ABSTRACT
Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays. Results: DBS eluates from convalescent individuals were compatible with RBD-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay, and commercial real-time PCR-based neutralization assay (NAB-Sure) but not cell-based pseudovirus assays or PRNT. The insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.
Subject(s)
COVID-19ABSTRACT
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.
Subject(s)
COVID-19ABSTRACT
COVID-19 convalescent plasma (CCP) received approval for use under an Emergency Use Authorization by the FDA for treatment of seriously ill patients. Use of CCP units with a signal-to-cutoff ratio of [≥]12 using the Ortho VITROS SARS-CoV-2 IgG test (OVSARS2IgG) is authorized. Little is known about the relationship between this ratio and the neutralizing capacity of plasma/sera against genuine SARS-CoV-2 virus. We measured the neutralizing capacity of 981 samples from 196 CCP donors 7-119 days post initial donation (DPID). Neutralizing capacity was assessed for 50% (PRNT50) and 90% (PRNT90) reduction of infectious virus using the gold standard plaque reduction neutralization test (PRNT). Importantly, while 32.7%/79.5% (PRNT90/PRNT50) of donations met the FDA minimum titer of 1:80 initially, only 14.0%/48.8% (PRNT90/PRNT50) met this cut-off [≥]85 DPID. A subset of 91 donations were evaluated using the OVSARS2IgG and compared to PRNT titers for diagnostic accuracy. The correlation of OVSARS2IgG results to neutralizing capacity allowed extrapolation to CCP therapy efficacy results. CCP with OVSARS2IgG ratios in the therapeutically beneficial group had neutralizing titers of [≥]1:640 (PRNT50) and/or [≥]1:80 (PRNT90). This information provides a new basis for refining the recommended properties of CCP that is used to treat severe COVID-19.
Subject(s)
COVID-19ABSTRACT
With the COVID-19 pandemic surpassing 12M confirmed cases and 550K deaths worldwide, defining the key components of the immune response to SARS-CoV-2 infection is critical. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present and validate a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike (S) protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). This test is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD and serves as a surrogate neutralization assay.