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Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay.

ABSTRACT: This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the 'gold standard' assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R² = 0.975, n = 25), demonstrating high sensitivity.

SUBMITTER: Shalash AO

PROVIDER: S-EPMC8705285 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection.MethodsWe recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background.ResultsThe optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative.ConclusionsWe conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Project description:Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

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Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay.

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