Throughput Assay for Monitoring COVID-19 Antibodies and Their Isotypes (Webinar)
Presenter:
Susan Zolla-Pazner, PhD
Professor of Medicine, Division of Infectious Diseases Icahn School of Medicine at Mount Sinai
Abstract:
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 COVID-19, 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 COVID-19 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 greater than 140 COVID-infected and uninfected individuals (p values less than 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 COVID-19 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.
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