A High-Throughput Assay for Circulating Antibodies Directed Against the S Protein of Severe Acute Respiratory Syndrome Coronavirus 2

Abstract

More than 24 million infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were confirmed globally by September 2020. While polymerase chain reaction-based assays are used for diagnosis, there is a need for high-throughput, rapid serologic methods. A Luminex binding assay was developed and used to assess simultaneously the presence of coronavirus disease 2019 (COVID-19)-specific antibodies in human serum and plasma. Clear differentiation was achieved between specimens from infected and uninfected subjects, and a wide range of serum/plasma antibody levels was delineated in infected subjects. All 25 specimens from 18 patients with COVID-19 were positive in the assays with both the trimeric spike and the receptor-binding domain proteins. None of the 13 specimens from uninfected subjects displayed antibodies to either antigen. There was a highly statistically significant difference between the antibody levels of COVID-19-infected and -uninfected specimens (P < .0001). This high-throughput antibody assay is accurate, requires only 2.5 hours, and uses 5 ng of antigen per test.

Keywords: COVID-19; SARS-CoV-2; antibodies; antibody assay; coronavirus.

Figure 1.

Screening for the presence of severe acute respiratory syndrome coronavirus 2 antibodies in specimens from coronavirus disease 2019–infected (pos.) and –uninfected (neg.) humans. Assays were run using the S protein produced in mammalian cells (mSpike, left) and mRBD (right). Results are shown using sera tested at a dilution of 1:200. For specimens from 4 patients (P1–P4) run against both antigens, the data shown are the mean + standard deviation of 2–5 experiments. For 4 patients (P5–P8), the specimens were run only against the mSpike in a single experiment. Abbreviations: COVID-19, coronavirus disease 2019; MFI, mean fluorescence intensity; mRBD, mammalian-derived Receptor Binding Domain polypeptide; mSpike, mammalian-derived Spike protein.

Figure 2.

Screening for the presence of severe acute respiratory syndrome coronavirus 2 antibodies in plasma from coronavirus disease 2019 (COVID-19)–positive subjects (COVID-19 pos.) and from plasma derived from the general blood bank plasma inventory (COVID-19 neg.). Assays were run using the S protein produced in mammalian cells: mSpike (left) and mRBD (right). Results are shown using plasma tested at a dilution of 1:200. Statistical analysis using the Mann–Whitney test is shown. Medians with error bars representing standard deviation are indicated. Abbreviations: COVID-19, coronavirus disease 2019; MFI, mean fluorescence intensity; mRBD, mammalian-derived Receptor Binding Domain polypeptide; mSpike, mammalian-derived Spike protein.

Figure 3.

Titration of coronavirus disease 2019 (COVID-19)–positive/negative sera. Specimens were diluted at 2-fold dilutions from 1:50 to 1:6400 and tested using the Luminex assay described herein. Titration curves are shown for sera from 11 specimens from 8 infected patients using the mSpike as antigen (left) and for sera from 3 patients tested vs mRBD (right). Abbreviations: COVID-19, coronavirus disease 2019; MFI, mean fluorescence intensity; mRBD, mammalian-derived Receptor Binding Domain polypeptide; mSpike, mammalian-derived Spike protein.

Figure 4.

Steps and time required for detection of antibodies to the mSpike or mRBD of severe acute respiratory syndrome coronavirus 2 antigens using the Luminex antibody binding assay described herein. Washing steps, delineated in the Materials and Methods, add approximately 30 minutes, resulting in a test requiring approximately 2.5 hours. Abbreviations: Ag, antigen; Ig, immunoglobulin; PE, phycoerythrin.