Development of ELISA-Based Assay for Detection of SARS-CoV-2 Neutralizing Antibody

Abstract

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulates the plasma B cells to secrete specific antibodies against the viral antigen. However, not all antibodies can prevent the virus from entering the cells. The subpopulation of antibodies which blocks the entry of the virus into host cells is termed neutralizing antibodies (NAbs). The gold standard test for the detection of NAbs is the viral plaque reduction and neutralization test; however, various other methods can also be utilized to detect NAbs. In this study, we have developed an Enzyme Linked Immunosobent Assay (ELISA)-based protocol for rapid detection of SARS CoV-2 NAb by inhibiting the binding of the spike protein receptor-binding domain to angiotensin converting enzyme 2 and compared it with cPASS neutralizing antibody kit, which was approved by the Food and Drug Administration (FDA). The results obtained suggest that the in-house ELISA developed for the detection of NAbs against SARS-CoV-2 is rapid and reliable. Compared to FDA-approved GenScript's cPass assay, the specificity and the sensitivity of the in-house-developed ELISA kit were 100% (95% confidence intervals of 69.15–100.00) and 96% (95% confidence intervals of 86.29–99.51), respectively. Thus, the ELISA protocol developed to test the neutralizing activities of antibodies is rapid, which requires a BSL-2 infrastructure facility and can be easily performed. It has very high potential applications in the rapid screening of NAb against SARS-CoV-2.

Introduction

The causative agent of coronavirus disease 2019 (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is known to be infectious in humans. The SARS-CoV-2 virus has a positive sense, single-stranded RNA genome, which has about 29,903 nucleotide sequence that makes about 14 genes that are transcribed into ∼27 proteins (Andersen et al., 2020; Zhou et al., 2020). It includes nonstructural proteins (nsp1–14), structural proteins, and accessory proteins. Structural proteins include spike (S), envelope (E), membrane protein (M), and nucleocapsid (N) proteins (Mishra et al., 2021; Satarker and Nampoothiri, 2020; Shereen et al., 2020).

The spike (S) protein is involved in the recognition of host cell receptors and the cell membrane fusion process. The spike protein consists of two subunits known as the S1 and S2. Out of the two subunits, S1 subunit of the spike protein contains the receptor-binding domain (RBD), which recognizes the host cell surface receptor, that is, angiotensin converting enzyme-2 (ACE2). Several studies have reported that the RBD of the virus spike protein displays a strong binding affinity to the human ACE2 receptor, which leads to endocytosis in the host cells (Lempp et al., 2021), on the contrary, the S2 subunit mediates the fusion of the viral and the host cell membranes by forming a six-helical bundle via the two heptad repeat domains (Jackson et al., 2022).

Human ACE2 is known to convert a peptide hormone known as angiotensin I to angiotensin 1–9 (Donoghue et al., 2000). The receptor protein ACE2 is reported to be expressed ubiquitously in the majority of tissues, including the heart, kidney, intestine, lungs, and the alveolar epithelial type II cells (Scialo et al., 2020; Zhang et al., 2020). It is also a known receptor for SARS-CoV. The SARS-CoV S protein binds with the ACE2 receptor (Hoffmann et al., 2020; Shang et al., 2020).

Infection with SARS-CoV-2 initiates an immune response, which includes the production of binding antibodies in the blood. Not all binding antibodies can block the entry of the virus into the cell. The subpopulation of the antibodies that can block virus entry into the cells is termed neutralizing antibodies (NAbs).

NAbs testing is limited to laboratories equipped with BSL-3 or 4 facilities with standard detection techniques, that is, Conventional Virus Neutralization Test (VNT) or Pseudovirus based VNT. All other assays such as ELISA and the Lateral Flow Assay detect quantitatively the level of total antibodies in the sample, but fail to differentiate between the levels of binding as well as the NAbs (Tan et al., 2020).

In the present study, we have established ELISA-based test method that detects NAbs without the need for any live virus and can be performed in a Biosafety level 2 facility. By using a commercially available RBD from the spike protein of the SARS-CoV-2 virus and the ACE2 receptor present on the host cell, our test method mimics the biological interaction between the virus-host in an ELISA plate. The interaction between the RBD–ACE2 is known to be inhibited by a type of antibodies known as NAbs, which are present in the sera of previously infected or vaccinated individuals. The ELISA test method was compared with Food and Drug Administration (FDA)-approved commercially available GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection kit for the detection of Neutralization antibodies (Genscript Biotech, Leiden, The Netherlands).

Materials and Methods

The recombinant human ACE2 was obtained from SignalChem (Cat. No. A51C2-G341F-100), Coronavirus 2019 Spike Glycoprotein-S1 RBD (319–541 a.a), Biotinylated was obtained from ProSpec (Cat. No. SARS-030), Streptavidin conjugated to horseradish peroxidase was obtained from Rockland (S000-03), Anti-2019-novel coronavirus spike NAb was obtained from Elabscience (Cat. No. E-AB-V1024), and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was obtained from Surmodics (Cat. No. TMBX-1000-01) to develop the neutralizing antibody ELISA kit. The assay diluent contains phosphate buffer saline with pH 6.6–7.2 (Cat. No. SM01-1000). The blocking buffer was obtained from Surmodics (Cat. No. ST01-0050). The wash buffer used for the kit was obtained from Surmodics, which contains Tris-buffered saline solution with 0.5% Tween 20 (Cat. No. WSHW-1000-01).

Ethics approval

The purpose and goal of the study were informed to the study participants, and they consented to participate by signing the consent form before taking part in the study.

Blood collection and serum separation

A total of 100 serum samples were analyzed from 50 vaccinated and/or naturally infected subjects and 50 true negative samples which were taken before December 2019 (sera samples stored at −80°C). In brief, 10 mL of blood was collected from the antecubital vein in unheparinized vacutainer blood collection tubes and stored at 4°C for an hour. The blood in the absence of an anticlotting agent resulted in the formation of a clot at the bottom with a clear layer of serum formed above the clotted blood. The serum was then gently collected using a micropipette in a sterile microcentrifuge tube and centrifuged for 10 min at 1,200 rpm. The serum was then again centrifuged to remove any residual amount of red blood cells if present. The clear serum was aliquoted in tubes and stored at −40°C for further experimentation.

Details of the procedures and methods developed for NAb ELISA of SARS-CoV-2

A 96-well ELISA plate was coated with recombinant hACE2 receptor protein (200 ng/well) and overnight incubated at 4°C. Plate was washed five times with wash buffer and then blocked for 2 h using blocking buffer at room temperature. S1-RBD-Biotin (300 ng/mL) was mixed with the positive control (Anti-2019-novel coronavirus spike NAb), negative control, and serum samples (serum samples were diluted in 1:1 ratio in assay diluent buffer) for 1.5 h at room temperature. After 1.5 h, the plate was washed five times with wash buffer and the mix was added to the wells and incubated for an hour.

After completion of incubation, the plate was again washed five times, and streptavidin-horseradish peroxidase (HRP) conjugate (500 ng/well) was added to the wells and further incubated for 1 h at room temperature. After washing, substrate TMB/H₂O₂ was added and color was developed, the reaction was stopped by the addition of 50 μL of the stop solution. After which the optical density (OD) was measured spectrophotometrically at 450 nm.

To check the quality of the test results, negative and positive controls were used. The average OD spectrophotometrically observed at 450 nm of the positive controls must be below 0.35 and negative control must be above 1.0.

The results were reported as Signal Inhibition (%) and were calculated using the following formula: Signal Inhibition (%) = (1−[OD of sample]/Mean OD of Negative control) × 100. If the percentage inhibition was >30%, it was considered positive, indicating the presence of S1-RBD NAb. If percentage inhibition was <30%, sample was considered negative, indicating S1-RBD NAb is absent or below the detection limit.

The assay was validated with FDA-approved cPass neutralizing antibody assay kit for sensitivity and specificity.

GenScript surrogate VNT

According to the manufacturer's instruction, the GenScript cPass SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript Biotech, Leiden, The Netherlands) assay was performed. In brief, the positive and negative controls and the serum samples of the tests were diluted to a ratio of 1:9 in the sample dilution buffer. After this, the diluted sample and the control were mixed with HRP-RBD working solution in a 1:1 ratio and incubated for 30 min at 37°C. In a 96-well plate precoated with the ACE2 receptor, 100 μL of the diluted control and the samples were added. The plate was then incubated for 15 min at 37°C and subsequently washed four times with 300 μL of wash buffer. After washing each well, 100 μL of substrate solution was added, after which the plate was incubated for 15 min in the dark at room temperature.

Finally, to stop the reaction after the color is developed, 50 μL of stop solution was added to each well and the OD was measured at 450 nm using ELISA-Reader. Signal inhibition was calculated using the following formula Inhibition (%) = (1–[Sample OD₄₅₀/Average Negative Control OD₄₅₀]) × 100.

Statistical analysis

The data obtained for percent inhibition by in-house neutralizing antibody ELISA kit and cPass kit were correlated with Pearson correlation coefficient (R). The data are considered significant at p < 0.05.

Results

Development of the ELISA test method for the rapid detection of neutralizing activity

The NAb known to be specific for the S1-RBD was used as a positive control and assay diluent was used as negative control and 100 serum samples were analyzed for validating and optimizing the ELISA kit for detection of NAb against SARS-CoV-2. We have optimized the ELISA for NAb against SARS-CoV-2 and standard plots were made (Fig. 1a, b). The percent inhibition of NAbs in vaccinated and/or naturally infected individuals and true negative serum samples were analyzed and are shown in Tables 1 and 2, respectively. The test results indicate that the optimal S1-RBD protein concentration was 300 ng/mL and that of ACE2 was 0.2 μg/mL.

FIG. 1.

(a) Standard curve for SARS-CoV-2 NAby. Anti-2019-nCoV Spike NAb can block 2019-nCoV spike protein (RBD). The mean of OD ± SE is plotted. (b) Showing % inhibition curve of Spike NAbs. Cutoff for presence of NAbs was set at 30% inhibition. The mean % inhibition ± SE is plotted. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; OD, optical density; RBD, receptor-binding domain; SE, standard error; NAbs, neutralizing antibodies.

Table 1.

Test Results of Serum Samples (n = 50) of Vaccinated and/or Naturally Infected Recovered Subjects Based on % Inhibition of In-House-Developed Neutralizing Antibody ELISA Kit

S. No.	Sample ID	Percent inhibition of vaccinated and/or naturally infected subjects	Test result for presence of NAbs
1	1	−6.24	Negative
2	5	98.18	Positive
3	6	94.07	Positive
4	7	98.54	Positive
5	8	96.15	Positive
6	9	54.47	Positive
7	10	94.10	Positive
8	11	96.48	Positive
9	12	97.78	Positive
10	13	93.14	Positive
11	14	94.31	Positive
12	15	95.18	Positive
13	16	39.54	Positive
14	17	65.07	Positive
15	18	79.38	Positive
16	19	91.36	Positive
17	20	87.07	Positive
18	21	90.68	Positive
19	22	83.85	Positive
20	23	78.21	Positive
21	24	97.16	Positive
22	25	90.57	Positive
23	26	2.41	Negative
24	27	78.97	Positive
25	28	80.31	Positive
26	29	79.90	Positive
27	30	70.57	Positive
28	32	85.42	Positive
29	33	95.25	Positive
30	34	95.84	Positive
31	35	94.32	Positive
32	36	91.85	Positive
33	37	89.92	Positive
34	38	89.26	Positive
35	39	79.85	Positive
36	40	85.62	Positive
37	41	93.35	Positive
38	42	93.14	Positive
39	43	91.17	Positive
40	44	91.14	Positive
41	45	94.43	Positive
42	46	95.81	Positive
43	47	93.95	Positive
44	48	94.16	Positive
45	49	92.40	Positive
46	50	95.68	Positive
47	51	88.55	Positive
48	52	91.83	Positive
49	53	90.75	Positive
50	54	49.92	Positive

ELISA, Enzyme Linked Immunosorbent Assay; NAbs, neutralizing antibodies.

Table 2.

Test Results of Serum Samples (n = 50) of True Negative Sera Samples Based on % Inhibition of In-House-Developed Neutralizing Antibody ELISA Kit

S. No.	Sample ID	Percent inhibition of true negative subjects	Test result for presence of NAbs
1	N1	12.77	Negative
2	N2	−56.60	Negative
3	N3	18.71	Negative
4	N4	21.58	Negative
5	N5	20.47	Negative
6	N6	18.00	Negative
7	N7	17.97	Negative
8	N8	9.83	Negative
9	N9	14.96	Negative
10	N10	18.61	Negative
11	N11	18.84	Negative
12	N12	20.06	Negative
13	N13	14.69	Negative
14	N14	29.45	Negative
15	N15	13.17	Negative
16	N16	23.51	Negative
17	N17	25.23	Negative
18	N18	25.03	Negative
19	N19	20.26	Negative
20	N20	30.40	Negative
21	N21	1.86	Negative
22	N22	22.59	Negative
23	N23	15.87	Negative
24	N24	21.99	Negative
25	N25	5.61	Negative
26	N26	−34.07	Negative
27	N27	13.08	Negative
28	N28	21.62	Negative
29	N29	13.28	Negative
30	N30	8.18	Negative
31	N31	20.20	Negative
32	N32	8.89	Negative
33	N33	−18.66	Negative
34	N34	−35.65	Negative
35	N35	2.09	Negative
36	N36	0.20	Negative
37	N37	−12.61	Negative
38	N38	−24.31	Negative
39	N39	9.80	Negative
40	N40	7.91	Negative
41	N41	6.60	Negative
42	N42	0.16	Negative
43	N43	5.02	Negative
44	N44	6.25	Negative
45	N45	−61.78	Negative
46	N46	−119.57	Negative
47	N47	23.04	Negative
48	N48	3.08	Negative
49	N49	23.16	Negative
50	N50	−53.52	Negative

The results observed with these doses were comparable to those obtained in the cPass kit. The cutoff value kept for negative samples was set at ≤30%. The true negative serum samples analyzed were all below 30%. Further, we analyzed the concentration of NAb using a standard plot, and cutoff 350 ng/mL was considered negative (Fig. 2).

FIG. 2.

Semiquantitative analysis of mean concentration of NAb of 50 vaccinated and 50 true negative samples was evaluated using the standard plot shown in figure 1a (OD vs. NAb concentration). The NAb concentration analyzed using in house ELISA kit of the vaccinated and/or naturally infected subjects was compared with the true negative sera samples. Cutoff 350 ng/mL considered as negative test result. ELISA, Enzyme Linked Immunosorbent Assay.

Correlation of % inhibition by in-house NAb ELISA with cPass kit

The data obtained for percent inhibition were correlated with cPass data, and the Pearson correlation coefficient (R) was observed as 0.716, which was highly significant (p < 0.001).

Sensitivity and specificity

To determine the sensitivity as well as the specificity of the NAb-ELISA, we thereby tested 50 serum samples collected from COVID-19 vaccinated and or naturally infected recovered subjects using our developed NAb-ELISA, out of 50 samples tested for the presence of NAb, 48 were positive (Table 1). Further, to examine the specificity of the NAb-ELISA, we tested 50 SARS-CoV-2-negative sera (collected before December 2019). The lack of a positive result in SARS-CoV-2 negative sera suggested 100% specificity, (Table 2). Next, 50 serum samples from COVID-19-vaccinated and/or naturally infected and recovered subjects and 10 true negative samples were tested using the NAb-ELISA and cPass kit.

All 50 serum samples from COVID-19-vaccinated and/or naturally infected and recovered subjects were found to be positive in the GenScript cPass test, while 48 sera samples tested positive using the in-house NAb ELISA method. The comparative data of both the kits are graphically represented in Figure 3. All 10 true negative sera samples were negative in both the test methods (Fig. 4). Compared to cPAss kit, the sensitivity of in-house NAb-ELISA was 96% (95% confidence intervals of 86.29–99.51) and specificity was 100% (95% confidence intervals of 69.15–100.00) (Table 3).

FIG. 3.

Comparison of percent inhibition of in-house ELISA kit with the GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection kit (grey line is of in-house ELISA result and dark grey line is of cPass kit, n = 50).

FIG. 4.

Serum samples of true negative subjects were analyzed and compared with FDA-approved cPass kit for NAb (n = 10). FDA, Food and Drug Administration.

Table 3.

Evaluation of Sensitivity and Specificity of In-House Neutralizing Antibody ELISA Kit with Comparator GenScript's cPass Kit

	Comparator
	Positive	Negative
Test kit
Positive	True positive	False positive
Negative	False negative	True negative
Test kit
Positive	48	0
Negative	2	10

Sensitivity = (True positive/true positive+false negative) × 100

= (48/48 + 2) × 100 = 96%.

Specificity = (True negative/true negative+false positive) × 100

= (10/10 + 0) × 100 = 100%.

Discussion

The humoral immune response against any viral infection leads to the production of NAbs that can prevent the viruses from infecting cells (Addetia et al., 2020; Huang et al., 2020). For the detection of NAbs against the infectious virus, viral plaque reduction and neutralization test is the gold standard method, which requires a live virus and laboratory setting of biosafety level 3 or 4 for performing the experiment (Okba et al., 2020; Pieri et al., 2022).

In the current study, we have developed a SARS-CoV-2 NAb ELISA to detect NAbs against SARS-CoV-2 using purified proteins. The assay is based on the interaction of the SARS-CoV-2 RBD with the host ACE2 receptor (hACE2) (Lan et al., 2020; Liu et al., 2022; Tai et al., 2020). The use of purified hACE2 protein-coated ELISA plates and biotin-conjugated RBD allows this test kit to assess the presence of NAbs that block the interaction of RBD-Biotin with hACE2 with high correlation to the GenScript's cPass kit (Lan et al., 2020; Tan et al., 2020).

In this study, we have compared the FDA-approved cPass kit for Neutralizing antibodies with an in-house-developed neutralizing antibody ELISA kit. The sensitivity and specificity of the in-house-developed ELISA kit were 96% and 100%, respectively, when compared with FDA-approved cPass kit. NAbs are essentially very important for virus clearance and protection of the host against SARS-CoV-2 (Pang et al., 2021). As mentioned earlier, only NAbs are known to inhibit the entry of virus inside the host cells, therefore, it becomes essential to quantitate the presence of NAbs after vaccination as the level of circulating NAbs is directly correlated to survival as well as virus control in vaccinated or infected patients (Dispensiri et al., 2021). The gold standard method of testing NAbs requires BSL-3/4 facility, which poses a risk to scientific researchers (Li et al., 2022). Therefore, a safe and reliable method needs to be developed for NAbs for the prevention of SARS-CoV-2 infection.

Tan et al. (2020) developed a surrogate SARS-CoV-2 (surrogate VNT) neutralization test that was based upon the antibody-mediated blockade of the ACE2–S protein interaction. It was approved by FDA and can be used as an alternative method for determining NAbs. Herein, we developed an ELISA method to test the presence of NAbs in the serum by making a standard curve of known concentration of NAbs and its reaction with S1-RBD biotin and negative control and sera samples of vaccinated individuals for the rapid detection of NAbs. In our method, the level of NAbs can also be determined semiquantitatively, indicating the level of NAb present in serum.

The in-house-developed neutralizing antibody ELISA kit's performance in terms of percent inhibition was compared with FDA-approved GenScript cPass kit and correlation analysis gave a Pearson coefficient value (R) of 0.716, which was significant (p < 0.001). However, when the concentration of NAb and percent inhibition of in-house-developed NAb ELISA was correlated, the Pearson coefficient R-value was only 0.3, although it was significant (p < 0.04). The gold standard method for detection of neutralizing antibody (Plaque reduction neutralization test) is always superior over any other alternate method for detection of NAbs. A comparative analysis of advantages and disadvantages of the in-house ELISA-based assay, FDA-approved GenScript's cPass assay and pseudotyped virus-based neutralization assays are elucidated in Table 4

Table 4.

Advantages and Disadvantages of Neutralization Assays

Detection method	Time	Advantage	Disadvantage
In-house ELISA-based assay	4–5 h	Easy to perform, no live or pseudo virus required, mass screening can be performed for both qualitative and quantitative analysis in ordinary laboratory setup	Lower level of neutralizing antibody less than 350 ng/mL is considered negative
FDA-approved GenScript cPass assay	1 h	Easy to perform, no live or pseudo virus required, mass screening can be performed for only qualitative analysis	Only qualitative assay
Pseudotyped-virus based neutralization assay	3–4 days	Only quantitative analysis	Complicated and time consuming procedure, requires skilled manpower

FDA, Food and Drug Administration.

In summary, our study reveals that the NAb ELISA detection assay is able to detect NAbs in human serum samples. Also, it is at par with FDA-approved GenScript cPass kit. The ELISA-based NAb testing kit developed in our laboratory can also semiquantitively measure the level of NAb in the serum. The ELISA-based NAb estimation will help in estimating the prevalence of NAbs and humoral immunity. Although the in-house-developed ELISA kit can be used for mass screening of individuals for the presence of NAbs, however, this method has some limitations, a sample containing a lower level of NAbs (<350 ng/mL) will be considered as negative, as the cutoff value for the test is 30%. Further, this method cannot assess the assay performance against variants of concern because we lack sequence data and strain typing on the subjects from which the sera were collected.

At the late stage of the pandemic, we know that the immunity induced by vaccination against SARS-CoV-2 results in improved outcomes following infection, which include reduced symptoms and severity of disease and reduced hospitalization rates (McLean et al., 2022). The analysis of NAb levels against SARS-CoV-2 using this ELISA method may be useful in determining the immune protection status at the population level.

Footnotes

Acknowledgment

The authors thank Ms. Asha D. Kushwaha, Ms. Jyotsana Bakshi, and Ms. Smriti Sudha for their support.

Authors' Contributions

Conceptualization, K.P.M., M.S., and D.S.; methodology: K.P.M., M.S., and D.S.; resources and materials: K.P.M., M.S., D.S., and S.N.S. Data analysis: K.P.M.; article writing: K.P.M.; editing: K.P.M., M.S., D.S., and S.N.S. All authors have read and agreed to the published version of the article.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by Defence Institute of Physiology and Allied Sciences, Delhi.

References

Addetia

, Crawford

, Dingens

, et al. Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with a high attack rate. J Clin Microbiol, 2020; 58(11):e02107-20; doi: 10.1128/JCM.02107-20

Andersen

, Rambaut

, Lipkin

, et al. The proximal origin of SARS-CoV-2. Nat Med, 2020; 26(4):450–452; doi: 10.1038/s41591-020-0820-9

Dispinseri

, Secchi

, Pirillo

, et al. Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival. Nat Commun, 2021; 12(1):2670; doi: 10.1038/s41467-021-22958-8

Donoghue

, Hsieh

, Baronas

, et al. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res, 2000; 87(5):E1–E9; doi: 10.1161/01.res.87.5.e1

Hoffmann

, Kleine-Weber

, Schroeder

, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell, 2020; 181(2):271–280.e8; doi: 10.1016/j.cell.2020.02.052

Huang

, Garcia-Carreras

, Hitchings

MDT

, et al. A systematic review of antibody mediated immunity to coronaviruses: Kinetics, correlates of protection, and association with severity. Nat Commun, 2020; 11(1):4704; doi: 10.1038/s41467-020-18450-4

Jackson

, Farzan

, Chen

, et al. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol, 2022; 23(1):3–20; doi: 10.1038/s41580-021-00418-x

Lan

, Ge

, Yu

, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature, 2020; 581(7807):215–220; doi: 10.1038/s41586-020-2180-5

Lempp

, Soriaga

, Montiel-Ruiz

, et al. Lectins enhance SARS-CoV-2 infection and influence neutralizing antibodies. Nature, 2021; 598(7880):342–347; doi: 10.1038/s41586-021-03925-1

10.

, Wang

, Wu

, et al. Development of a rapid neutralizing antibody test for SARS-CoV-2 and its application for neutralizing antibody screening and vaccinated serum testing. Infect Med, 2022; 1(2):95–102; doi: 10.1016/j.imj.2022.04.003

11.

Liu

, Yang

, Zhang

, et al. SARS-CoV-2 tetrameric RBD protein blocks viral infection and induces potent neutralizing antibody response. Front Immunol, 2022; 13:960094; doi: 10.3389/fimmu.2022.960094

12.

McLean

, Kamil

, Lee

, et al. The impact of evolving SARS-CoV-2 mutations and variants on COVID-19 vaccines. mBio, 2022; 13(2):e0297921; doi: 10.1128/mbio.02979-21

13.

Mishra

, Singh

. Hyperinflammation and immune response generation in COVID-19. Neuroimmunomodulation, 2021; 27(2):80–86; doi: 10.1159/000513

14.

Okba

NMA

, Müller

, Li

, et al. Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients. Emerg Infect Dis, 2020; 26(7):1478–1488; doi: 10.3201/eid2607.200841

15.

Pang

, Pang

, Chow

, et al. Understanding neutralising antibodies against SARS-CoV-2 and their implications in clinical practice. Mil Med Res, 2021; 8(1):47; doi: 10.1186/s40779-021-00342-3

16.

Pieri

, Infantino

, Manfredi

, et al. Performance evaluation of four surrogate Virus Neutralization Tests (sVNTs) in comparison to the in vivo gold standard test. Front Biosci (Landmark Ed), 2022; 27(2):74; doi: 10.31083/j.fbl2702074

17.

Satarker

, Nampoothiri

. Structural proteins in severe acute respiratory syndrome coronavirus-2. Arch Med Res, 2020; 51(6):482–491; doi: 10.1016/j.arcmed.2020.05.012

18.

Scialo

, Daniele

, Amato

, et al. ACE2: The major cell entry receptor for SARS-CoV-2. Lung, 2020; 198(6):867–877; doi: 10.1007/s00408-020-00408-4

19.

Shang

, Wan

, Luo

, et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A, 2020; 117(21):11727–11734; doi: 10.1073/pnas.2003138117

20.

Shereen

, Khan

, Kazmi

, et al. COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses. J Adv Res, 2020; 24:91–98; doi: 10.1007/s12274-021-3949-z

21.

Tai

, He

, Zhang

, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: Implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol, 2020; 17(6):613–620; doi: 10.1038/s41423-020-0400-4

22.

Tan

, Chia

, Qin

, et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat Biotechnol, 2020; 38(9):1073–1078; doi: 10.1038/s41587-020-0631-z

23.

Zhang

, Penninger

, Li

, et al. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: Molecular mechanisms and potential therapeutic target. Intensive Care Med, 2020; 46:586–590; doi: 10.1007/s00134-020-05985-9

24.

Zhou

, Yang

, Wang

, et al. Addendum: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020; 588(7836):E6; doi: 10.1038/s41586-020-2951-z