A High Through-put Assay for Circulating Antibodies Directed against the S Protein of Severe Acute Respiratory Syndrome Corona virus 2

Abstract

Background: More than one million infections with the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) have been confirmed. While PCR-based assays are used for diagnosis, high through-put serologic methods are needed to detect antibodies for seroserveillance and for identification of seroconversion, potential plasma donors, and the nature of the immune response to this pathogen.

Methods: A Luminex binding assay was used to assess the presence of antibodies in human sera from COVID-19-infected and -uninfected individuals specific for two recombinant proteins of SARS-CoV-2.

Findings: Fluorochrome-labeled beads were coated with a recombinant soluble stabilized trimeric SARS-CoV-2 S protein ectodomain or its central portion, the receptor binding domain (RBD). Coated beads were incubated with sera, followed by incubation with biotinylated anti-human total Ig antibodies and phycoerythrin (PE)-labeled streptavidin. Readout using a Luminex analyzer clearly differentiated between sera of the infected and uninfected subject, delineating a wide range of serum antibody levels in infected subjects.

Interpretation: Antibody assays of sera can identify individuals who are infected with SARS-CoV-2 and have seroconverted, as well as subjects who have been infected and recovered. The use of the Luminex binding Ab assay has the advantage that it can be run in approximately 2.5 hours, uses very little antigen, and permits a high through-put of samples/day.

Funding: NIAID contracts and grants, Department of Veterans Affairs grants, the Microbiology Laboratory Clinical Services, Translational Science Hub, and Personalized Virology Initiative, and Department of Medicine of Mount Sinai Health System and Icahn School of Medicine at Mount Sinai.

Figure 1.

Screening for the presence of SARS-CoV-2 antibodies in specimens from COVID-19-infected and uninfected (neg) humans. Assays were run using the S protein produced in mammalian cells (mSpike, left) with sera from eight infected patients and with sera from four of these patients with mRBD (right). Results are shown using sera tested at a dilution of 1:200. For specimens from four patients (P#1, 2, 3 and 4) run against both antigens, the data shown are the mean + S. D. of two to five experiments. For four patients (P#5–8), the specimens were run only against the mSpike in a single experiment.

Figure 2.

Titration of COVID-19-positive and negative sera. Specimens were diluted at 2-fold dilutions from 1:50 – 1:6,400 and tested using the Luminex assay described herein. Titration curves are shown for sera from 11 specimens from eight infected patients using the mSpike as antigen (left) and for sera from three patients tested vs. mRBD (right).

Figure 3.

Steps and time required for detection of antibodies to the mSpike or mRBD of SARS-CoV-2 antigens using the Luminex antibody binding assay described herein. Washing steps, delineated in Methods, add approximately 30 minutes, resulting in a test requiring approximately 2.5 hr.