key: cord-353099-38bz0acw authors: Tang, Mei San; Case, James Brett; Franks, Caroline E.; Chen, Rita E.; Anderson, Neil W.; Henderson, Jeffrey P.; Diamond, Michael S.; Gronowski, Ann M.; Farnsworth, Christopher W. title: Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays date: 2020-07-02 journal: bioRxiv DOI: 10.1101/2020.07.01.182220 sha: doc_id: 353099 cord_uid: 38bz0acw Introduction Commercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there is limited published data associating the results from commercial assays with neutralizing antibodies. Methods 67 specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes. Results The correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46 respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) & 56% (30-80); Abbott was 96% (88-99) & 69% (44-86); and EUROIMMUN was 91% (80-96) & 81% (57-93) for distinguishing neutralizing antibodies. Patients who died, were intubated, or had a cardiac injury from COVID-19 infection had significantly higher neutralizing titers relative to those with mild symptoms. Conclusion COVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization. comparisons, all specimens were >d10 post-symptom onset. All statistical analyses 149 were performed with GraphPad Prism 8 (GraphPad). The correlation of the SARS-CoV-2 neutralizing titer with the ratio reported by the 162 Roche, Abbott, and EI assays was 0.29, 0.47, and 0.46 respectively (Figure 2A-C) . 163 Higher neutralizing titers were generally associated with a higher ratio as measured by 164 all three assays. At a cutoff of 1:32 for the neutralizing assay, the concordance kappa 165 with Roche was 0.61 (95% CI; 0.35-0.86), with Abbott was 0.65 (0.42-0.88), and with 166 EI was 0.69 (0.49-0.89). For all three assays, the concordance decreased with an 167 increased threshold for neutralizing titers. contrast, no significant difference in ratio was observed between patients that died from 199 COVID-19 compared to those that survived using the Roche, Abbott, or EI assays. 200 Increased neutralizing antibody titers were also higher in patients that were intubated, 201 had cardiac injury, or AKI relative to those with milder COVID-19 symptoms ( Figure 202 4B-D). In contrast, no significant differences were noted between the groups regardless 203 of outcomes when using the Roche, Abbott, and EI assays. However, similar non-204 significant trends (i.e., increase in ratio) were observed in patients who were intubated, 205 had cardiac injury, or AKI with the EI assay. Neutralizing titers trended higher in male 206 patients and patients >60 years old, although this was not statistically significant. 207 Similar trends were observed with the serology assay ratios as well (Supplemental 208 were no significant differences in outcomes between patients. However, there was an 210 increase in the ratio observed in high neutralizing titer patients (6.3, 95% CI; 5.7-6.9) 211 compared to low titer patients (5.1, 95% CI; 4.1-6.1) on the Abbott assay and the EI 212 assay (8.2, 7.1-9.2 vs. 6.1, 4.6-7.6) (Supplemental Table 1 all three commercial assays correlated with higher neutralizing titers, this was not 246 universally true. Consistent with this, the correlation between neutralizing titers and 247 serological results were <0.5 on all three commercial assays. These findings are neutralizing SARS-CoV-2 titers with anti-RBD IgG or anti-S IgG using laboratory 250 developed ELISAs (23). Nonetheless, we found that higher ratios reported by all three 251 commercial assays was associated with higher neutralizing titers. Importantly, all three 252 serological assays used in this study currently have Emergency Use Authorization 253 (EUA) to qualitatively determine the presence of antibodies against SARS-CoV-2. 254 While a negative result on SARS-CoV-2 serological assays is likely to be associated 255 with the absence of neutralizing antibody titers, a positive result is not reliable for 256 predicting the presence of neutralizing antibodies. Furthermore, since these assays are 257 under the EUA, they cannot be modified by the laboratory to report quantitative units. 258 Our results argue for a potential utility in reporting the ratio calculated for commercially 259 available assays relative to the calibrator. We, along with others, have previously patients (23). Our findings are also consistent with a study assessing the agreement 299 between the EI IgG result and neutralizing titers on predominantly non-hospitalized 300 convalescent plasma donors (33). The authors demonstrated that at a neutralizing titer 301 of 1:320, the PPA and NPA were 96% and 32% respectively and that neutralizing titers 302 were higher in a small cohort of hospitalized patients. Similarly, we demonstrate higher 303 neutralizing titers among patients with worse outcomes in an almost entirely 304 hospitalized cohort. Unique to this study, we also compare commercial tests head-to- SARS-CoV-2 cell entry depends on ace2 and tmprss2 and is blocked by a 361 clinically proven protease inhibitor The humoral immune response Janeway's Immunobiology A 366 SARS-CoV-infection model in mice demonstrates protection by neutralizing 367 antibodies Potent neutralizing antibodies from COVID-19 patients define multiple 370 targets of vulnerability Tang 18 Clinical performance of two SARS-CoV-2 serologic assays 17. CDC. Symptoms of coronavirus Neutralizing 412 antibody and soluble ace2 inhibition of a replication-competent VSV-SARS-CoV-413 2 and a clinical isolate of SARS-CoV-2 A highly conserved 416 cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV 20. World Health Organization Tang 16