Going beyond clinical routine in SARS CoV 2 antibody testing A multiplex corona virus antibody test for the evaluation of cross reactivity to endemic coronavirus antigens

Given the importance of the humoral immune response to SARS-CoV-2 as a global benchmark for immunity, a detailed analysis is needed to monitor seroconversion in the general population, understand manifestation and progression of COVID-19 disease, and ultimately predict the outcome of vaccine development. In contrast to currently available serological assays, which are only able to resolve the SARS-CoV-2 antibody response on an individual antigen level, we developed a multiplex immunoassay, for which we included spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses (NL63, OC43, 229E, HKU1) in an expanded antigen panel. Compared to three commercial in vitro diagnostic tests, our MULTICOV-AB assay achieved the highest sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. Simultaneously, high IgG responses against endemic coronaviruses became apparent throughout all samples, but no consistent cross-reactive IgG response patterns could be defined. In summary, we have established and validated, a robust, high-content-enabled, and antigen-saving multiplex assay MULTICOV-AB, which is highly suited to monitor vaccination studies and will facilitate epidemiologic screenings for the humoral immunity toward pandemic as well as endemic coronaviruses. ### Competing Interest Statement Yes there is potential Competing Interest. Dr Thomas Joos is a scientific advisor for Luminex. Dr Nicole Schneiderhan-Marra was a speaker at Luminex user meetings in the past. The Natural and Medical Sciences Institute at the University of Tuebingen is involved in applied research projects as a fee for services with Luminex. ### Funding Statement This work has received funding from the European Unions Horizon 2020 research and innovation programme under grant agreement No 101003480 - CORESMA. ### 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: A total of 1176 sera and plasma samples was used for CoVi-plex development. Ethical approval was granted from the Ethics Committee of Hannover Medical School (#9122\_BO\_K2020). Only de-identified samples were used for CoVi-plex development. All samples were pre-existing. Cohort age was 5-88 years; age was not known for 161 samples. 310 samples were from COVID-19 patients or convalescents. Samples were classified as SARS-CoV-2 infected, if a positive SARS-CoV-2 RT-PCR was reported and/ or if hospitalization/ quarantine for COVID-19 was indicated as part of the samples metadata. ΔT defined as time between PCR test or symptom onset and blood draw was 0-73 days (median= 38 d; n=258). ΔT was not provided for 52 samples. SARS-CoV-2 infected samples used in this study were collected after ethical review (9001\_BO\_K, Hannover Medical School; 179/2020/BO2, University Hospital Tuebingen). 866 control samples were from non-SARS-CoV-2 infected individuals and were classified as non-infected as they were obtained prior to the emergence of SARS-CoV-2 in December 2019 or because they were taken from individuals who had not reported cold symptoms since the beginning of 2020. The majority of non-SARS-COV-2 infected samples was randomly selected and consistent of prepandemic blood donors, commercially available (Central BioHub GmbH, Berlin, Germany and BBI Solutions, Crumlin, UK) or bio-banked specimens. 365 samples were from the Memory and Morbidity in Augsburg Elderly (MEMO) study (a subcohort of the MONICA S2 cohort (WHO 1988)) and were included based on available serological titers for HSV-1, HSV-2, HHV-6 and EBV23. 88 samples were obtained from transplanted patients with chronic respiratory conditions. Collection of non-SARS-CoV-2 infected control samples had been approved by several ethic votes: 3232-2016 (Ethics Committee of Hannover Medical School); 62/20 (Ethics Committees of the Medical Faculty of the Saarland University at the Saarland Aerztekammer); WUM 17.02.1997 (Joint ethics committee of the University of Muenster and the Westphalian Chamber of Physicians). 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 Raw data and analysis files are available for download as supplemental files of this manuscript