Sensitive and Specific Detection of SARS-CoV-2 Antibodies Using a High-Throughput, Fully Automated Liquid-Handling Robotic System

Abstract

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.

Keywords: PCR; agglutination; antibody; anti–spike protein; liquid handling.

Figure 1.

Instrument deck layout, based on the Microlab ADAP STAR.

Figure 2.

Antibody detection by agglutination (ADAP) differs from traditional immunoassays in that it is a pure solution-phase assay and is well-suited to detecting sensitive antibodies. The automated SARS-CoV-2 total antibody assay on the ADAP STAR is a completely hands-free workflow that uses a pair of antigen-DNA conjugates to probe target antibodies within a given sample (circle 1). If antibodies are present, then the antibody and antigen agglutinate, and close proximity allows the DNA conjugates to ligate (circle 2), leading to the formation of specific DNA amplicons that can then be quantified offline using qPCR (Circle 3).

Figure 3.

High correlation between the automated and manually performed antibody detection by agglutination assays. The assay cutoffs were 1.40 for both manual and automated methods.

Figure 4.

Bland-Altman plot to compare signals from the automated and manual antibody detection by agglutination assays. The middle dashed and dotted line is the mean difference, whereas the dotted lines to either side (top and bottom) mark the limits of two standard deviations from the mean.