-529 of Sequence ID No.1(GenBank ID: YP_009724390.1; amino acid sequence 333-529) and the human ACE2 protein is represented by amino acid sequence 19 to 805 of Sequence ID No.2. (GenBank ID: NM_021804.1; amino acid sequence 19-805). In an embodiment, the cells are adherent cell lines which do not express ACE2 protein, however, post stable transfection (chromosomal integration) with the expressing cassette of ACE2, the cells express the ACE2 protein on the cell surface. Preferred cell lines are from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, COS-7, A549 which do not express ACE2 protein. Other cell lines which express ACE2 naturally such as Vero E6, VERO 81, VERO SLAM, MA104, LLC-MK2, BGM, and Caco-2 etc. also could be used directly without need of ACE2 constitutive expression cassette stable transfection. More preferably, the cell line is adherent HEK293 cells. Benefit of using cell lines which does not express naturally ACE2 is that any background due to non-specific interaction of RBD- detection-element on the cell surface could be detected by running a neutralization reaction with ACE2 untransfected cells and the background could be eliminated by appropriate stringent condition or cut-off. Adherent HEK293 cell line is used in this assay instead of suspension cell Expi293 as used in previous published report (4). It is very easy to handle and to remove any debris or dead cell by washing with PBS and / or changing media. The cell line also could be maintained by low cost Minimum Essential media as well as easy to transfection with ordinary transfection reagent with high efficacy. The cells are first cultured in an animal cell culture media with fetal bovine serum (Gibco, USA). Post culturing, the plasmids comprising a constitutive expression cassette encoding ACE2 protein are transfected using standard transfection reagents (eg. lipofectamine 2000, lipofectamine 3000, Effectene transfection reagent etc.) into the cultured cells and propagated in the presence of selection marker to obtain stable transfected cells. The plasmid is selected from a pCEP4-myc-ACE2 plasmid or hACE2 or pcDNA3.1-hACE2 or any other DNA vector having human ACE2 constitutive expression cassette comprising a mammalian selection marker. The stable transfected cells expressing ACE2 protein are resuspended in a reaction buffer comprising Sodium Chloride and Ammonium Chloride at a concentration of 0.140 M and 0.025 M, respectively at pH 7.4 in a cell culture flask, followed by dislodging the cells from the flask by scrapping and passing through a cell strainer to making single cell suspension and further the cell concentration is determined by SPARK Multifunctional Microplate Reader, TECAN using “Dual chamber chip for automated cell counting” .Alternatively any other reliable cell counting methods could be used like flowcytometer,TC20 automated cell counter etc. 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. The single cell suspension was prepared by pipetting the cell suspension against the wall of the culture flask followed by passing through cell strainer to remove any cell clump. This transfected cell line is very loosely attached and form very fragile cell clump. Therefore, 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 (7). As K+in standard Dulbecco′s Phosphate Buffered Saline (DPBS) facilitate endocytosis of receptor and ligand complex regular PBS buffer was replaced for final cell washing and resuspension. Instead cells were washed and resuspended by 1X Reaction Buffer containing 25 mM ammonium chloride (pH7.4) and 140 mMNaCl. Additionally, Ammonium chloride inhibit endocytosis by preventing acidification of endosome (8-10). 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 are processed in ready to use freezing format. The cells are freezed in freezing buffer containing reaction buffer and 10% dimethyl sulfoxide (DMSO) in aliquots at -80°C to -150°C as a concentrated stock in ready to use format before the assay. Ammonium buffer and freezing inhibit endocytosis of cell surface receptor ligand complex making RBD-detection entity ACE2 complex stable on cell surface. As the ready to use frozen cell will be used in the assay there will be further reduction of metabolic activity for endocytosis. This will result more stable cell surface interaction of RBD- GFP with ACE2 with better signal stability at least for 3 hours in ice. Additionally, freezing cells in DMSO containing buffer make pore formation in the cell surface enabling staining of cell nucleus with DNA binding dye (11) for downstream flow cytometry. 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 from culture for every assay set up. The frozen cell is 75 times diluted by cell resuspension buffer containing reaction buffer and DNA binding florescent dye. The DNA binding dye which stain nucleus will be used for identification of total intact cell population during downstream flowcytometry. The reaction buffer comprises Sodium Chloride and Ammonium Chloride at a concentration of 0.140 M and 0.025 M, respectively at pH7.4. In an embodiment, the DNA binding florescent dye is selected from Propidium Iodide, 7- aminoactinomycin D and SYBR Green. Preferably, Propidium Iodide stock solution at a concentration of 0.3-0.7 mg / ml is used. Propidium Iodide (PI) which is nuclear stain is used for staining dead cells and cannot normally penetrate live cell membrane. PI is added in the range of 0.3-0.7 µg / ml, more specifically 0.5 µg / ml to the 1X reaction buffer to make cell resuspension buffer for cell re-suspension as well as nuclear staining. Normally PI is used for staining dead cell nucleus. PI cannot penetrate the cell membrane of fresh live cell. As cells were frozen with DMSO 10% in freezing buffer it will create pore formation in the cell membrane thereby, helping in permealization of the cells enabling penetration of PI within the cells for nuclear staining. So it helps gating only nucleated cells in flow cytometry removing the debris or any other non-cellular component or any precipitate which may arise from serum. 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 laboratory does not use flow cytometry machine having UV laser. PI could be measured using ordinary single lesser basic flow cytometry machine which is commonly available in ordinary clinical laboratory. In another embodiment of the present disclosure, the SARS-CoV-2 spike protein receptor binding domain (RBD) for binding to human ACE2 proteins tagged with a detection entity. The detection entity is selected from super folded green fluorescent protein (sfGFP), red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), near–infrared fluorescent protein 702 (iRFP702). Preferably, super folded green fluorescent protein (sfGFP) is used as described in plasmid pcDNA3-SARS-CoV-2-S-RBD-sfGFP (4). In another embodiment of the present disclosure, the SARS-CoV-2 spike protein receptor binding domain (RBD) with detection entity was tagged with N terminally cleavable signal sequence for extracellular secretion of the recombinant fusion protein. The signal peptide is selected from the cleavable signal peptide of the influenza hemagglutinin (Sequence ID No.3), signal peptide of the mammalian secreted protein, resistin (Sequence ID No.4). Preferably signal peptide of the influenza hemagglutinin is used as described in plasmid pcDNA3-SARS- CoV-2-S-RBD-sfGFP (4). Green florescent protein (sfGFP) tagged RBD COVID-19 spike protein (RBD-sfGFP) is expressed and secreted in the media, from another cell line which already has been stably transfected with the expressing cassette of (RBD-sfGFP). Stable transfection provides the assay more reproducibility, cost effective, more robustness of the assay. There is no requirement of transfection before every assay as like previous assay design. The cells are first cultured in a complete Minimum Essential Medium (MEM) with 10 % fetal bovine serum. The cells do not express RBD domain, however post transfection, the cells express the detection entity tagged RBD domain of the SARS-CoV-2 spike protein. Such cells are selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, COS-7. Cell lines which express ACE2 naturally are avoided for this purpose to prevent self-binding of RBD fusion protein on the cell surface upon secretion. More preferably, the cells are adherent HEK293 cells. Post culturing, the plasmids comprising a constitutive expression cassette encoding RBD domain of the SARS- CoV-2 spike protein are transfected using standard transfection reagents (eg. lipofectamine 2000, lipofectamine 3000, Effectene transfection reagent etc.) into the cultured cells and propagated in the presence of selection marker to obtain stable transfected cells. The C terminal of the receptor binding domain is tagged with super-folded green fluorescent protein (RBD-sfGFP) and N terminal is tagged with signal peptide from Influenza virus hemagglutinin. This N terminal signal sequence tag will help to secrete the RBD-sfGFP into extra cellular media while the signal sequence will be cleaved during the process. The coding nucleotide sequence of N terminal signal peptide, RBD and sfGFP were inserted into the plasmid sequentially in triplet coding frame of the respective coding sequences to express a fusion protein of 51.2kDa. The plasmid is a pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmid comprising a mammalian selection marker (4). The stable transfected cells are further incubated in minimal essential media for 3-5 days at 37°C in CO2 incubator. The media is then centrifuged at 10000 RPM for 10 minute at 4oC to obtain a clear supernatant without any dead cell / debris that is then diluted in 1X reaction buffer comprising Sodium Chloride and Ammonium Chloride at a concentration of 0.140M and 0.025 M, respectively at pH7.4, such a way that the resultant concentration of the supernatant will be 4-5X (4-5 times than the final concentration to be used) followed by freezing the media at - 20°C to -80°C. The RBD-sfGFP is secreted by the transfected cells into a media, preferably an animal cell culture media frozen at -20°C to -80°C. More Preferably, Minimal essential Medium comprising basal minimal essential medium mixed with 10% fetal bovine can be used as an animal cell culture media. In yet another embodiment of the present disclosure, an in vitro assay for analysing a sample for presence of neutralizing antibody against SARS-CoV-2 is provided. The sample is first incubated with a media comprising secretedSARS-CoV-2 spike protein receptor binding domain (RBD) of a human ACE2 protein, tagged with a detection entity followed dilution of the frozen media by 1 X reaction buffer, at room temperature for 30-90 minutes followed by incubation on ice for 10-30 minutes. Frozen transfected cells expressing human ACE2 protein are then thawed, followed by dilution in a cell resuspension buffer comprising 0.3-0.7 µg / ml of DNA binding dye to obtain thawed cells. The thawed cells are then contacted with the sample incubated with the media for 60- 90minutes on ice bath to form a reaction mixture. The reaction mixture is diluted by adding the reaction mixture into an ice cold reaction buffer, and the intensity of green fluorescence in the cells. If there is no neutralizing antibody in the test serum or no inhibitor of the interaction, the cell is stained green on its surface due to binding of GFP tagged RBD with its cell surface ACE- 2 receptor. The percentage of stained cells is evaluated by flow cytometry. Any blocking of the spike protein RBD due to the presence of neutralizing antibody / inhibitor will cause / reduce binding of the RBD on to the cell surface resulting low percentage of green positive cells. Percentage of inhibition will be expressed as proportion of RBD binding without any serum. A fluorescence intensity greater than the cut-off value indicated absence of neutralization antibody and a fluorescence intensity less than the cut-off value indicated the presence of neutralization antibody. Preferably, the cut- off value is in the range of 20-30% and depends on the negative control. Percentage of neutralization will be determined by the formula as below: % of RBD binding in antibody treated samples–background Neutralization (%NT) = 1 – ×100 Adherent 293T cell line is used % in R tBhiDs- aGssFaPy b iinnsdteinagd i onf u snutsrepaetnesdio cnon cterloll– Ebxapcik2g9r3ou ansd used in previous published report (7). It is very easy to handle and to remove any debris or dead cell Background: Percent of cells (samples without incubating RBD-GFP) above the threshold for GFP positivity The positive control of the kit comprises cocktail of serum samples from post vaccinated (with Covishield / Sputnik V / Covaxin or other anti SARS-CoV-2 vaccine) individuals (healthy volunteers) having neutralizing antibody against SARS-CoV-2. Presence of neutralizing antibody in the final serum cocktail could be determined by any standard SARS-CoV-2 live virus neutralizing assay (e.g. PRNT, cVNA, any PRNT validated surrogate neutralization test) and antibody titter should be at limiting serum dilution preferable of 1:640 or higher. Negative control comprises cocktail of serum samples (from healthy volunteers) without neutralizing antibody against SARS-CoV-2, determined by any standard SARS-CoV-2 live virus neutralizing assay (e.g. PRNT / cVNA, any PRNT validated surrogate neutralization test). As the present disclosure used stably transfected ACE2 expressing cells which express ACE2 on its surface consistently with a same high percentage resulting consistent percent of RBD- GFP binding upon assaying with different batches of ACE2 expressing cells. So in this protocol it is possible to omit step of staining ACE2 with antibody. 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. This strategy of using no staining antibody in the assay provide further robustness of the assay and reduce the cost. The assay avoids any florescent tagged antibody for staining ACE2 and the concentration of PI and RBD-GFP was optimized such a way it provided negligible background and washing steps were omitted, making the assay further robust cell based assay. In this way the assay achieves 100% neutralization using SARS-CoV-2 immune serum. Unlike previous protocol, as per the present disclosure the components could be packaged as standalone ready to use kit without need of cell culture by the end user. The present disclosure thereby offers an economical and low cost kit and assay for analysing the presence of neutralising antibody. As the recombinant genetic materials containing expression cassette of ACE2 genes and S-RBD-GFP (SARS-CoV-2 spike protein receptor binding domain tagged with GFP) are already chromosomally integrated within the cell lines, there is no requirement of transfection for every batch of the assay kit preparation using expensive transfection 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. As like manufacturing ELISA based assay kits, which need series of procedures like purification of recombinant spike protein RBD and ACE2, the protein conjugation with detection systems, special reagents for detection of signal is not required in this assay. 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. More over optimized reaction buffer and protocol were developed to reduce the assay variations which happen in any other cell based assay system. Now, the present disclosure will be described through illustrative examples. The examples given are non-limiting in nature, merely illustrative of the uses, processes and products claimed in this invention, and the practice of the invention itself is not restricted to or by the examples described. EXAMPLES Source of the chemicals and biological materials used in the present invention: 1. HEK 293 cells: ATCC, USA. 2. Propidium iodide: Merck, USA 3. Plasmids used for transfection: pursue through addgene (https: / / www.addgene.org / 141184 / sequences / ) and any other similar plasmid / viral vector with desirable expression cassette and having mammalian selection marker may also use. 4. Minimum essential media: Sigma, USA 5. DMSO: Sigma, USA 6. Ammonium chloride: Sigma, USA 7. Sodium chloride: Sigma, USA Example 1: Preparation of RBD-GFP containing media Steps of preparation of components have been described in schematically in figure 9. An adherent cell line (e.g. HEK 293 cell line grown in MEM media with 10 % fetal bovine serum, incubated at 37oC with 5% CO2) was stably transfected (integrated into the genome of the cell line) with a plasmid containing expression cassette of SARS-CoV-2 RBD-GFP with signal sequence for its extracellular release and having mammalian selection marker. The plasmid (pcDNA3-SARS-CoV-2-S-RBD-sfGFP) was already described in document (3) and distributed through addgene (https: / / www.addgene.org / 141184 / sequences / ). Any other similar plasmid / viral vector with RBD-GFP expression cassette and having mammalian selection marker may also work. In the plasmid the RBD of SARS-CoV-2 (strain ID 2697049) could be replaced by any variant of RBD from any newer SARS-CoV-2 strains (in protein coding frame of upstream N-terminal signal sequence and downstream C-terminal sfGFP) to detect neutralizing antibody against the newer SARS-CoV-2 strains. The Kit could be updated as per emergence of newer strains, while rest of the protocol will be remaining same. For stable transfection the RBD-GFP expression plasmid was first transfected by lipofectamin 3000 then after 48 hrs media was replaced by fresh media containing selection antibiotic Genticin 400µg / ml (G418). After two weeks of incubation in selection media, single green fluorescent colonies were identified, separated and re plated into 96 well plate. Following regrowth, the well was screened for most populated green fluorescent cells under fluorescent microscope. Further screening was done by checking supernatant of the wells having green florescence cells, florescence was measured using an appropriate filter for GFP (e.g. excitation: 488nm / emission 530nm). The colony which gave the best green florescence in the supernatant was propagated and amplified for preparing optimized GFP-RBD containing media. Particularly the cell line was grown upto 70 % confluence. Fresh MEM media was added 2ml for 25cm2flask and 6 ml media for 75cm2flask. After 4 days of incubation media was collected. The media was then centrifuged and supernatant was collected and fluorescence was adjusted to 60-25 U diluting with 1X reaction buffer, at this range of florescence level, assay would be more linear and would have increase detection limit in lower range. The media could be further calibrated with a positive control (PC) reference serum (post vaccinated / convalescent), neutralizing antibody titre of which has been predetermined by gold standard assay (PRNT). Dilution of the media could be adjusted so that titre of the PC by the new test closely matched with the titre of gold standard PRNT assay while the % of RBD-GFP positive cells of untreated control is kept above 80%. The diluted media was then dispensed in 2ml tubes and kept in -20oC. For calibration of new batch of RBD-GFP containing media, activity of RBD-GFP was calibrated by diluting the media, adjusting % of GFP positive cells and mean florescence of single cell of the previous standardized batch. Example 2: Preparation of ACE2 Receptor cell line Steps of preparation of components has been described schematically in figure 9. An adherent cell line which don’t express ACE2 (e.g. HEK 293 cell line grown in MEM media with 10 % fetal bovine serum, incubated at 37oC with 5% CO2) was stably transfected (integrated into the genome of the cell line) with a ACE2 expression cassette / plasmid having mammalian selection marker. The plasmid (pCEP4-myc-ACE2) has been already described in previous report (3) and distributed through addgene (https: / / www.addgene.org / 141185 / sequences / ). Any other similar plasmid / viral vector with ACE2 expression cassette and having mammalian selection marker may also will work. For stable transfection the ACE2 expression plasmid was first transfected by lipofectamin3000 then after 48 hrs media was replaced by fresh media containing selection antibiotic Hygromyocin 150µg / ml. After two week single colonies were separated and re-plated into 96 well plate. Following re-growth functional screening was carried out by observing RBD-GFP binding activity under florescent microscope. Colony with optimal binding of RBD-GFP was propagated for the assay. All the cell washing and resuspension was conducted in reaction buffer. The reaction buffer should 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 NH4Cl pH 7.4 (endosytosis blocker as well as alternative buffer system). Any other buffer like HEPES and blocker also could be evaluated. Reaction buffer will stabilize RBD-ACE2 complex on cell surface preventing endocytosis). The cells were washed with reaction buffer and dislodged from the cell culture flask by scrapping by cell scraper and single cell suspension was prepared by pipetting in reaction buffer and was cleared passing through cell strainer. The cells were then centrifuged at 180-300g for 5 min and supernatant was removed and resuspended in 1X reaction buffer. The cells were then counted, centrifuged and supernatant was removed. The cells were then resuspended in cell freezing buffer (reaction buffer with 10% DMSO) at a desired concentration (e.g.50 Lakh cell / ml). The cells were then dispensed in 20µl per 2 ml tube sufficient for 10 reactions and was kept at -80oC till it is used. Different cell concentration and volume could be used for different purpose and size of the kit. Example 3: Preparation of other components of the Kit 10X Reaction buffer was prepared comprising 1.40 M NaCl, 0.25M NH4Cl, pH7.4: The buffer was made by dissolving 13.37g of Ammonium Chloride(NH4Cl) and 81.81g Sodium Chloride(NaCl) dissolved in 800 ml distilled water, pH was adjusted to 7.4 with the help of liquor ammonia and final volume was make-up to 1000ml. 1000X propidium iodide stock was prepared by diluting commercial stock of 1mg / ml Propidium Iodide (PI) to 0.5mg / ml in water. The working stock was used at a concentration 1µl / ml of reaction buffer for making cell dilution buffer. Positive control (PC) reference serum: Blood samples of individuals (healthy volunteers) who are already vaccinated with SARS-CoV-2 vaccine (with Covishield / Sputnik V / Covaxin or other anti SARS-CoV-2 vaccine and within 1-4 month following the booster dose) were collected without anticoagulant by vein puncture. Serum was isolated following standard method (i.e.1-hour incubation at room temperature followed by centrifugation at 1000-2000×g for 10 minute and serum was collected from supernatant). The serum samples were screened for presence of high titre of the SARS-CoV-2 neutralizing antibody using cPass SARS-CoV-2 Neutralizing Antibody Detection Kit, (GeneScript) which is already validated with the live virus neutralization assay, ‘PRNT’ and approved by FDA for emergency use. The assay was performed as per the manufacturer’s protocol ( https: / / www.fda.gov / media / 143583 / download). Sample could also be screened by PRNT / cVNA. Equal volume of 10 serum samples (could be included more) showing highest neutralization titre were mixed and the final serum titre was determined by SARS-CoV-2 live virus neutralization test (cVNA) as described in reference 16 (alternatively it could be detected by PRNT or any PRNT validated surrogate neutralization test) The standardized cocktail of serum sample was stored in aliquot at -20oC and is used for subsequent test as a standard. Final neutralizing antibody concentration / titre should be 1:640 or more by PRNT or equivalent concentration. Negative control (NC) reference serum: Using the similar protocol as described in PC, 10 negative serum samples (could be included more) which have been selected following screening of serum samples from healthy volunteers by cPass SARS-CoV-2 Neutralizing Antibody Detection Kit, were mixed in equal volume and reconfirmed by cVNA(12)(alternatively PRNT / any PRNT validated surrogate neutralization test). The cocktail serum was stored at -20oC in aliquot and used as negative control standard for any subsequent test. Example 4: In vitro assay using kit of the present disclosure Blood was collected without any anti-coagulant by vein puncture and serum was separated by centrifugation at 1000-2000×g for 10 min following incubation for 1 hours at room temperature (25oC). Supernatant (serum) was collected, stored at 4oC for short term use or at -20oC for long term storage. 10X reaction buffer was diluted to 1X concentration using distilled water. Cell dilution buffer was made by adding 1µl / ml 1000X PI into the 1X reaction buffer. Serum was then serially diluted with the 1X reaction buffer.50 µl of frozen standardized media containing GFP-RBD was taken into 96 well plate.5 µl of 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 transfected cell expressing ACE2 protein was thawed on ice and diluted 75 times with pre cooled cell resuspension buffer containing 0.5µg / ml Propidium iodide (e.g. for 20 µl frozen cells in 1.5 ml cell resuspension buffer). 150µl of ice cold cell suspension was added into each well. In this way 10k cell was distributed in each reaction. The reaction mixture was mixed properly and kept for 60-90 minutes into the ice bath. Following incubation, the reaction mixture was pipetted to remove any cell clump and the whole reaction mixture was added into ice cold 500µl RB kept into flow cytometry tubes. Tubes were kept into mini cooler (-20oC) till flow cytometry measurement was done. For flow cytometry analysis GFP was detected by FL1 and PI was detected by FL3 Chanel. First single intact cell population was gated form SSC-FSC plot. Then PI stained cells were gated from SSC-PI plot. In a GFP-PI quadrant plot threshold was 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 HEK 293 ACE2 cells was indicated by % of GFP positive cells. Binding of RBD-GFP to the cells without incubation with any serum samples or inhibitor was considered as 100% binding. Any reduction of binding as will be detected as reduced GFP positive cells, following incubation of the RBD-GFP with any serum samples or inhibitor will be the measure of neutralization. Accordingly, neutralization will have measured by the following formula: (1- % GFP positive cells in treated samples / GFP positive cells in control untreated sample). Method of analysis by flow cytometry is demonstrated in Fig 4. Interpretation of the result: If % neutralization was > 20% (Cut-off 20%) at 1:10 serum dilution the serum is considered positive for presence of Neutralization antibody against SARS-CoV-2 and if it is <20% it is considered negative or no neutralization antibody against SARS-CoV-2 (Figure 4). Method of cut-off determination: Total 41 pre COVID-19 serum samples, collected before march 31, 2020 were used to determine the assay cut-off. Mean+2SD was determined to be 16% (-2%±18). So for practical purpose 20% was used as cut-off making it towards more stringent (figure 4). Within the linear range of the assay (20%-80%) cut-off value was 2-6% (table 4). Figure 1 depicts microscopic view of surrogate cell based neutralising assay (CENA). 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 2 hours with human serum (control serum: collected before the COVID-19 pandemic, test serum: Serum collected from subject already vaccinated with two dose of Covishield). Cells incubated with control sample shows green ring outside the cell membrane indicating binding of RBD-S-GFP on cell surface. Upon incubation with serum from vaccinated subject, the green hallows disappeared indicating blockage of interacting domain of RBD of SARS-CoV-2 Spike protein. Same observation was replicated when the corresponding samples were analysed by flow cytometry. While control serum treated samples showed 50% RBD-GFP binding while incubation of vaccinated serum showed only 2% binding close to background. Example 5: Selection of optimum reaction buffer condition The buffers as shown in table 1 were tested for binding efficacy of RBD-GFP to the HEK 293 cells expressing ACE2. Following first reading after dilution of the reaction with the corresponding Reaction Buffer (RB) at 2 hours, 2ndreading was taken after 20 minutes on ice following the first reading. Figure 2 demonstrates that reaction buffer RB6 showed the highest RBD-GFP binding efficiency and stability to ACE2 expressing 293 cells among the tested buffers. Table 1 Reaction Composition Remarks Buffer RB1 Dulbecco's Phosphate Buffered Saline, Contain K+:Facilitate Ph7.4 (DPBS) endocytosis RB2 Phosphate Buffered Saline (PBS) Ph7.4, No K+140 mMNaCl RB3 Phosphate Buffered Saline (PBS) pH8, No K+140 mMNaCl RB4 10 mM HEPES buffer pH7.4, 140 _ mMNaCl RB5 10 mM HEPES buffer Ph8, 140 _ mMNaCl RB6 25 mM Ammonium Chloride pH7.4, 140 Ammonium buffer inhibit mMNaCl endocytosis by increasing pH of endosome RB7 25 Mm Ammonium Chloride Ph8, 140 Ammonium buffer inhibit mMNaCl endocytosis by increasing pH of endosome Example 6: Analysing the effect of DMSO and cell freezing The effect of DMSO and cell freezing on live cell Propidium Iodide staining and RBD-GFP binding with the ACE2 expressing HEK 293 cells were evaluated at different temperatures. DMSO was incorporated in the buffer and the ACE2 expressing cells were frozen at -80°C, demonstrated improved Propidium iodide staining of the cells (Figure 3A). Further, freezing of the ACE2 expressing cells at -80°C in DMSO improved the RBD-GFP binding of the cells (Figure 3B). Example 7: Comparative analysis of the present in vitro assay with conventional assays 7.1 Comparative clinical agreement of CENA and conventional CPE-based SARS-CoV- 2 viral neutralization assay (cVNA) Comparative analysis was carried out between in-vitro assay of the present disclosure (CENA) with conventional CPE-based SARS-CoV-2 viral neutralization assay (cVNA). A total of 15 serum samples were used for the comparative test as follows: 1. Post vaccinated serum (12 samples) 2. Serum collected before (before March 2020) COVID-19 pandemic (1 samples) 3. Cocktail of serums from 10 COVID-19 vaccinated individual (PC) (1 sample) 4. Cocktail of serums from individuals having no reported RT- PCR positive COVID-19 infection and no vaccination (NC) (1 sample). The serum samples were serially two fold diluted and each was tested for neutralisation by CENA as described above in the present disclosure and by cVNT as described previously (12).Serum titre was determined by CENA as the highest limit of serum dilution which produced at least >50% neutralization (CENA50). If CENA50 was <1:40 serum dilution, sample was considered negative (table 2). Serum titre was determined by cVNT as the highest limit serum dilution which produce 100% neutralization (cVNT100). If cVNT100 was <1:40 serum dilution, sample was considered negative (table 2). The comparative profile by both the test is shown in table 3. The two tests show 100% agreement to detect positive and negative samples (table 4). Table 2. Cut-off conditions for comparative analysis of CENA and cVNA for defining positive or negative by each assay. Sample Positive: Negative: Remark dilution cut-off Cell based SARS-CoV-2 1:40 Cut-off Cut- CENA50 Neutralizing Antibody detection >50% NT off<50% NT assay (CENA) Conventional CPE based Virus 1:40 Cut-off Cut-off cVNA 100 Neutralization Assay (cVNA) 100% NT <100% NT CPE: Cytopathic effect; NT: Neutralization Test result expressed as % of neutralization of SARS-CoV-2 RBD to bind with the receptor ACE2 by the respective assay. Table 3: Comparative clinical agreement with serum titre (limit of serum dilution which gives positive result) measured by CENA50 vs cVNT100 (live virus Neutralization Test) Sample description Cell based SARS-CoV-2 Live Virus Neutralizing ID Neutralizing Antibody Antibody detection assay detection assay (CENA50) (cVNT100) %NT@ Interpretation 100% NT@ 1:40 1:40 P / N (>50% NT) 1 vaccinated * 20% Negative Negative 2 vaccinated * 100% Positive Positive 3 vaccinated * 30% Negative Negative 4 vaccinated * 17% Negative Negative 5 vaccinated * 94% Positive Positive 6 vaccinated * 74% Positive Positive 7 vaccinated * 44% Negative Negative 8 vaccinated * 76% Positive Positive 9 vaccinated * 100% Positive Positive 10 vaccinated * 15% Negative Negative 11 Uninfected, -2% Negative Negative unvaccinated** 12 vaccinated * 100% Positive Positive 13 vaccinated * 100% Positive Positive 14 PC 99% Positive Positive 15 NC -3% Negative Negative Table 4: Clinical agreement with CENA50 and cVNA100 CPE based Live Virus neutralization assay (cVNA100) Positive Negative 8 7 Cell based SARS-CoV- Positive 8 0 2Neutralizing Antibody Negative 0 7 detection assay (CENA50) Over all % of 100% agreement: Cohen’s k 1 In the given condition the result of CENA have 100% agreement with the conventional SARS- CoV-2 live virus neutralization assay. Figure 5 depicts linear Correlation of CENA50 with the conventional CPE based SARS-CoV- 2 live virus neutralization assay(cVNA100). Neutralization Test, was carried out using serial two-fold dilution of serum samples and measured by either CENA50 and cVNT100 to find out serum titre (limit of serum dilution which gives positive result). Log of the serum titre by the respective test (CENA50 / cVNT100) were plotted in Y and X axis 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). 7.2 Comparative correlation of CENA and commercially available cPass SARS-CoV-2 Neutralizing Antibody Detection Kit. Serum samples of subjects who already completed two doses of COVID-19 vaccine within 7 months (53 samples) were taken for analysis. cPass SARS-CoV-2 Neutralizing Antibody Detection Kit, (Genescript) is the FDA (Emergency Use Authorization) approved well validated and internationally widely used SARS-CoV-2 neutralization antibody detection kit. The test was performed as per manufacturer’s protocol duplicate at a serum dilution of 1:20 (as per recommendation). The reading was taken in SPARK multimode plate reader (TECAN). % of Neutralization (% Signal Inhibition) was calculated as per the formula provided by the manufacturer. The cell based Neutralization assay (CENA) was carried out using the same protocol described in the invention at 1:20 dilution by flow cytometry. % Neutralisation was determined as described before in the invention. Figure 6 depicts correlation analysis of the 53 post SARS-CoV-2 vaccinated serum samples having different levels of neutralizing antibody (Nab) by CENA and cPass SARS-CoV-2 Nab Detection Kit (Genescript) at a serum dilution 1:20. % of Neutralization (% NT) by the respective assay was plotted along X and Y axis. Pearson’s Correlation coefficient, linear regression was carried out in excel. Statistical significance (p value) was calculated by two tailed test. The data is the mean of duplicate test. Figure 6 showed CENA have good linear correlation with the cPass SARS-CoV-2 kit (R 0.94, p<0.00001). Figure 7 depicts a dot plot showing comparative performance of CENA and cPass (Genescript) kit to detect neutralizing antibody in serum samples. 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: >30% positive &<30% negative, Cut-off for CENA NT: >50% positive &<50% negative, N: 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; dotted 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. Table 5: Cut-off condition for comparing CENA and cPass SARS-CoV-2 Neutralizing Antibody Detection Kit Sample Positive Negative Remark Name of the assay dilution cut-off Cell based SARS-CoV-2 1:20 Cut off >20% Cut off CENA20 Neutralizing Antibody NT* <20% NT* detection assay (CENA) cPass SARS-CoV-2 1:20 Cut off >30% Cut off sVNT30 Neutralizing Antibody NT** <30% NT** Detection Kit (sVNT) (Genescript) *Determination of Cut-off 20%NT for CENA: Neutralization test(NT) by CENA using pre COVID-19 serum samples(20 samples, collected before March 2020) was performed. %NT(Mean +3SD) =20% was used as cut-off for CENA; ** Cut-off 30% NT** for cPass kit was provided as per manufacturer’s protocol. Table 6. 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 Negative (38) (15) Total Result of Cell Positive 37 3 40 based SARS- Negative 1 12 13 CoV-2 Total 38 15 53 Neutralizing % of total 92.4% Antibody agreement detection assay Cohen’s kappa 0.806 (interpretation: Almost perfect (CENA) (k)* agreement**) Neutralization measured at a dilution 1:20. *Cohen’s kappa (k) was determined using a web based software ‘Cohen’s kappa free calculator” 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 Example 8: Determination of Sensitivity and specificity of CENA The sensitivity and specificity of CENA of the present disclosure was determined using the standard commercially available cPass SARS-CoV-2 Neutralizing Antibody Detection Kit, (GeneScript) which is already validated with the live virus neutralization assay, ‘PRNT’ and approved by FDA for emergency use), as known standard test for detecting positive samples and pre COVID-19 (collected before March 2020) serum as negative samples as per the manufacturer’s protocol(https: / / www.fda.gov / media / 143583 / download). The results are shown in Table 7. Table 7 Positive*: Negative: post SARS-CoV- pre COVID-19 2 vaccinated (collected before serum * March 2020) serum Cell based Positive 37 0 SARS-CoV-2 Negative 1 20 Neutralizing Total 38 20 Antibody Test Sensitivity: 97% Specificity: 100% detection Performance assay(CENA) * Positive samples Determined by FDA approved validated cPass SARS-CoV-2 NT kit. Example 9: Determination of response to different brands of COVID-19 vaccines in a population. Serum samples of two healthy individuals before and one month after COVID-19 vaccination were collected for the assay. One subject was vaccinated with Sputnic V and another subject was vaccinated with Covishield. Neutralization assay was carried using the same protocol described in the invention at serial two-fold dilution starting from 1:10 till it reaches to baseline. 4PL graph was plotted log of dilution vs neutralization. IC50 was determined. Result is shown in figure 8. 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 (IC5078 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. Example 10: Community Survey of Post COVID-19 vaccination neutralizing antibody status. Serum samples of 53 health staffs who already completed two doses of COVID-19 vaccine within 7 months were collected for the assay. Neutralization assay was carried using the same protocol described in the invention at 1:20 dilution. Cut-off was set at 20% (mean+3SD of %NT of 20 pre COVID-19 samples). Out of 53 samples only 40 showed detectable neutralizing antibody against SARS-CoV-2 at a serum dilution 1:20 while the neutralizing antibody was not detectable in 13 subjects (Table 6). This application will help low budget survey on different target population for determining level neutralizing antibody. The data could be used for taking any preventive policy making decision including decision on need for booster dose. This low cost assay could be used by employers to take policy for booster dosing for the employees who will be assigned to work in front line, having high risk of exposure to the SARS-CoV-2 infection. References 1. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Müller MA, Drosten C, Pöhlmann S. SARS- CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020 Apr 16;181(2):271-280.e8. doi: 10.1016 / j.cell.2020.02.052. Epub 2020 Mar 5. 2. Tan, C.W., Chia, W.N., Qin, X. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction. Nat Biotechnol 38, 1073–1078 (2020). https: / / doi.org / 10.1038 / s41587-020-0631-z. 3. Abe KT, Li Z, Samson R, Samavarchi-Tehrani P, Valcourt EJ, Wood H, Budylowski P, Dupuis AP 2nd, Girardin RC, Rathod B, Wang JH, Barrios-Rodiles M, Colwill K, McGeer AJ, Mubareka S, Gommerman JL, Durocher Y, Ostrowski M, McDonough KA, Drebot MA, Drews SJ, Rini JM, Gingras AC. A simple protein-based surrogate neutralization assay for SARS-CoV-2. JCI Insight. 2020 Oct 2;5(19):e142362. doi: 10.1172 / jci.insight.142362. PMID: 32870820; PMCID: PMC7566699. 4. Chan KK, Dorosky D, Sharma P, Abbasi SA, Dye JM, Kranz DM, Herbert AS, Procko E. Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2. Science.2020 Sep 4;369(6508):1261-1265. 5. pcDNA3-SARS-CoV-2-S-RBD-sfGFP Sequences from adgene: https: / / www.addgene.org / 141184 / sequences / 6. pCEP4-myc-ACE2: https: / / www.addgene.org / 141185 / sequences / 7. Burkard C, Verheije MH, Wicht O, van Kasteren SI, van Kuppeveld FJ, Haagmans BL, Pelkmans L, Rottier PJ, Bosch BJ, de Haan CA. 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. 8. Larkin JM, Brown MS, Goldstein JL, Anderson RG. Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell. 1983 May;33(1):273-85. doi: 10.1016 / 0092-8674(83)90356-2. PMID: 6147196. 9. Wang, H., Yang, P., Liu, K. et al. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 18, 290–301 (2008). https: / / doi.org / 10.1038 / cr.2008.15 10. Nicola AV, McEvoy AM, Straus SE. Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells. J Virol. 2003;77(9):5324- 5332. doi:10.1128 / jvi.77.9.5324-5332.2003. 11. Zhang, N., Fan, Y., Li, C. et al. Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts. Appl Microbiol Biotechnol 102, 4183–4191 (2018). https: / / doi.org / 10.1007 / s00253-018-8906-8 12. Malladi SK, Singh R, Pandey S, Gayathri S, Kanjo K, Ahmed S, Khan MS, Kalita P, Girish N, Upadhyaya A, Reddy P, Pramanick I, Bhasin M, Mani S, Bhattacharyya S, Joseph J, Thankamani K, Raj VS, Dutta S, Singh R, Nadig G, Varadarajan R. Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment. J Biol Chem. 2021 Jan- Jun;296:100025. doi: 10.1074 / jbc.RA120.016284.

Claims

CLAIMS:

1. A kit for detecting neutralizing antibody in vitro against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in a sample comprising: (i) a frozen media comprising secreted SARS-CoV-2 spike protein receptor binding domain (RBD) for binding to human ACE2 protein tagged with a detection entity; (ii) a ready to use frozen transfected cells expressing human ACE2 protein; (iii) a reaction buffer; (iv) a DNA binding florescent dye; (v) a Positive control; and (vi) a Negative control 2. The kit as claimed in claim 1, wherein the receptor binding domain (RBD) is represented by amino acid sequence 333 to 529 of Sequence ID No.1 and the human ACE2 protein is represented by amino acid sequence 19 to 805 of Sequence ID No.

2.

3. The kit as claimed in claim 1, wherein the frozen media comprising SARS-CoV-2 spike protein receptor binding domain (RBD) is a supernatant from culturing of transfected cells expressing SARS-CoV-2 spike protein receptor binding domain (RBD) in an animal cell culture medium and diluted in the reaction buffer, followed by freezing at-20°C to - 80°C.

4. The kit as claimed in claim 1, wherein the animal cell culture medium is selected from Minimum Essential Medium comprising basal Minimal Essential Medium (MEM) mixed with 10% fetal bovine serum (FBS).

5. The kit as claimed in claim 1, wherein the detection entity is selected from super folded green fluorescent protein (sGFP), red fluorescent protein (RFP), monomeric near- infrared red fluorescent protein 670 (miRFP 670), near–infrared fluorescent protein 702 (iRFP702).

6. The kit as claimed in claim 1, wherein the cell lines are selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, COS-7.

7. The kit as claimed in claim 1, wherein the reaction buffer comprises Sodium Chloride and Ammonium Chloride at a concentration of 0.140M and 0.025 M, respectively at pH7.

4.

8. The kit as claimed in claim 1, wherein the DNA binding florescent dye is selected from Propidium Iodide, 7-aminoactinomycin D and SYBR Green.

9. The kit as claimed in claim 1, wherein the kit comprises Propidium Iodide stock solution at a concentration of 0.3-0.7 mg / ml.

10. The kit as claimed in claim 1, wherein the cells expressing human ACE2 protein are resuspended in the reaction buffer with 10% DMSO and frozen at -80oC.

11. The kit as claimed in claim 1, wherein the positive control comprises cocktail of serum samples from post vaccinated individual having neutralizing antibody against SARS- CoV-2 and negative control comprises cocktail of serum without neutralizing antibody against SARS-CoV-2.

12. The kit as claimed in claim 1, wherein the sample is selected from the group consisting of serum, blood, cerebrospinal fluid, sweat, semen, saliva, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, otitis fluid, gastric fluid, and breast milk.

13. A method for preparing frozen cell line expressing human ACE2 protein as claimed in claim 1, said method comprising: (i) culturing of cells in an animal cell culture media with 10 % fetal bovine serum to obtain cultured cells; (ii) transfecting a plasmid comprising a constitutive expression cassette encoding ACE2 protein into the cultured cells which does not express ACE2 and propagating in presence of selection marker to obtain stable transfected cells; (iii)resuspending the stable transfected cells expressing ACE2 protein or cells that naturally express ACE2 protein, in a reaction buffer in a cell culture flask, followed by dislodging the cells from the flask and passing through a cell strainer to making single cell suspension and determining cell concentration; (iv) storing the cells in a freezing buffer consisting of reaction buffer, and 10% dimethyl sulfoxide at -80°C to -150°C as a concentrated stock, and wherein the reaction buffer comprises Sodium Chloride and Ammonium Chloride at a concentration of 0.140M and 0.025 M, respectively at pH7.

4.

14. The method as claimed in claim 13, wherein the cells that are transfected are selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, COS-7, A549 and the cells naturally expressing ACE2 are selected from adherent cells Vero E6, Vero 81, MA104, LLC-MK2, BGM, and Caco-2.

15. The method as claimed in claim 13, wherein the plasmid is selected from a pCEP4-myc- ACE2 plasmid or hACE2 or pcDNA3.1-hACE2 or any other DNA vector having human ACE2 constitutive expression cassette comprising a mammalian selection marker.

16. A method for preparing a frozen media comprising secreted SARS-CoV-2 spike protein receptor binding domain (RBD) of a human ACE2 protein as claimed in claim 1, said method comprising: (i) culturing of cells in a Minimum Essential Medium (MEM) with 10 % fetal bovine serum to obtain cultured cells; (ii) transfecting a plasmid comprising an expression cassette encoding SARS-CoV-2 receptor binding domain, wherein the C terminal of the receptor binding domain is tagged with super-folded green fluorescent protein (RBD-sGFP) and N terminal is tagged with signal peptide from influenza HA into the cultured cells and propagating the transfected cells in presence of a selection marker to obtain stable transfected cells; (iii)incubating the stable transfected cells in minimum essential media for 3-5 days at 37℃ in CO2incubator; (iv) centrifuging the media following incubation to obtain a clear supernatant; (v) diluting the supernatant in 1X reaction buffer comprising Sodium Chloride and Ammonium Chloride at a concentration of 0.140M and 0.025 M, respectively at pH7.4, to obtain a final media as claimed in claim 3 and claim 4, such a way that the concentration of the supernatant will be 4-5 times than the final concentration to be used and (vi) freezing the final media at -20°C to -80°C.

17. The method as claimed in claim 16, wherein the cells are from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, COS-7.

18. The method as claimed in claim 17, wherein the plasmid is a pcDNA3-SARS-CoV-2-S- RBD-sfGFP plasmid comprising a mammalian selection marker 19. An in vitro assay for analysing a sample for presence of neutralizing antibody against SARS-CoV-2 comprising: (i) incubating a sample with a media comprising secreted SARS-CoV-2 spike protein receptor binding domain (RBD) of a human ACE2 protein, tagged with a detection entity followed dilution of the frozen media by 1 X reaction buffer, at room temperature for 30-90 minute followed by incubation on ice for 10-30 minutes;(ii) separately, thawing frozen transfected cells expressing human ACE2 protein, followed by dilution in a cell dilution buffer comprising 0.3-0.7 µg / ml of DNA binding florescent dye to obtain thawed cells; (iii)contacting the thawed cells of step (ii) with the sample incubated with the media of step (i) for 60-90 min on ice bath to form a reaction mixture; (iv) diluting the reaction mixture by adding the reaction mixture into an ice cold reaction buffer, and (v) determining the intensity of green fluorescence in cells by flow cytometry. wherein a fluorescence intensity greater than 20%-30% indicates absence of neutralization antibody and a fluorescence intensity less than 20%-30% indicates the presence of neutralization antibody.

20. The method as claimed in claim 19, wherein the sample is selected from the group consisting of human serum, blood, plasma, cerebrospinal fluid, sweat, semen, saliva, tears, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, nasal fluid, otitis fluid, gastric fluid, and breast milk.

21. The method as claimed in claim 19, wherein the media is a minimum essential media, wherein the detection entity is selected from green fluorescent protein, red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), near–infrared fluorescent protein 702 (iRFP702); wherein the DNA binding florescent dye is selected from Propidium Iodide, 7- aminoactinomycin D and SYBR Green, and wherein the cell dilution buffer comprises 1X reaction buffer having sodium chloride and ammonium chloride at a concentration of 0.140M and 0.025 M, respectively at pH7.4 with PI at a concentration of 0.3-0.7ug / ml.

22. The method as claimed in claim 19, wherein the detection entity detects the interaction between the human ACE2 protein and SARS-CoV-2 spike protein receptor binding domain, and wherein the % of green fluorescence is proportional to the percentage binding of GFP tagged RBD with cell surface ACE2 receptor.

Images