COVID-19 Serology in New York State Using a Multiplex Microsphere Immunoassay

The emergence of SARS-CoV-2, leading to COVID-19, necessitated the development of new molecular and serological tests. Here, we describe a multiplexed serological assay developed as the global pandemic moved into New York State in the spring of 2020. The original microsphere immunoassay used a target antigen from the SARS-CoV-1 virus responsible for the 2003 SARS outbreak, but evolved to incorporate multiple SARS-CoV-2 protein antigens (nucleocapsid, spike and spike domains, spike and nucleocapsid proteins from seasonal human coronaviruses). Besides being highly versatile due to multiplex capabilities, the assay was highly specific and sensitive and adaptable to measuring both total antibodies and antibody isotypes. While determining the assay performance characteristics, we were able to identify antibody production patterns (e.g., kinetics of isotypes, individual variations) for total antibodies and individual antibody classes. Overall, the results provide insights into the laboratory response to new serology needs, and how the evolution and fine-tuning of a serology assay helped contribute to a better understanding of the antibody response to SARS-CoV-2.

Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

COVID-19 is associated with a wide spectrum of disease severity, ranging from asymptomatic to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity, and the levels of circulating SARS-CoV-2-specific antibodies, including virus neutralizing titers. Through a serological analysis of serum samples from 536 convalescent healthcare workers, we found that SARS-CoV-2-specific and virus-neutralizing antibody levels were indeed elevated in individuals that experienced severe disease. The severity-associated increase in SARS-CoV-2-specific antibody was dominated by IgG, with an IgG subclass ratio skewed towards elevated receptor binding domain (RBD)- and S1-specific IgG3. However, RBD- and S1-specific IgG1, rather than IgG3 were best correlated with virus-neutralizing titers. We propose that Spike-specific IgG3 subclass utilization contributes to COVID-19 disease severity through potent Fc-mediated effector functions. These results have significant implications for SARS-CoV-2 vaccine design, and convalescent plasma therapy.

Neutralizing Antibody Responses in COVID-19 Convalescent Sera

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administrations (FDA) guidelines for convalescent plasma recommends target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at low (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was [~]93% (PRNT50) and [~]54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 ELISA titers) showed maximal activity, but not all high titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.