A Combination of N and S Antigens With IgA and IgG Measurement Strengthens the Accuracy of SARS-CoV-2 Serodiagnostics

Abstract

Background Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. Methods We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. Results The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. Conclusions The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.

A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies

“…The SARS-CoV-2 EIA used in this study has previously been described in detail ( 15 ). We measured IgG, IgA, IgM, and total Ig antibodies against SARS-CoV-2 S1 and N proteins from sera diluted 1:300.…”

“…The assay is based on an FMIA previously described by Trivedi et al ( 13 ) We have recently reported the performance of the FMIA for measuring IgG antibodies against SARS-CoV-2 nucleoprotein ( 14 ). Here, we report our findings on the analytical and clinical performance of FMIA and compare its IgG, IgA, and IgM assay results to another laboratory’s in-house enzyme immunoassay (EIA) ( 15 , 16 ), both calibrated for IgG with the WHO international standard ( 17 ). Furthermore, we compare FMIA antibody levels to neutralizing antibody (NAb) titers ( 14 , 18 ) and NAb titers of microneutralization tests (MNTs) between two separate laboratories.…”

SARS-CoV-2 Isolates Show Impaired Replication in Human Immune Cells but Differential Ability to Replicate and Induce Innate Immunity in Lung Epithelial Cells

“…SARS-CoV N protein- and SARS-CoV-2 S1 protein-specific rabbit antibodies were prepared against baculovirus-expressed preparative SDS-PAGE-purified SARS-CoV N protein ( 27 ) or HEK293 cell-produced S1 ( 64 ). Three New Zealand White rabbits were immunized four times with 50 μg/dose/rabbit of N or S1 protein at 4- or 3-week intervals, respectively.…”

COVID-19 adenovirus vaccine triggers antibodies against PF4 complexes to activate complement and platelets

“…Cut-off values for HAdV-2 hexon and ChAdOx1 were calculated as the mean EIA units plus three times standard deviations (SDs) of 20 negative serum samples. Positive control specimens included samples with high antibody levels against SARS-CoV-2 S1 and N proteins [12] and adenovirus hexon proteins [10] , assigned EIA unit values of 100, respectively.…”

“…96-well plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated with 4 μg/mL AdV2 hexon protein and 6 μg/mL ChAdOx1 nCoV-19 vaccine (Vaxzevria®, AstraZeneca) in PBS for 16 h at +4 • C. HAdV-2 was cultivated in HeLa cells, and hexon protein was purified with anion-exchange liquid chromatography [11]. SARS-CoV-2 nucleoprotein (N) and spike protein (S1)-specific IgG antibodies were detected with an in-house EIA described previously [12]. Cut-off values for HAdV-2 hexon and ChAdOx1 were calculated as the mean EIA units plus three times standard deviations (SDs) of 20 negative serum samples.…”