Serum samples were collected at dpi 0 (baseline), 1, 3, 5, 7, 10, 12, 14, 17 and 21. response triggered LAG3 by SARS-CoV-2 infection. IgG antibody cross-reactivity with SARS-CoV-1 and MERS-CoV spike proteins (Figure 5B), by combining serology data from pre-2019 ARIC sera and SARS-CoV-2 PCR negative subjects, and comparing to SARS-CoV-2 PCR positive subjects. IgG antibody levels detected with all five -CoV spike proteins in the SARS-CoV-2 PCR positive patient serum samples were significantly higher than those in pre-2019 ARIC serum samples and SARS-CoV-2 negative patient serum samples (Figure 6A-E). These levels of SARS-CoV-1, MERS-CoV, HKU1 and OC43 reactive IgG antibodies after SARS-CoV-2 infection provide compelling evidence that SARS-CoV-2 stimulates a polyclonal antibody response containing cross-reactive antibodies that can bind to spike proteins from closely and distantly-related -CoVs. Further, a MMIA analysis of available longitudinal serum samples provided a novel glimpse of potential differences in cross-reactive antibody responses. Maximum OC43 antibody levels (blue arrow) are reached at earlier dpso after SARS-CoV-2 infection than are SARS-CoV-1 and MERS-CoV-2 levels (red arrow), suggesting that SARS-CoV-2 stimulates an OC43 humoral memory response in some subjects, independent of the SARS-CoV-2 IgG response that is cross-reactive with MERS-CoV and SARS-CoV-1 spike proteins (Figure 7A-D). Further, OC43 reactive IgG responses were observed to Dihydrexidine wane in parallel with SARS-CoV-2 IgG (Figure 7B-C), suggesting a temporal linkage between these -CoV specific IgG responses. Discussion In this study, we have demonstrated that use of a multiplex microsphere-based immunoassay (MMIA) built using Luminex xMAP-based technology in which individual microspheres are bound to pre-fusion stabilized S glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and each of the two seasonal -CoVs enables highly sensitive and specific detection of SARS-CoV-2 IgG antibodies. In contrast to commercial ELISA and lateral flow assays for SARS-CoV-2 IgG, which typically have a sensitivity in the range of 65C70% up to 14 days after symptom onset (10), the MMIA has a sensitivity of 98% at just 10 days after symptom onset in PCR-confirmed cases of SARS-CoV-2 infection. Use of this highly sensitive MMIA will enhance studies on the kinetics of humoral responses to SARS-CoV-2 infection. We hypothesize that the high sensitivity and specificity of this assay for SARS-CoV-2 serology is due both to the Dihydrexidine physics of the Luminex xMAP-based Dihydrexidine platform, which enables a high dynamic range of measurement, as well as its multiplexing design with CoV S glycoproteins. Multiplex microsphere-based immunoassays have been shown to be more sensitive than standard ELISA for SARS-CoV-2 antibody detection (25) and several other virus infections, including Lassa virus, Ebola virus, and simian immunodeficiency virus (11C13). Additionally, and perhaps more importantly, simultaneously incubating serum against spike proteins of seasonal HCoVs appears to enable the establishment of a lower threshold of positivity for detection of SARS-CoV-2 specific antibodies. Given the presence of cross-reactive antibodies, assays that only test for antibodies against the SARS-CoV-2 spike protein may have to utilize a high signal threshold as a cut-off for positivity to reduce false positive rates. By incubating serum against multiple CoV spike proteins, the MMIA platform would allow for the preferential binding of cross-reactive antibodies to the antigens of the CoV against which they were initially induced, thus enabling a lower, and apparently more sensitive, cut-off for the detection of SARS-CoV-2 specific Dihydrexidine antibodies. Early in the COVID-19 pandemic, SARS-CoV-2 IgG seroconversion was surprisingly detected early after exposure and sometimes in parallel with IgM seroconversion (26C28). The temporal Dihydrexidine window to capture SARS-CoV-2 IgM is shorter than IgG, and with IgG seroconversion occurring 10 dpi in NHP SARS-CoV-2 disease models, and detectable as early as 7 dpso in subjects, there appears little benefit for continued SARS-CoV-2 IgM detection (Table S2). As we placed no upper limit on the dpso of the first serum collection included in performance analysis, the IgM sensitivity is lower than IgG, driven by outpatient enrollments in the EPICC, IDCRP-085 protocol with an average 28 dpso that were IgG positive, but IgM negative. Although less sensitive and less specific than IgG detection, the benefit of IgM detection may lay in its ability to place a temporal window on SARS-CoV-2 exposure in asymptomatic IgG positive individuals, which would be.
