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
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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.
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SciScore for 10.1101/2020.09.15.20192765: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding The results were scored as positive or negative for IgM and IgG by two independent readers blinded to donor sample status. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources The plates were again washed three times with PBS containing 0.05% Tween 20 (PBST) and banged on absorbent paper towels, and immediately anti-human horseradish peroxidase (HRP)-conjugated secondary antibodies for IgG (cat#A18817, Thermo Fisher, 1:2000), IgM (cat#A18841, Thermo Fisher, 1:8000), and IgA (Jackson Immunoresearch, cat#109-035-011, 1:2000) diluted in casein blocking … SciScore for 10.1101/2020.09.15.20192765: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding The results were scored as positive or negative for IgM and IgG by two independent readers blinded to donor sample status. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources The plates were again washed three times with PBS containing 0.05% Tween 20 (PBST) and banged on absorbent paper towels, and immediately anti-human horseradish peroxidase (HRP)-conjugated secondary antibodies for IgG (cat#A18817, Thermo Fisher, 1:2000), IgM (cat#A18841, Thermo Fisher, 1:8000), and IgA (Jackson Immunoresearch, cat#109-035-011, 1:2000) diluted in casein blocking buffer were added to the plates at 50μl per well for 30 minutes at room temperature. anti-human horseradish peroxidase ( HRP)-conjugated secondary antibodies for IgGsuggested: Nonecat#A18817suggested: (Thermo Fisher Scientific Cat# A18841, RRID:AB_2535618)cat#A18841suggested: (Jackson ImmunoResearch Labs Cat# 109-035-011, RRID:AB_2337580)Determination of the presence versus absence of SARS-CoV-2 reactive antibodies in samples and of Arbitrary Unit Values: First, the average ODs of corresponding ‘blank’ wells (sample diluent only in buffer only coated or antigen coated) on a given plate was subtracted from all wells with samples. antigen coatedsuggested: Nones DISCOVID IgM IgG LFD test was used to detect SARS-CoV-2 RBD specific IgM and IgG antibodies following manufacturer instructions. IgGsuggested: NoneSoftware and Algorithms Sentences Resources Determination of Arbitrary Units: Data were analyzed using GraphPad Prism 8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:However, a limitation of this assay is that it is currently considerably lower in throughput compared to other serological platforms. This protocol requires an operator for the manual wash steps, limiting the number of plates that can be run compared to automated methods, however, there is potential for throughput increase if automated washers/ELISA systems can be adapted to more closely mimic this protocol. Other important features of our approach include the inclusion of paired sample dilutions with buffer only coated wells to enable detection of true antigen-reactive signal and adjustment of the length of substrate incubation time based on standard curve development for OD standardization to enable direct comparison between samples on different plates. Quantification of relative antibody levels via Arbitrary Units (AU) or a similar method will be imperative for determining which convalescent samples have antibody levels sufficient for effective plasma transfer as well as other applications. However, while we believe this is a preferred approach for determining of relative output values within all samples, it is critical to note that the unique dynamics of the panoply of antibodies of varying affinities and isotypes within a given specimen causes inherent confounding factors to serologic readouts. For example, a specimen with a high level of SARS-CoV-2 RBD reactive IgM antibodies could have a lower detected signal for IgG and IgA due to IgM’s pentameric conformation blockin...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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