Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection

Abstract

The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this ‘BU ELISA’ method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.

The impact of maternal SARS‐CoV‐2 vaccination and first trimester infection on feto‐maternal immune responses

“…Antibodies reactive to SARS‐CoV‐2 receptor binding domain (RBD) of the spike protein and nucleocapsid protein (N‐protein) were assayed from plasma following the BU ELISA protocol as previously described. 29 Subject maternal or infant (cord/neonatal) serum or dilutions of monoclonal SARS‐CoV‐2 reactive antibodies (RBD IgG, clone CR3022, gift from the Alter lab at Ragon Institute) were added to 96‐well plates coated with the appropriate SARS‐CoV‐2 protein. The secondary antibody was anti‐human horseradish peroxidase (HRP)‐conjugated antibody against IgG (cat#A18817, Thermo Fisher, 1:2000), and detection was performed with 3,3′,5,5′‐Tetramethylbenzidine (TMB)‐ELISA substrate solution (Thermo Fisher Scientific, cat# 34029) with accompanying stop solution for TMB (Thermo Fisher Scientific, cat#N600).…”

Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection Associates With Unstable Housing and Occurs in the Presence of Antibodies

“…SARS-CoV-2 antibody levels were quantified using a previously described enzyme-linked immunosorbent assay protocol [ 24 ]. CR3022 antibody (Abcam 273073) served as a positive control against SARS-CoV-2 receptor-binding domain (RBD).…”

On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination

“…Moreover, the level of antibodies directed to RBD strongly correlates with a neutralizing response 14 , 26 , 27 , providing useful information when handling the viral particle is not possible. Furthermore, the combination of three antibody isotypes (IgG, IgA and IgM) improves the efficiency of using the test for diagnostic purposes 28 . While IgM may represent a recent infection complementing the molecular diagnosis for SARS-CoV-2 29 , a recent study showed a high incidence of IgA response in PCR + samples, even when they did not show IgM reactivity 30 .…”