SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing

Background To determine how serologic antibody testing outcome links with virus neutralization of SARS-CoV-2 to ascertain immune protection status, we evaluated a unique set of individuals for SARS-CoV-2 antibody detection and viral neutralization. Methods Herein, we compare several analytic platforms with 15 positive and 30 negative SARS-CoV-2 infected controls followed by viral neutralization assessment. We then applied these platforms in a clinically relevant population: 114 individuals with unknown histories of SARS-CoV-2 infection. Results In control populations, the best performing antibody detection assays were SARS-CoV-2 receptor binding domain (RBD) IgG (specificity 87%, sensitivity 100%, PPV 100%, NPV 93%), spike IgG3 (specificity 93%, sensitivity 97%, PPV 93%, NPV 97%), and nucleocapsid (NP) protein IgG (specificity 93%, sensitivity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. 20 unknown individuals had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. The antibody assays that best predicted virus neutralization were RBD IgG (misidentified 2), spike IgG3 (misidentified 1), and NP IgG (misidentified 2). Conclusion These data suggest that meaningful evaluation of antibody assay performance requires testing in an unknown population. Further, these results indicate coupling of virus neutralization analysis to a positive antibody test is required to categorize patients based on SARS-CoV-2 immune protection status following virus exposure or vaccine administration. One of the antibody detection platforms identified in this study followed by the pseudoneutralization or focus reduction assay would provide a practical testing strategy to assess for SARS-CoV-2 antibodies with optimal prediction of correlates to neutralizing immunity. Funding Supported by NIH grants AI148684, AI151698, AI145296, and UW funds to the Center for Innate Immunity and Immune Disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Supported by NIH grants AI148684, AI151698, AI145296, and UW funds to the Center for Innate Immunity and Immune Disease. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Approved by UW IRB: Study 00009810 All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data that is not included in the manuscript and supplemental material is available upon request to interested parties. Data will not be released if it violates the privacy of our subjects as required by our IRB.