ABSTRACT
SARS-CoV-2 has infected millions of people and is on a trajectory to kill more than one million globally. Virus entry depends on the receptor-binding domain (RBD) of the spike protein. Although previous studies demonstrated anti-spike and -RBD antibodies as essential for protection and convalescent plasma as a promising therapeutic option, little is known about the immunoglobulin (Ig) isotypes capable of blocking virus entry. Here, we studied spike- and RBD-specific Ig isotypes in plasma/sera from two acutely infected and 29 convalescent individuals. Spike- and RBD-specific IgM, IgG1, and IgA1 antibodies were produced by all or nearly all subjects at varying levels and detected at 7-8 days post-disease onset. IgG2, IgG3, IgG4, and IgA2 were also present but at much lower levels. All samples also displayed neutralizing activity. IgM, IgG, and IgA were capable of mediating neutralization, but neutralization titers correlated better with binding levels of IgM and IgA1 than IgG.
ABSTRACT
BackgroundMore 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. MethodsA 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. FindingsFluorochrome-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 subjects, delineating a wide range of serum antibody levels in infected subjects. InterpretationAntibody 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. FundingNIAID 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. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe outbreak of infections with SARS-CoV-2 began in late 2019. Specimens from nasopharyngeal swabs are being used in PCR-based assays to test for the presence of the virus. Until the first week in April, 2020 there were no licensed tests for the presence of serum antibodies against proteins of the virus. The first approved tests are now becoming available, but none use a format that can be scaled up for mass screening which is now needed for implementing various public health measures. As per a recent Pubmed search, less than 10 studies using serologic assays have been published and none are high through-put. Added value of this studyHigh through-put antibody tests are needed in order to identify seroconversion, to perform serosurveys, identify potential donors for plasma therapy, assess the prevalence of infection in populations, identify healthcare workers who may be immune to SARS-CoV-2, and to study the nature of the immune response to this pathogen. The method described for detecting antibodies in SARS-CoV-2-infected patients can be applied in hospital and reference labs, allowing the assessment of present and past infection in a much higher number of donors per unit of time than assays described heretofore. Implications of all the available evidenceThis study shows that a test in which magnetic beads are coated with soluble forms of the spike protein of SARS-CoV-2 can be used to test for the presence of antibodies targeting this pathogen. The platform allows for the efficient testing of multiple specimens simultaneously using as little as 5 nanograms of antigen per test. This test affords the possibility of large scale, economical and efficient antibody testing.
