Development of an in- house IgM and IgG ELISA for detection of exposure to SARS-COV2

Abstract

Background: Coronavirus disease of 2019 (COVID-19), proved a fatal respiratory disease and affected millions globally. Due to the faster transmission nature of the virus and any other predicted forms of viral mutations, countries should strengthen the diagnostic testing preparedness. Reliable and accurate diagnostic tools are urgently needed to aid in massive screening and epidemiological surveillances. Serological techniques are vital not only for diagnosis but surveillance of immunity to natural exposure and vaccine responses. Methods: In this study, we developed a SARS-CoV-2 spike (S1) protein IgM and IgG indirect enzyme-linked immunosorbent assay (ELISA) protocol to detect SARS-CoV-2 specific antibodies. For all optimization procedures, we utilized commercially procured items and fully characterized sera samples stored in IBHR3AU laboratories to support SARS-COV-2 diagnostics and research purposes. Results: Four commercial anti-SARS-COV-2 antibodies were characterised to obtain an in house positive control. Invitrogen monoclonal antibody from ebiosciences was selected as the in-house control with a high mean OD values at 2.9 for pre-coated and in-house coated micro well plates. Spike antigen S1 protein was detected at 100ng/well, peaking at 500ng/well and 1500ng/well for IgG and IgM antibodies respectively. Phosphate buffered saline (0.05% tween 20 PBS) was ideal for protein dilution, with overnight antigen coating at 4oC and 10% Bovine serum albumin demonstrated best blocking properties with mean OD values of 1.27. Conclusion: SARS-COV-2 Spike protein antigen concentration of 500ng/well and 1500ng/well for IgG and IgM antibodies respectively were optimal for micro-well plate coating, with a 1:50 serum sample dilutions of 1mg/ml, for detection of antibodies to SARS-COV-2 in human serum.