High-Throughput Assay for Monitoring COVID19 Antibodies and Their Isotypes

The 2020 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to rage. While PCR-based assays are used for viral diagnosis, high through-put, rapid serologic methods are needed to assess the prevalence of COVID19, the disease caused by SARS-CoV-2, and to identify persons previously infected.  In particular, high through-put antibody (Ab) assays are needed for mass screening.  A Luminex binding assay is described that simultaneously assesses the presence and level of circulating Abs specific for the S protein of SARS-CoV-2 and for its receptor binding domain (RBD). For this, fluorochrome-labeled magnetic beads are coated with a recombinant soluble stabilized trimeric form of the SARS-CoV-2 S protein ectodomain or with the RBD. Coated beads are incubated with serum or plasma, biotinylated anti-human Abs specific for total immunoglobulins, and phycoerythrin-labeled streptavidin, followed by a read-out using a Luminex analyzer. Isotypes of the Abs are quantified by using isotype-specific biotinylated secondary Abs (anti-IgG, -IgG1, -IgG2, -IgG3, -IgG4, -IgA1, -IgA2, and -IgM). The COVID19 Luminex Ab assay is both qualitative (binary) and quantitative. There was a statistically significant difference in total immunoglobulin anti-S and anti-RBD Ab levels when comparing serum or plasma from >140 COVID-infected and uninfected individuals (p<0.0001). The advantages of the Luminex over the ELISA platform include: (a) the use of 20-fold less antigen per test, (b) a 2.5-fold faster run time, (c) a turn-around time of ½ day vs. 2 days, (d) performance of the RBD screen and S protein titration simultaneously rather than sequentially, and (e) a sensitivity 5-10x greater than that of ELISA, resulting in far fewer false negative results.  Delineation of COVID19 Ab isotypes using the Luminex platform also showed  differences in the patterns of IgG, IgA, and IgM from patient-to-patient, and IgA and IgM Ab response rates which are higher than IgG response rates.

Learning Objectives:

1. Impart need for and ways to perform mass COVID19 antibody screening

2. Educate about the isotypes of antibodies being induced by SARS-CoV-2 infections