Kit and assay for analyzing presence of neutralizing antibodies against SARS-CoV-2

Abstract

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

Description

Technical Field

[0001] This disclosure relates to the in vitro identification of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Specifically, this disclosure relates to a kit and an in vitro assay for analyzing the presence of neutralizing antibodies / inhibitors that can block or inactivate the receptor-binding domain of the spike protein of SARS-CoV-2. Background Technology

[0002] The outbreak of novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has created a global health crisis. Controlling the spread of the virus and developing effective treatments and vaccines are crucial to combating the epidemic. In the context of COVID-19, the protective immune response against the SARS-CoV-2 pathogen involves the production of antibodies against the spike protein. However, only a subset of functional antibodies targeting specific sites at the receptor-binding domain (RBD) of the spike protein play a key role in neutralizing the virus's binding to its receptor ACE2 and blocking viral entry into human cells, thereby preventing infection (1). Neutralizing antibodies in patients or vaccinated individuals typically monitor disease outcomes and determine vaccine efficacy, and are crucial for evaluating herd immunity. Numerous methods have been developed to identify neutralizing antibodies against SARS-CoV-2, such as micro-neutralization assays, SARS-CoV-2 pseudovirus assays, enzyme-linked immunosorbent assays (ELISA), and rapid lateral chromatography assays. Given the variety of serological assays used to quantify neutralizing antibody titers, comparing results from different assays remains a challenge. Furthermore, in 2020, the World Health Organization introduced the first international standards as universal units for defining neutralizing antibody titers and antibody responses against SARS-CoV-2. These standards can be used to compare results from different assays and laboratories.

[0003] However, these assays have several limitations in clinical laboratories for direct use as neutralization assays in ready-to-use formats and at an affordable cost. The Plaque Reduction Neutralization Test (PRNT) is considered the gold standard for measuring neutralizing antibodies against SARS-CoV-2, but it is time-consuming and limited to 6-well or 24-well plate formats. Therefore, it is unsuitable for emergency responses and large-scale serological studies, and safety concerns exist because it requires exposure to live virus in a biosafety level 3 laboratory. ELISA-based serological assays are protein-based alternatives to SARS-CoV-2 neutralization assays (2,3), and are commercially available. While based on ELISA technology, the kits are very expensive, limiting their widespread clinical and research use. Many components of the kits need to be purchased from third parties, increasing the product cost. Pseudovirus-based SARS-CoV-2 neutralization assays are time-consuming, requiring at least 2 days to obtain results for candidate samples even with pre-prepared pseudovirus stock solutions. Therefore, using alternative assays to determine neutralizing antibodies is a cell-based alternative that closely approximates the gold standard, but most cell-based assays have high coefficients of performance (CV) due to biological variables, making them unsuitable for diagnostic assays. Cell-based RBD-ACE2 binding assays, based on the interaction between ACE2 and its receptor-binding domain (RBD), involve transiently transfecting suspension Expi293 cells with the plasmids pcDNA3-SARS-CoV-2-S-RBD-sfGFP (4,5) and pCEP4-myc-ACE2 (4,6). This results in increased assay costs, reduced reproducibility, and inconvenience for packaging as a kit for diagnostic testing. In suspension cells, cells that die after transfection or due to culture proliferation are difficult to remove. Furthermore, maintaining cell lines is impractical in routine clinical laboratories. For each new batch of ACE2-transfected cells, cell concentration must be determined, and all assay parameters must be read just before the assay begins, based on transfection efficiency. This makes these assays unsuitable as affordable and ideal diagnostic assay kits to be performed by undertrained healthcare personnel in routine clinical laboratories.

[0004] Therefore, considering all the points above and closely monitoring the current situation worldwide, antibody neutralization assays appear to need updating to meet current requirements. Consequently, there is a need for robust, stable, and simple assays in any laboratory to analyze neutralizing antibodies or inhibitors without further standardization.

[0005] Purpose of the invention Therefore, the purpose of this disclosure is to provide a kit and assay for in vitro analysis of the presence of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).

[0006] The purpose of this disclosure is to provide a low-cost, cell-based, virus-free, ready-to-use kit and assay for the semi-quantitative analysis of neutralizing / blocking functional antibodies that can neutralize / block the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the corresponding human receptor ACE2.

[0007] Another object of this disclosure is to provide a kit for identifying neutralizing antibodies that is ready to use and does not require complex cell culture equipment.

[0008] Another objective of this disclosure is to provide an in vitro assay for identifying neutralizing antibodies against SARS-CoV-2 that is more reproducible and comparable and can be performed in a short time.

[0009] These and other objects and advantages of this disclosure will become apparent from the following description. Summary of the Invention

[0010] Therefore, this disclosure provides a kit for in vitro identification of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in samples, the kit comprising: a frozen culture medium containing a secreted SARS-CoV-2 spike protein receptor-binding domain (RBD) for binding to human ACE2 protein labeled with the test entity; ready-to-use frozen transfected cells expressing human ACE2 protein; a reaction buffer; a DNA fluorescent binding dye; a positive control; and a negative control.

[0011] In one aspect of this disclosure, a method for preparing a frozen cell line expressing human ACE2 protein is provided.

[0012] In another aspect of this disclosure, a method for preparing a frozen culture medium comprising the secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) of the human ACE2 protein is provided.

[0013] In another aspect of this disclosure, an in vitro assay is provided for analyzing the presence of neutralizing antibodies against SARS-CoV-2 in a sample. Attached Figure Description

[0014] Figure 1 Microscopic views of the neutralization assay based on alternative cells (CENA) of this disclosure are provided.

[0015] Figure 2 The effects of different reaction buffers on the binding stability of receptor-binding domain-green fluorescent protein (RBD-GFP) over time were depicted.

[0016] Figure 3 The figure illustrates the effects of DMSO and cell freezing on live cell propidium iodide staining and the binding of RBD-GFP to HEK293 cells expressing ACE2.

[0017] Figure 4 An analysis was provided on the neutralization of the RBD of the SARS-CoV-2 spike protein using flow cytometry via the CENA method.

[0018] Figure 5 The figure shows the linear correlation between CENA 50 and the conventional CPE-based SARS-CoV-2 live virus neutralization assay (cVNA 100).

[0019] Figure 6 The correlation between CENA and the commercially available cPass SARS-CoV-2 neutralizing antibody assay kit is shown.

[0020] Figure 7 This is a dot plot showing the comparative performance of CENA and the cPass (Genescript) kit for detecting neutralizing antibodies in serum samples.

[0021] Figure 8 The neutralizing antibody titers against the RBD spike protein of SARS-CoV-2 were demonstrated in serum after vaccination with a vaccine against SARS-CoV-2 by assay (CENA) as disclosed herein.

[0022] Figure 9 The diagram illustrates components 1 and 2 of the kit manufactured on-site by the manufacturer. Figure 10 The procedure for assaying by allowing the different components of the kit to interact at the end-user site is described. Detailed Implementation

[0023] In this document, the word "exemplary" is used to mean "serving as an example, instance, or illustration." Any implementation or embodiment of this subject matter described as "exemplary" herein is not necessarily to be construed as preferred or advantageous relative to other implementations.

[0024] While this disclosure is susceptible to various modifications and alternatives, its specific implementation has been illustrated by way of example in the accompanying drawings and will be described in detail below. However, it should be understood that this disclosure is not intended to limit it to the disclosed forms; rather, it will cover all modifications, equivalents, and alternatives falling within the scope of this disclosure.

[0025] The terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations. Unless the context otherwise requires, the singular forms “a,” “an,” and “described” as used herein also include the plural forms.

[0026] It should also be understood that, when used herein, the terms “comprising,” “including,” “comprises,” and / or “includes” specify the presence of the said feature, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof.

[0027] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which the exemplary embodiments pertain. It should also be understood that terms, such as those defined in common dictionaries, should be interpreted as having meanings consistent with their meanings in the context of the relevant field and should not be interpreted in an idealized or overly formal sense, unless expressly defined herein.

[0028] Neutralizing antibodies against SARS-CoV-2 are functional antibodies that directly block the binding of the virus to its ACE2 receptor by blocking the interacting epitope of the receptor-binding domain (RBD) of the viral spike protein. The presence of these antibodies after natural infection or vaccination can prevent reinfection. Therefore, the detection of these antibodies in biological fluids via neutralizing antibody assays is important for evaluating vaccine efficacy. This neutralizing antibody assay differs from routine serological antibody assays used to detect binding antibodies in the general population.

[0029] Therefore, this disclosure provides a cell-based in vitro assay for analyzing the presence of neutralizing antibodies against SARS-CoV-2 in a sample using flow cytometry. Unlike other multi-step cell-based assays that use pseudoviruses, this assay can be completed in two steps within 3 hours and does not require any washing steps to eliminate background as is required in other serological assays. Furthermore, the assay can be packaged into a stand-alone kit, and the kit eliminates the need for any cell culture prior to the assay.

[0030] In one embodiment, this disclosure provides a kit for in vitro detection of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in a sample, the kit comprising: (i) A frozen culture medium containing a secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) for binding to human ACE2 protein labeled with the test entity; (ii) Ready-to-use frozen transfected cells expressing human ACE2 protein; (iii) Reaction buffer; (iv) DNA binds to fluorescent dyes; (v) Positive control; and (vi) Negative control.

[0031] In one embodiment, the sample is a biological sample 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 media fluid, gastric juice, and breast milk. Preferably, the sample is human serum or plasma. More preferably, the serum is from individuals previously exposed to SARS-CoV-2 or vaccinated against COVID-19, or both.

[0032] In one 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 19-805 of sequence ID No. 2 (GenBank ID: NM_021804.1; amino acid sequence 19-805).

[0033] In one embodiment, the cells are adherent cell lines that do not express the ACE2 protein; however, after stable transfection with an ACE2 expression cassette (chromosomal integration), the cells express the ACE2 protein on their cell surface. Preferred cell lines are adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, COS-7, and A549 cells that do not express the ACE2 protein. Other naturally ACE2-expressing cell lines (such as Vero E6, VERO 81, VERO SLAM, MA104, LLC-MK2, BGM, and Caco-2) can also be used directly without stable transfection with an ACE2 constitutive expression cassette. More preferably, the cell line is adherent HEK293 cells. The advantage of using cell lines that do not naturally express ACE2 is that any background arising from non-specific interactions of the RBD detection element on the cell surface can be detected by neutralization reactions with ACE2-untransfected cells, and this background can be eliminated by appropriate stringent conditions or cut-off values.

[0034] This assay used adherent HEK293 cell line instead of suspension cells Expi293 as used in the previously published report (4). Any debris or dead cells could be easily handled and removed by washing with PBS and / or changing the medium. The cell line can also be maintained with low-cost, minimally necessitated medium and is readily transfected efficiently with common transfection reagents.

[0035] First, cells were cultured in animal cell culture medium containing fetal bovine serum (Gibco, USA). After culture, a plasmid containing a constitutive expression cassette encoding the ACE2 protein was transfected into the cultured cells using standard transfection reagents (e.g., lipofectamine 2000, lipofectamine 3000, Effectene transfection reagent, etc.), and the cells were proliferated in the presence of a selection marker to obtain stable transfected cells. The plasmid was selected from pCEP4-myc-ACE2 plasmid, hACE2, pcDNA3.1-hACE2, or any other DNA vector containing a human ACE2 constitutive expression cassette containing a mammalian selection marker.

[0036] Stable transfected cells expressing ACE2 protein were resuspended in cell culture flasks in reaction buffer containing 0.140 M and 0.025 M sodium chloride and ammonium chloride, respectively, at pH 7.4. Cells were then scraped from the flasks and passed through a cell filter to prepare a single-cell suspension. Cell concentration was further determined using a TECAN SPARK multi-well microplate reader with a dual-chamber chip for automated cell counting. Alternatively, any other reliable cell counting method can be used, such as flow cytometry, the TC20 automated cell counter, etc.

[0037] Because cells transfected with the ACE2 gene expression cassette express ACE2 on their cell surface, and ACE2 requires intact ACE2 cells to interact with the spike protein, cells are scraped off the plate instead of the trypsin digestion method typically used to remove adherent cells. Single-cell suspensions are prepared by pipetting the cell suspension onto the culture flask wall and then removing any cell clumps through a cell filter. This transfected cell line adheres very loosely and forms very fragile cell clumps. Therefore, single-cell suspensions can be easily prepared by gently pipetting a few times against the flask wall.

[0038] It has been previously established that SARS-CoV-2 enters cells via pH-dependent and receptor-dependent endocytosis (7). This is due to the presence of K+ in standard Dulbecco phosphate-buffered saline (DPBS). +It facilitates endocytosis of the receptor and ligand complex, therefore, for final cell washing and resuspension, replace the regular PBS buffer. Instead, wash and resuspend cells with 1× reaction buffer (pH 7.4) containing 25 mM ammonium chloride and 140 mM NaCl. Furthermore, ammonium chloride inhibits endocytosis by preventing endosome acidification (8-10).

[0039] To facilitate direct measurement by users and to enable measurement in any laboratory without cell culture facilities with minimal steps, cells were processed in a ready-to-use frozen format. Prior to measurement, cells were aliquoted and frozen in a freezing buffer containing reaction buffer and 10% dimethyl sulfoxide (DMSO) at -80°C to -150°C as a concentrated stock solution for the ready-to-use format. The ammonium buffer and freezing inhibit endocytosis of the cell surface receptor-ligand complex, thereby stabilizing the RBD detection entity ACE2 complex on the cell surface. Since ready-to-use frozen cells will be used in the measurement, metabolic activity for endocytosis will be further reduced. This will result in a more stable cell surface interaction between RBD-GFP and ACE2, better signal stability, and preservation in ice for at least 3 hours. Furthermore, freezing cells in a DMSO-containing buffer creates pores on the cell surface, allowing for staining of the nucleus with a DNA-binding dye (11) for downstream flow cytometry. Furthermore, assays using frozen cell systems can be packaged into kit formats for cell-based assays, which would be impossible if fresh cultured cells were required for each experimental procedure.

[0040] Frozen cells were diluted 75-fold with cell resuspension buffer containing reaction buffer and a DNA-binding fluorescent dye. The DNA-binding dye used to stain the cell nuclei will be used to identify the total intact cell population during downstream flow cytometry. The reaction buffer contained sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, at pH 7.4.

[0041] In one embodiment, the DNA-binding fluorescent dye is selected from propidium iodide, 7-aminoactinomycin D, and SYBR Green. Preferably, a propidium iodide stock solution with a concentration of 0.3-0.7 mg / ml is used.

[0042] Propidium iodide (PI), used as a nuclear staining agent, is used to stain dead cells and generally cannot penetrate the membranes of living cells. To prepare a cell resuspension buffer, 0.3–0.7 µg / ml, more specifically 0.5 µg / ml, is added to 1× reaction buffer for cell resuspension and nuclear staining. Typically, PI is used to stain the nuclei of dead cells. PI cannot penetrate the cell membranes of fresh, living cells. When cells are frozen in a freezing buffer containing 10% DMSO, pores are formed in the cell membrane, facilitating cell penetration and allowing PI to penetrate into the cell for nuclear staining. Therefore, it helps to gate only nucleated cells in flow cytometry, removing debris or any other non-cellular components or precipitates that may be generated from serum. While some important nuclear staining agents (such as Hoechst 33342) can be used directly to stain fresh cells without the need for DMSO in the buffer, this requires specialized flow cytometry using a UV-range laser. Most laboratories do not use flow cytometers equipped with UV lasers. PIs can be measured using a standard, small-scale, commercially available flow cytometer in a typical clinical laboratory.

[0043] In another embodiment of this disclosure, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein for binding to human ACE2 protein is labeled with a detection entity. The detection entity is selected from superfolded green fluorescent protein (sfGFP), red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), and near-infrared fluorescent protein 702 (iRFP702). Preferably, as described in plasmid pcDNA3-SARS-CoV-2-S-RBD-sfGFP (4), superfolded green fluorescent protein (sfGFP) is used.

[0044] In another embodiment of this disclosure, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein containing the detection entity is labeled with an N-terminal cleavable signal sequence for extracellular secretion of the recombinant fusion protein. The signal peptide is selected from the cleavable signal peptide of influenza hemagglutinin (Sequence ID No. 3) and the signal peptide of a mammalian secreted protein (resistin) (Sequence ID No. 4). Preferably, the signal peptide (4) of influenza hemagglutinin is used as described in plasmid pcDNA3-SARS-CoV-2-S-RBD-sfGFP.

[0045] The RBD COVID-19 spike protein (RBD-sfGFP) labeled with green fluorescent protein (sfGFP) was expressed and secreted in culture medium by another cell line stably transfected with an expression cassette of (RBD-sfGFP). Stable transfection makes the assay more reproducible, cost-effective, and robust. Unlike previous assay designs, no transfection is required before each assay.

[0046] Cells were first cultured in complete minimum essential medium (MEM) containing 10% fetal bovine serum. The cells did not express the RBD domain; however, after transfection, they expressed the RBD domain of the SARS-CoV-2 spike protein, as detected by physical markers. These cells were selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, and COS-7. For this purpose, cell lines naturally expressing ACE2 were avoided to prevent self-binding of the RBD fusion protein to the cell surface after secretion. More preferably, adherent HEK293 cells were used.

[0047] After culturing, plasmids containing constitutive expression cassettes encoding the RBD domain of the SARS-CoV-2 spike protein were transfected into cultured cells using standard transfection reagents (e.g., lipofectamine 2000, lipofectamine 3000, Effectene transfection reagent, etc.) and allowed to proliferate in the presence of selection markers to obtain stable transfected cells. The C-terminus of the receptor-binding domain was labeled with hyperfolded green fluorescent protein (RBD sfGFP), and the N-terminus was labeled with a signal peptide from influenza virus hemagglutinin. This N-terminal signal sequence labeling facilitates the secretion of RBD sfGFP into the extracellular culture medium, while the signal sequence is cleaved during the process. The encoding nucleotide sequences of the N-terminal signal peptide, RBD, and sfGFP were sequentially inserted into the plasmid in a triplet coding frame of the corresponding coding sequences to express a 51.2 kDa fusion protein. The plasmid was pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmid containing mammalian selection markers (4).

[0048] Stable transfected cells were further incubated in minimum essential medium at 37°C in a CO2 incubator for 3–5 days. The medium was then centrifuged at 10,000 RPM for 10 minutes at 4°C to obtain a clear supernatant free of dead cells / debris. This supernatant was then diluted with 1× reaction buffer containing 0.140 M and 0.025 M sodium chloride and ammonium chloride, pH 7.4, to a final concentration of 4–5× (4–5 times the final concentration to be used). The medium was then frozen at -20°C to -80°C.

[0049] RBD sfGFP is secreted into a culture medium by transfected cells, preferably in an animal cell culture medium frozen at -20°C to -80°C. More preferably, a minimum essential medium comprising a basal minimum essential medium mixed with 10% fetal bovine serum can be used as the animal cell culture medium.

[0050] In another embodiment of this disclosure, an in vitro assay is provided for analyzing the presence of neutralizing antibodies against SARS-CoV-2 in a sample. After diluting the frozen culture medium with 1× reaction buffer, the sample is first incubated at room temperature for 30-90 minutes with a medium containing the receptor-binding domain (RBD) of the secreted ARS-CoV-2 spike protein labeled with the human ACE2 protein of the detection entity, followed by incubation on ice for 10-30 minutes.

[0051] Frozen transfected cells expressing human ACE2 protein were then thawed and diluted in cell resuspension buffer containing 0.3–0.7 µg / ml DNA-binding dye to obtain thawed cells. The thawed cells were then contacted with the culture-incubated sample in an ice bath for 60–90 minutes to form a reaction mixture. The reaction mixture was diluted by adding it to ice-cold reaction buffer, and the intensity of green fluorescence in the cells was determined. If no neutralizing antibody or inhibitor of interaction was present in the test serum, the cells would be stained green on their surface due to the binding of the GFP-labeled RBD to its ACE-2 receptor. The percentage of stained cells was evaluated by flow cytometry. Any blockage of the spike protein RBD in the presence of neutralizing antibody / inhibitor would result in / reduce RBD binding to the cell surface, resulting in a low proportion of green-positive cells. The inhibition percentage is expressed as the proportion of RBD binding in the absence of any serum.

[0052] A fluorescence intensity greater than the cutoff value indicates the absence of neutralizing antibodies, while a fluorescence intensity less than the cutoff value indicates the presence of neutralizing antibodies. Preferably, the cutoff value is in the range of 20-30%, and depends on the negative control.

[0053] The neutralization percentage is determined by the following formula:

[0054] The positive control of the kit comprises a mixture of serum samples from individuals (healthy volunteers) who have been vaccinated (with Covishield / Sputnik V / Covaxin or other anti-SARS-CoV-2 vaccines) and have neutralizing antibodies against SARS-CoV-2. The presence of neutralizing antibodies in the final serum mixture can be determined by any standard SARS-CoV-2 live virus neutralization assay (e.g., PRNT, cVNA, any PRNT-validated alternative neutralization test), and the antibody titer should be at a limiting serum dilution, preferably 1:640 or higher. The negative control comprises a mixture of serum samples (from healthy volunteers) that do not contain neutralizing antibodies against SARS-CoV-2, determined by any standard SARS-CoV-2 live virus neutralization assay (e.g., PRNT / cVNA, any PRNT-validated alternative neutralization test).

[0055] Because this disclosure uses stably transfected ACE2-expressing cells, which are capable of consistently expressing ACE2 at the same high percentage on their surface, a consistent RBD-GFP binding percentage is achieved when assays are performed using different batches of ACE2-expressing cells. Therefore, the step of staining ACE2 with an antibody can be omitted in this protocol. This also reduces potential steric hindrance to antibody binding to the ACE2 receptor and lowers assay costs.

[0056] This strategy of not using staining antibodies in the assay provides further robustness and reduces costs.

[0057] The assay avoids any fluorescently labeled antibodies used for staining ACE2 and optimizes the concentrations of PI and RBD-GFP to provide negligible background and eliminates the need for a washing step, thus making the assay a more robust cell-based assay. In this way, the assay achieves 100% neutralization using SARS-CoV-2 immune serum.

[0058] Unlike previous approaches, this disclosure allows components to be packaged into stand-alone, ready-to-use kits without requiring end-users to perform cell culture.

[0059] Therefore, this disclosure provides an economical and low-cost kit and assay for analyzing the presence of neutralizing antibodies. Since the recombinant genetic material, containing the expression cassette of the ACE2 gene and S-RBD-GFP (the SARS-CoV-2 spike protein receptor-binding domain labeled with GFP), has been integrated into the chromosome of the cell line, expensive transfection reagents are not required during each batch of kit preparation. For large-scale production, simple propagation of stably transfected cell lines using standard cell culture media is sufficient. This reduces the manufacturing cost of the assay and minimizes biological variability. Unlike the manufacture of ELISA-based assay kits, which require a series of procedures such as purification of the recombinant spike protein RBD and ACE2, and conjugation of the protein to the detection system, no special reagents are required for signal detection in this assay. In this assay, the GFP signal, as an indicator of RBD binding to ACE2, can be directly detected by flow cytometry without any additional reagents. Therefore, the manufacturing cost of the reagents only includes very inexpensive cell culture media, buffers, cell culture flasks, etc. Furthermore, optimized reaction buffers and protocols have been developed to reduce assay variability that occurs in any other cell-based assay system.

[0060] This disclosure will now be described by way of illustrative embodiments. The embodiments given are non-limiting in nature and are merely illustrative of the uses, methods, and products claimed in this invention, and the practice of the invention itself is not limited to or restricted by the described embodiments.

[0061] Example Sources of chemicals and biological materials used in this invention: 1. HEK 293 cells: American Type Culture Collection (ATCC).

[0062] 2. Propidine iodide: Merck & Co., Ltd. 3. Plasmids used for transfection: obtained via addgene ( https: / / www.addgene.org / 141184 / sequences / Alternatively, any other similar plasmid / viral vector with the desired expression cassette and mammalian selection markers can be used.

[0063] 4. Minimum Required Culture Medium: Sigma-Aldrich (USA) 5. DMSO: Sigma Corporation, USA 6. Ammonium chloride: Sigma-Aldrich, USA 7. Sodium chloride: Sigma-Aldrich, USA Example 1: Preparation of RBD-GFP-containing culture medium Figure 9The preparation steps of the components are illustrated in the diagram. Adherent cell lines (e.g., HEK 293 cells grown in MEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2) were stably transfected (integrated into the genome of the cell line) with a plasmid containing an expression cassette of SARS-CoV-2RBD-GFP. The plasmid has a signal sequence for its extracellular release and contains a mammalian selection marker. The plasmid (pcDNA3-SARS-CoV-2-S-RBD-sfGFP) has been described in reference (3) and is available from addgene (https: / / www.addgene.org / 141184 / sequences / ). Any other similar plasmid / viral vector containing an RBD-GFP expression cassette and a mammalian selection marker is also applicable. In the plasmid, the RBD of SARS-CoV-2 (strain ID 2697049) can be replaced by any variant of the RBD from any newer SARS-CoV-2 strain (in the protein coding frame of the upstream N-terminal signal sequence and the downstream C-terminal sfGFP) to detect neutralizing antibodies against the newer SARS-CoV-2 strain. The kit can be updated based on the emergence of newer strains, while the rest of the protocol remains unchanged.

[0064] To ensure stable transfection, the RBD-GFP expression plasmid was first transfected with lipofectamin 3000, and then the medium was replaced with fresh medium containing the selective antibiotic Genticin 400 µg / ml (G418) after 48 hours. After incubation in the selective medium for two weeks, single green fluorescent colonies were identified, isolated, and replate into 96-well plates. After regrowth, wells with the densest concentration of green fluorescent cells were screened under a fluorescence microscope. Further screening was performed by examining the supernatant from wells containing green fluorescent cells, and fluorescence was measured using an appropriate GFP filter (e.g., excitation: 488 nm / emission: 530 nm). The colonies with the best green fluorescence in the supernatant were propagated and amplified to prepare the optimized GFP-RBD-containing medium.

[0065] Specifically, the cell line was grown to 70% confluence. Towards 25 cm... 2 Add 2 ml of fresh MEM culture medium to the flask, and pour into a 75 cm column. 2 Add 6 ml of culture medium to the flask. After incubation for 4 days, collect the culture medium.

[0066] The culture medium was then centrifuged, the supernatant was collected, and diluted with 1× reaction buffer to adjust the fluorescence to 60-25 U. Within this fluorescence level range, the assay will be more linear, and the detection limit will be improved in the lower range.

[0067] The culture medium can be further calibrated using positive control (PC) reference serum (post-vaccination / convalescent phase), and the neutralizing antibody titer has been pre-determined using gold standard assay (PRNT). The dilution of the culture medium can be adjusted so that the PC titer obtained through the new test closely matches the titer determined by the gold standard PRNT, while maintaining the percentage of RBD-GFP positive cells in the untreated control above 80%.

[0068] The diluted culture medium was then dispensed into 2 ml test tubes and kept at -20°C.

[0069] To calibrate the new batch of RBD-GFP-containing culture medium, the activity of RBD-GFP was calibrated by diluting the medium, adjusting the percentage of GFP-positive cells, and comparing the average fluorescence intensity of single cells from the previous standardized batch.

[0070] Example 2: Preparation of ACE2 receptor cell lines exist Figure 9 The preparation steps of the components are illustrated in the diagram. Adherent cell lines that do not express ACE2 (e.g., HEK 293 cells grown in MEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2) were stably transfected (integrated into the genome of the cell line) with an ACE2 expression cassette / plasmid containing a mammalian selection marker. The plasmid (pCEP4-myc-ACE2) has been described in a previous report (3) and is available via AddGene (https: / / www.addgene.org / 141185 / sequences / ). Any other similar plasmid / viral vector containing an ACE2 expression cassette and a mammalian selection marker is also applicable. For stable transfection, the ACE2 expression plasmid was first transfected with lipofectamin 3000, and then the medium was replaced with fresh medium containing the selective antibiotic lipofectamin 50 µg / ml after 48 hours. After two weeks, single colonies were isolated and replate into 96-well plates. Regeneration functional screening was performed by observing RBD-GFP binding activity under a fluorescence microscope. Colonies with optimal RBD-GFP binding were then propagated for assay purposes.

[0071] All cell washing and resuspension were performed in the reaction buffer. The reaction buffer should consist of a near-isotonic concentration of NaCl, along with an endocytosis inhibitor and a phosphate-free buffer system, such as 140 mM NaCl and 25 mM NH4Cl at pH 7.4 (for endocytosis inhibitors and alternative buffer systems). Any other buffers (such as HEPES) and inhibitors may also be evaluated. The reaction buffer will stabilize the RBD-ACE2 complex on the cell surface, thereby preventing endocytosis.

[0072] The cells were washed with reaction buffer and scraped off with a cell scraper to remove them from the cell culture flask. A single-cell suspension was prepared in reaction buffer using a pipette and clarified through a cell filter.

[0073] The cells were then centrifuged at 180–300 g for 5 minutes, the supernatant was removed, and the cells were resuspended in 1× reaction buffer. The cells were then counted, centrifuged, and the supernatant was removed.

[0074] Cells are then resuspended at the desired concentration (e.g., 5 million cells / ml) in cell freezing buffer (reaction buffer containing 10% DMSO). Cells are then dispensed at 20 µl per 2 ml tube (enough for 10 reactions) and kept at -80°C until use. Different cell concentrations and volumes are available for different purposes and kit sizes.

[0075] Example 3: Preparation of other components of the kit Prepare a 10× reaction buffer containing 1.40 M NaCl, 0.25 M NH4Cl, and pH 7.4. The buffer was prepared by dissolving 13.37 g of ammonium chloride (NH4Cl) and 81.81 g of sodium chloride (NaCl) in 800 ml of distilled water, adjusting the pH to 7.4 with ammonia, and bringing the final volume to 1000 ml.

[0076] A 1000× propidium iodide stock solution was prepared by diluting a commercial stock solution of 1 mg / ml propidium iodide (PI) to 0.5 mg / ml in water. When preparing the cell dilution buffer, the working stock solution was used at a concentration of 1 µl / ml reaction buffer.

[0077] Positive control (PC) reference serum: Blood samples were collected via venipuncture without anticoagulants from individuals (healthy volunteers) who had received a SARS-CoV-2 vaccine (with Covishield / Sputnik V / Covaxin or other anti-SARS-CoV-2 vaccines, and within 1–4 months after a booster dose). Serum was separated according to standard procedures (i.e., incubated at room temperature for 1 hour, followed by centrifugation at 1000–2000 × g for 10 minutes, and serum was collected from the supernatant). The presence of high titers of SARS-CoV-2 neutralizing antibodies in serum samples was screened using the cPass SARS-CoV-2 Neutralizing Antibody Detection Kit (GeneScript), which has been validated with the live virus neutralization assay “PRNT” and has been FDA approved for emergency use. The assay was performed according to the manufacturer’s protocol. https: / / www.fda.gov / media / 143583 / downloadSamples can also be screened using PRNT / cVNA. Ten serum samples (and potentially more) showing the highest neutralizing titers are mixed in equal volumes, and the final serum titer is determined using the SARS-CoV-2 live virus neutralization test (cVNA) as described in Reference 16 (alternatively, it can be detected using PRNT or any PRNT-validated alternative neutralization test). The mixture of standardized serum samples is stored aliquoted at -20°C and used as a standard for subsequent testing. The final neutralizing antibody concentration / titer should be greater than 1:640 using PRNT or equivalent concentrations.

[0078] Negative Control (NC) Reference Serum: Using a similar protocol as described in PC, ten negative serum samples (which may include more) selected after screening serum samples from healthy volunteers with the cPass SARS-CoV-2 Neutralizing Antibody Assay Kit were mixed in equal volumes and reconfirmed by cVNA(12) (alternatively, PRNT / any PRNT-validated alternative neutralization test). The mixed serum was stored aliquoted at -20°C and used as a negative control standard for any subsequent tests.

[0079] Example 4: In vitro assay using the kit disclosed herein Blood was collected via venipuncture without any anticoagulant. After incubation at room temperature (25°C) for 1 hour, serum was separated by centrifugation at 1000-2000×g for 10 minutes. The supernatant (serum) was collected and stored for short-term storage at 4°C or long-term storage at -20°C.

[0080] Dilute the 10× reaction buffer to a 1× concentration using distilled water. Prepare the cell dilution buffer by adding 1 µl / ml of 1000×PI to the 1× reaction buffer.

[0081] The serum was then serially diluted with 1× reaction buffer. 50 µl of frozen, standardized medium containing GFP-RBD was placed in a 96-well plate. 5 µl of serum sample or its dilution was added to the corresponding well. After incubating at room temperature for 1 hour, the plate was placed on ice for another 15 minutes.

[0082] Frozen, transfected cells expressing ACE2 protein were thawed on ice and diluted 75-fold with pre-chilled cell resuspension buffer containing 0.5 µg / ml propidium iodide (e.g., 1.5 ml cell resuspension buffer for 20 µl of frozen cells). 150 µl of the chilled cell suspension was added to each well. In this way, 10 k cells were distributed for each reaction. The reaction mixture was properly mixed and incubated on ice for 60–90 minutes.

[0083] After incubation, pipette the reaction mixture to remove any cell clumps and add the entire reaction mixture to a 500 µl RB container kept in the flow cytometer tube at room temperature. Place the tube in a microcooler (-20°C) until flow cytometry measurements are performed.

[0084] For flow cytometry analysis, GFP was detected via the FL1 channel and PI via the FL3 channel. First, a single intact cell population was selected from the SSC-FSC plot. Then, PI-stained cells were selected from the SSC-PI plot. In the GFP-PI four-quadrant plot, the threshold was adjusted by running PI-stained cells without RBD-GFP and set to 0–2% as the threshold for either the FL1 channel or GFP. The percentage of RBD-GFP binding to HEK 293 ACE2 cells is expressed as the percentage of GFP-positive cells. Binding of RBD-GFP to cells without incubation with any serum sample or inhibitor was considered 100%. Any reduction in binding after incubation of RBD-GFP with any serum sample or inhibitor was detected as a reduction in GFP-positive cells and served as a measure of neutralization. Therefore, neutralization was measured using the following formula: (1 – % GFP-positive cells in the treated sample / GFP-positive cells in the untreated control sample). Figure 4 The paper demonstrates the analytical method using flow cytometry.

[0085] Interpretation of results: If the neutralization percentage (%neutralization) is >20% (cutoff value 20%) at a 1:10 serum dilution, the presence of neutralizing antibodies against SARS-CoV-2 is considered positive; if the neutralization percentage is <20%, it is considered negative or that there are no neutralizing antibodies against SARS-CoV-2. Figure 4 ).

[0086] Cutoff value determination method: The cutoff value was determined using a total of 41 pre-COVID-19 serum samples collected before March 31, 2020. The mean +2SD was determined to be 16% (-2% ± 18%). Therefore, for practical purposes, 20% was used as the cutoff value to make it more stringent. Figure 4 Within the measured linear range (20%-80%), the cutoff value is 2-6% (Table 4).

[0087] Figure 1Microscopic views of a cell-based neutralization assay (CENA) were depicted. HEK 293 cells stably transfected with the human ACE2 receptor gene expression cassette in a plasmid were 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: collected from subjects who had received two doses of Covishield). Cells incubated with the control sample showed a green ring outside the cell membrane, indicating that RBD-S-GFP bound to the cell surface. After incubation with serum from vaccinated subjects, the green ring disappeared, indicating that the RBD-interacting domain of the SARS-CoV-2 spike protein was blocked. The same observations were repeated when the corresponding samples were analyzed by flow cytometry. Samples treated with control serum showed 50% RBD-GFP binding, while incubation with vaccinated serum showed only 2% binding, close to background.

[0088] Example 5: Selection of optimal reaction buffer conditions The binding efficacy of RBD-GFP to HEK 293 cells expressing ACE2 was tested using the buffers shown in Table 1. The first reading was taken 2 hours after dilution with the appropriate reaction buffer (RB), followed by a second reading after 20 minutes of incubation on ice. Figure 2 The results showed that, in the tested buffer, reaction buffer RB6 exhibited the highest RBD-GFP binding efficacy and stability in 293 cells expressing ACE2.

[0089] Table 1

[0090] Example 6: Analysis of the effects of DMSO and cell freezing The effects of DMSO and cell freezing on propidium iodide staining in live cells and the binding of RBD-GFP to HEK 293 cells expressing ACE2 were evaluated at different temperatures. Adding DMSO to the buffer and freezing ACE2-expressing cells at -80°C showed improved propidium iodide staining. Figure 3 (A in the middle).

[0091] Furthermore, freezing cells expressing ACE2 at -80°C in DMSO improved RBD-GFP binding in the cells. Figure 3 (B in the middle).

[0092] Example 7: Comparative Analysis of In Vitro Assays and Conventional Assays 7.1 Clinical concordance between CENA and conventional CPE-based SARS-CoV-2 virus neutralization assay (cVNA) A comparative analysis was performed between the in vitro assay (CENA) of this disclosure and the conventional CPE-based SARS-CoV-2 virus neutralization assay (cVNA).

[0093] The following tests were conducted using a total of 15 serum samples for comparison: 1. Serum samples after vaccination (12 samples) 2. Serum (1 sample) collected before the COVID-19 pandemic (before March 2020) 3. Serum mixtures from 10 individuals (PCs) who received the COVID-19 vaccine (1 sample) 4. A mixture of serum from individuals who have not reported positive RT-PCR COVID-19 infection and have not been vaccinated (NC) (1 sample).

[0094] Serum samples were serially diluted twofold and subjected to neutralization tests, respectively, using CENA as described in this disclosure and cVNT (12) as previously described. Serum titers were determined by CENA as the highest limit for serum dilutions producing at least >50% neutralization (CENA50). If CENA50 < 1:40 serum dilution, the sample was considered negative (Table 2). Serum titers were determined by cVNT as the highest limit for serum dilutions producing 100% neutralization (cVNT100). If cVNT100 < 1:40 serum dilution, the sample was considered negative (Table 2). A comparative overview of the two tests is shown in Table 3. Both tests showed 100% concordance in detecting positive and negative samples (Table 4).

[0095] Table 2. Cutoff conditions for determining positive or negative CENA and cVNA comparative analysis for each assay.

[0096] CPE: Cytopathic effect; NT: Neutralization test result, expressed as the neutralization rate of SARS-CoV-2 RBD binding to receptor ACE2 in each assay.

[0097] Table 3: Clinical Consistency Comparison of Serum Titers (Limits of Serum Dilutions for Positive Results) Measured by CENA50 and cVNT100 (Live Virus Neutralization Assay)

[0098] Table 4: Clinical Consistency between CENA50 and cVNA100

[0099] Under the given conditions, the results of CENA showed 100% concordance with conventional SARS-CoV-2 live virus neutralization assays.

[0100] Figure 5 The linear correlation between CENA50 and the conventional CPE-based SARS-CoV-2 live virus neutralization assay (cVNA100) was plotted. The neutralization test was performed using serially 2-fold diluted serum samples and measured using both CENA50 and cVNT100 to determine the serum titer (the limit of serum dilution for a positive result). The logarithms of the serum titers for the corresponding tests (CENA50 / cVNT100) were plotted on the Y and X axes, respectively. Correlation analysis of the serum titers measured by the two tests showed a good correlation between CENA50 and cVNA100 (R 0.9, p < 0.0016).

[0101] 7.2 Correlation comparison between CENA and commercially available cPass SARS-CoV-2 neutralizing antibody test kits.

[0102] Serum samples (53 samples) from subjects who had completed two doses of the COVID-19 vaccine within 7 months were analyzed.

[0103] The cPass SARS-CoV-2 Neutralizing Antibody Detection Kit (Genescript) is an FDA-approved (Emergency Use Authorization) fully validated and widely used internationally for SARS-CoV-2 neutralizing antibody testing. Perform the test in duplicate at a 1:20 serum dilution (as recommended) according to the manufacturer's protocol (https: / / www.fda.gov / media / 143583 / download). Take readings using a Spark multi-mode plate reader (TECAN). Calculate the percentage of neutralization (% signal inhibition) according to the formula provided by the manufacturer.

[0104] Using the same protocol described in this invention, cell-based neutralization assays (CENA) were performed by flow cytometry at a dilution of 1:20. The percentage of neutralization was determined as previously described in this invention.

[0105] Figure 6 This study depicts the correlation analysis of 53 serum samples with different neutralizing antibody (Nab) levels after SARS-CoV-2 vaccination using the CENA and cPass SARS-CoV-2 Nab detection kit (Genescript) at a serum dilution of 1:20. The percentage of neutralization (%NT) through the corresponding assays is plotted along the X and Y axes. Pearson correlation coefficients and linear regression analyses were performed in Excel. Statistical significance (p-value) was calculated using a two-tailed test. Data are the mean of repeated tests. Figure 6 The results showed that CENA and the cPass SARS-CoV-2 kit had a good linear correlation (R 0.94, p<0.00001).

[0106] Figure 7 A dot plot illustrating the comparative performance of the CENA and cPass (Genescript) kits for detecting neutralizing antibodies in serum samples is presented. A total of 53 post-SARS-CoV-2 vaccination samples (PVAC) and 20 pre-COVID-19 samples (collected before March 2020) were simultaneously tested at a serum dilution of 1:20 using CENA and the cPass kit for detecting SARS-CoV-2 neutralization. Cutoff values ​​for cPass NT: >30% positive, <30% negative; Cutoff values ​​for CENA NT: >50% positive, <50% negative; N: NT negative; P: NT positive; CENA-PVAC: %NT distribution in PVAC samples via CENA; CENA-pre-CoV: %NT distribution in pre-CoV samples via CENA; cPass-PVAC: %NT distribution in PVAC samples via cPass; cPass-pre-CoV: %NT distribution in pre-CoV samples via cPass; Dashed lines indicate the corresponding cutoff values ​​for the tests shown in the figure; Brown circles: Samples that tested positive for the corresponding test; Blue circles: Samples that tested negative for the corresponding test.

[0107] Table 5: Cutoff conditions for comparing the CENA and cPass SARS-CoV-2 neutralizing antibody detection kits

[0108] *Determination of the 20% NT cutoff value for CENA: Neutralization test (NT) was performed using pre-COVID-19 serum samples (20 samples, collected before March 2020) via CENA. %NT (mean + 3SD) = 20% was used as the cutoff value for CENA; **The 30% NT cutoff value for the cPass kit** is provided according to the manufacturer's protocol.

[0109] Table 6. Clinical concordance of the CENA and cPass SARS-CoV-2 neutralizing antibody detection kits used for serological classification as NT-positive or negative after SARS-CoV-2 vaccination.

[0110] Neutralization as measured at a dilution of 1:20.

[0111] *Cohen's κ (k) was determined using the web-based software “Cohen's κ Free Calculator” described by Landis, JR & Koch, GG (1977) for measuring observer consistency in categorical data (https: / / idostatistics.com / cohen-kappa-free-calculator / #risultati).

[0112] **Explanation of Cohen's κ (k):** 0.01-0.20 Weak consistency 0.21-0.40 General consistency 0.41-0.60 Moderate consistency 0.61-0.80 High consistency 0.81-1.00 Near-perfect or perfect consistency Example 8: Determination of the sensitivity and specificity of CENA The sensitivity and specificity of the CENA assay disclosed herein were determined using the standard commercially available cPass SARS-CoV-2 neutralizing antibody test kit (GeneScript), which has been validated by the live virus neutralization assay “PRNT” and approved by the FDA for emergency use, according to the manufacturer’s protocol (https: / / www.fda.gov / media / 143583 / download) for detecting positive samples and pre-COVID-19 (collected before March 2020) serum as a known standard test for negative samples. The results are shown in Table 7.

[0113] Table 7

[0114] * Positive samples identified by the FDA-approved, empirically validated cPass SARS-CoV-2NT kit.

[0115] Example 9: Determining population response to different brands of COVID-19 vaccines Serum samples were collected from two healthy individuals before and one month after COVID-19 vaccination for assays. One subject received the Sputnic V vaccine, and the other received the Covishield vaccine. Neutralization assays were performed using the same protocol described in this invention, with sequential 2-fold dilutions starting at 1:10 until baseline was reached. A 4PL plot was generated as the logarithm of dilution versus neutralization. The IC50 was determined. Results were presented in... Figure 8As shown in the figure, comparing two experimental subjects, the subject who received the Sputnic V vaccine had a lower immune response after a single dose than the subject who received the Covishield vaccine (IC50 of 78 vs. 134). By investigating neutralizing antibody titer responses to different brands of COVID-19 vaccines in a population, a larger-scale vaccination policy can be implemented for the population. Alternatively, the same examples measured could be used for personal protective equipment. If a particular brand of vaccine fails to produce a protective response, an alternative brand could be tried if national protocols permit.

[0116] Example 10: Community survey of neutralizing antibody status after COVID-19 vaccination.

[0117] Serum samples were collected from 53 healthcare workers who had completed two doses of COVID-19 vaccination within 7 months for assays. Neutralization assays were performed at a 1:20 dilution using the same protocol described in this invention. The cutoff value was set at 20% (mean of %NT of 20 pre-COVID-19 samples + 3SD). Of the 53 samples, only 40 showed detectable neutralizing antibodies against SARS-CoV-2 at a serum dilution of 1:20, while no neutralizing antibodies were detected in 13 subjects (Table 6).

[0118] This application will facilitate low-budget surveys of different target populations to determine neutralizing antibody levels. The data can be used to implement any preventative policies, including decisions on whether booster doses are necessary. This low-cost assay can be used by employers to develop booster dose strategies for employees at high risk of SARS-CoV-2 infection who will be assigned to frontline work.

[0119] 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. October 2, 2020;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. September 4, 2020;369(6508):1261-1265. 5. pcDNA3-SARS-CoV-2-S-RBD-sfGFP Sequences from an 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 KuppeveldFJ, 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 Pathology. 6 / 11 / 2014;10(11):e1004502. doi: 10.1371 / journal.ppat.1004502. Error in: PLoS Pathog. Feb 2015;11(2):e1004709.PMID: 25375324; PMCID: PMC4223067. [ Abstract ] 8. Larkin JM, Brown MS, Goldstein JL, Anderson RG. Depletion ofintracellular potassium arrests coated pit formation and receptor-mediateddendocytosis in fibroblasts. Cell. May 1983;33(1):273-85. doi: 10.1016 / 0092-8674(83)90356-2. PMID: 6147196. [ PMC free article ] [ PubMed ] 9. Wang, H., Yang, P., Liu, K.Entry of SARS coronavirus into hostcells through a novel clathrin- and caveolae-independent endocyticpathway. Cell Res Rev. 18,290–301 (2008). https: / / doi.org / 10.1038 / cr.2008.15 10. Nicola AV, McEvoy AM, Straus SE. Roles for endocytosis and low pHin 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 DNAstaining 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, KhanMS, 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-June; 296:100025. doi: 10.1074 / jbc.RA120.016284. Claims (as amended under Article 19 of the Treaty) 1. A kit for in vitro detection of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in samples, comprising: (i) A frozen culture medium containing a secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) for binding to human ACE2 protein labeled with the test entity; (ii) Ready-to-use frozen transfected cells expressing human ACE2 protein; (iii) Reaction buffer; (iv) DNA binds to fluorescent dyes; (v) Positive control; and (vi) Negative control; The frozen medium containing the SARS-CoV-2 spike protein receptor-binding domain (RBD) is derived from the supernatant of transfected cells expressing the SARS-CoV-2 spike protein receptor-binding domain (RBD) cultured in animal cell culture medium, diluted in reaction buffer, and then frozen at -20°C to -80°C. The cells expressing human ACE2 protein were resuspended in a reaction buffer containing 10% DMSO and frozen at -80°C. The reaction buffer solution contains sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, and has a pH of 7.4. 2. The kit according to claim 1, wherein the receptor-binding domain (RBD) is represented by amino acid sequences 333 to 529 of sequence ID No. 1, and the human ACE2 protein is represented by amino acid sequences 19 to 805 of sequence ID No. 2. 3. The kit according to claim 1, wherein the animal cell culture medium is selected from minimum essential culture media, the minimum essential culture medium comprising basal minimum essential medium (MEM) mixed with 10% fetal bovine serum (FBS). 4. The kit according to claim 1, wherein the detection entity is selected from superfolded green fluorescent protein (sGFP), red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), and near-infrared fluorescent protein 702 (iRFP702). 5. The kit according to claim 1, wherein the cell line is selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, and COS-7. 6. The kit according to claim 1, wherein the DNA-binding fluorescent dye is selected from propidium iodide, 7-aminoactinomycin D, and SYBR Green. 7. The kit according to claim 1, wherein the kit comprises a propidium iodide stock solution at a concentration of 0.3-0.7 mg / ml. 8. The kit of claim 1, wherein the positive control comprises a mixture of serum samples from a vaccinated individual having neutralizing antibodies against SARS-CoV-2, and the negative control comprises a mixture of serum samples not having neutralizing antibodies against SARS-CoV-2. 9. The kit according to 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 media fluid, gastric juice, and breast milk. 10. A method for preparing a frozen cell line expressing human ACE2 protein according to claim 1, the method comprising: (i) Cells were cultured in an animal cell culture medium containing 10% fetal bovine serum to obtain cultured cells; (ii) Transfect a plasmid containing a constitutive expression cassette encoding the ACE2 protein into the cultured cells that do not express ACE2 and multiply them in the presence of a selection marker to obtain stable transfected cells; (iii) Resuspend stable transfected cells expressing ACE2 protein or cells naturally expressing ACE2 protein in reaction buffer in a cell culture flask, then remove the cells from the flask and pass them through a cell filter to prepare a single-cell suspension and determine the cell concentration. (iv) The cells were stored as a concentrated stock solution in a cryogenic buffer consisting of reaction buffer and 10% dimethyl sulfoxide at temperatures ranging from -80°C to -150°C. The reaction buffer solution contains sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, and has a pH of 7.4. 11. The method of claim 10, wherein the transfected cells are selected from adherent HEK293 cells, HEK293T cells, MDCK cells, HeLa, Hep2C, L20B, COS-7, and A549, and the cells naturally expressing ACE2 are selected from adherent cells Vero E6, Vero 81, MA104, LLC-MK2, BGM, and Caco-2. 12. The method of claim 10, wherein the plasmid is selected from pCEP4-myc-ACE2 plasmid or hACE2 or pcDNA3.1-hACE2 or any other DNA vector having a human ACE2 constitutive expression cassette containing mammalian selection markers. 13. A method for preparing a frozen culture medium comprising the secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) of the human ACE2 protein according to claim 1, the method comprising: (i) Cells were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum to obtain cultured cells; (ii) Transfecting the cultured cells with a plasmid containing an expression cassette encoding a SARS-CoV-2 receptor-binding domain, wherein the C-terminus of the receptor-binding domain is labeled with superfolded green fluorescent protein (RBD-sGFP) and the N-terminus is labeled with a signal peptide from influenza HA, and propagating the transfected cells in the presence of the selection marker to obtain stable transfected cells; (iii) Incubate the stable transfected cells in a CO2 incubator at 37°C in minimum essential medium for 3-5 days; (iv) After incubation, the culture medium is centrifuged to obtain a clear supernatant; (v) The supernatant is diluted with a 1× reaction buffer containing sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, at pH 7.4, to obtain the final culture medium according to claims 3 and 4, such that the concentration of the supernatant is 4-5 times the final concentration used; and (vi) The final culture medium is frozen at -20°C to -80°C. 14. The method according to claim 13, wherein the cells are selected from adherent HEK293 cells, HEK293T cells, MDCK cells, HeLa, Hep2C, L20B, A549, and COS-7. 15. The method of claim 14, wherein the plasmid is a pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmid containing mammalian selection markers. 16. An in vitro assay for analyzing the presence of neutralizing antibodies against SARS-CoV-2 in a sample, comprising: (i) After diluting the frozen medium with 1× reaction buffer, incubate the sample with a medium containing the receptor-binding domain (RBD) of the secretory SARS-CoV-2 spike protein labeled with human ACE2 protein of the test entity at room temperature for 30-90 minutes, followed by incubation on ice for 10-30 minutes. (ii) Frozen transfected cells expressing human ACE2 protein were thawed separately and then diluted in cell dilution buffer containing 0.3–0.7 µg / ml DNA-binding fluorescent dye to obtain thawed cells; (iii) Contact the thawed cells of step (ii) with the sample incubated with the culture medium of step (i) on an ice bath for 60-90 minutes to form a reaction mixture; (iv) The reaction mixture was diluted by adding it to an ice-cold reaction buffer, and (v) The intensity of green fluorescence and the percentage of fluorescently positive cells in cells were determined by flow cytometry and neutralization. The neutralization was measured using the following formula: (1% of GFP-positive cells in the treated sample / GFP-positive cells in the untreated control sample), where a neutralization percentage greater than 20%-30% indicated the presence of neutralizing antibodies, and a neutralization percentage less than 20%-30% indicated the absence of neutralizing antibodies. The cell dilution buffer contains 1× reaction buffer with concentrations of 0.140 M and 0.025 M sodium chloride and ammonium chloride, pH 7.4, and PI at a concentration of 0.3-0.7 ug / ml. 17. The method according to claim 16, 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 media fluid, gastric juice, and breast milk. 18. The method of claim 16, wherein the culture medium is the minimum necessary culture medium. The detection entity is selected from green fluorescent protein, red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), and near-infrared fluorescent protein 702 (iRFP702). The DNA-binding fluorescent dye is selected from propidium iodide, 7-aminoactinomycin D, and SYBR Green. 19. The method of claim 16, wherein the detection entity detects the interaction between the human ACE2 protein and the SARS-CoV-2 spike protein receptor-binding domain, and wherein the percentage of green fluorescence is proportional to the percentage of GFP-labeled RBD binding to the cell surface ACE2 receptor.

Claims

1. A kit for in vitro detection of neutralizing antibodies against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in samples, comprising: (i) A frozen culture medium containing a secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) for binding to human ACE2 protein labeled with the test entity; (ii) Ready-to-use frozen transfected cells expressing human ACE2 protein; (iii) Reaction buffer; (iv) DNA binds to fluorescent dyes; (v) Positive control; and (vi) Negative control.

2. The kit according to claim 1, wherein the receptor-binding domain (RBD) is represented by amino acid sequences 333 to 529 of sequence ID No. 1, and the human ACE2 protein is represented by amino acid sequences 19 to 805 of sequence ID No.

2.

3. The kit according to claim 1, wherein the frozen medium containing the SARS-CoV-2 spike protein receptor-binding domain (RBD) is derived from the supernatant of transfected cells expressing the SARS-CoV-2 spike protein receptor-binding domain (RBD) cultured in animal cell culture medium, diluted in reaction buffer, and then frozen at -20°C to -80°C.

4. The kit according to claim 1, wherein the animal cell culture medium is selected from minimum essential culture media, the minimum essential culture medium comprising basal minimum essential culture medium (MEM) mixed with 10% fetal bovine serum (FBS).

5. The kit according to claim 1, wherein the detection entity is selected from superfolded green fluorescent protein (sGFP), red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), and near-infrared fluorescent protein 702 (iRFP702).

6. The kit according to claim 1, wherein the cell line is selected from adherent HEK293 cells, HEK293T, MDCK cells, HeLa, Hep2C, L20B, A549, and COS-7.

7. The kit according to claim 1, wherein the reaction buffer comprises sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, at pH 7.

4.

8. The kit according to claim 1, wherein the DNA-binding fluorescent dye is selected from propidium iodide, 7-aminoactinomycin D, and SYBR Green.

9. The kit according to claim 1, wherein the kit comprises a propidium iodide stock solution at a concentration of 0.3-0.7 mg / ml.

10. The kit according to claim 1, wherein the cells expressing human ACE2 protein are resuspended in a reaction buffer containing 10% DMSO and frozen at -80°C.

11. The kit of claim 1, wherein the positive control comprises a mixture of serum samples from a vaccinated individual having neutralizing antibodies against SARS-CoV-2, and the negative control comprises a mixture of serum samples not having neutralizing antibodies against SARS-CoV-2.

12. The kit according to 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 media fluid, gastric juice, and breast milk.

13. A method for preparing a frozen cell line expressing human ACE2 protein according to claim 1, the method comprising: (i) Cells were cultured in an animal cell culture medium containing 10% fetal bovine serum to obtain cultured cells; (ii) Transfect a plasmid containing a constitutive expression cassette encoding the ACE2 protein into the cultured cells that do not express ACE2 and multiply them in the presence of a selection marker to obtain stable transfected cells; (iii) Resuspend stable transfected cells expressing ACE2 protein or cells naturally expressing ACE2 protein in reaction buffer in a cell culture flask, then remove the cells from the flask and pass them through a cell filter to prepare a single-cell suspension and determine the cell concentration. (iv) The cells were stored as a concentrated stock solution in a cryogenic buffer consisting of reaction buffer and 10% dimethyl sulfoxide at temperatures ranging from -80°C to -150°C. The reaction buffer solution contains sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, and has a pH of 7.

4.

14. The method of claim 13, wherein the transfected cells are selected from adherent HEK293 cells, HEK293T cells, MDCK cells, HeLa, Hep2C, L20B, COS-7, and 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 of claim 13, wherein the plasmid is selected from pCEP4-myc-ACE2 plasmid or hACE2 or pcDNA3.1-hACE2 or any other DNA vector having a human ACE2 constitutive expression cassette containing mammalian selection markers.

16. A method for preparing a frozen culture medium comprising the secretory SARS-CoV-2 spike protein receptor-binding domain (RBD) of the human ACE2 protein according to claim 1, the method comprising: (i) Cells were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum to obtain cultured cells; (ii) Transfecting the cultured cells with a plasmid containing an expression cassette encoding a SARS-CoV-2 receptor-binding domain, wherein the C-terminus of the receptor-binding domain is labeled with superfolded green fluorescent protein (RBD-sGFP) and the N-terminus is labeled with a signal peptide from influenza HA, and propagating the transfected cells in the presence of the selection marker to obtain stable transfected cells; (iii) Incubate the stable transfected cells in a CO2 incubator at 37°C in minimum essential medium for 3-5 days; (iv) After incubation, the culture medium is centrifuged to obtain a clear supernatant; (v) The supernatant is diluted with a 1× reaction buffer containing sodium chloride and ammonium chloride at concentrations of 0.140 M and 0.025 M, respectively, at pH 7.4, to obtain the final culture medium according to claims 3 and 4, such that the concentration of the supernatant is 4-5 times that of the final concentration used; and (vi) The final culture medium is frozen at -20°C to -80°C.

17. The method according to claim 16, wherein the cells are selected from adherent HEK293 cells, HEK293T cells, MDCK cells, HeLa, Hep2C, L20B, A549, and COS-7.

18. The method of claim 17, wherein the plasmid is a pcDNA3-SARS-CoV-2-S-RBD-sfGFP plasmid containing mammalian selection markers.

19. An in vitro assay for analyzing the presence of neutralizing antibodies against SARS-CoV-2 in a sample, comprising: (i) After diluting the frozen medium with 1× reaction buffer, incubate the sample with a medium containing the receptor-binding domain (RBD) of the secretory SARS-CoV-2 spike protein labeled with human ACE2 protein of the test entity at room temperature for 30-90 minutes, followed by incubation on ice for 10-30 minutes. (ii) Frozen transfected cells expressing human ACE2 protein were thawed separately and then diluted in cell dilution buffer containing 0.3–0.7 µg / ml DNA-binding fluorescent dye to obtain thawed cells; (iii) Contact the thawed cells of step (ii) with the sample incubated with the culture medium of step (i) on an ice bath for 60-90 minutes to form a reaction mixture; (iv) The reaction mixture was diluted by adding it to an ice-cold reaction buffer, and (v) The intensity of green fluorescence in cells was determined by flow cytometry. A fluorescence intensity greater than 20%-30% indicates the absence of neutralizing antibodies, while a fluorescence intensity less than 20%-30% indicates the presence of neutralizing antibodies.

20. The method according to 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 media fluid, gastric juice, and breast milk.

21. The method of claim 19, wherein the culture medium is the minimum necessary culture medium. The detection entity is selected from green fluorescent protein, red fluorescent protein (RFP), monomeric near-infrared red fluorescent protein 670 (miRFP 670), and near-infrared fluorescent protein 702 (iRFP702). The DNA-binding fluorescent dye is selected from propidium iodide, 7-aminoactinomycin D, and SYBR Green. The cell dilution buffer contains 1× reaction buffer with concentrations of 0.140 M and 0.025 M sodium chloride and ammonium chloride, pH 7.4, and PI at a concentration of 0.3-0.7 ug / ml.

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

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