Thread by @KSadtler

Our ELISA validation Breakdown for #SARSCoV2, in short, we validated six (6) ELISAs: spike & RBD IgG, IgM and IgA (check out @cispt2’s summary too!) (1/9) medrxiv.org/content/10.110…

Great ELISA starts with great protein: @domespo @FredNatLab generated two spike and two RBD constructs from different plasmids, and we found that @NIAIDNews VRC spike ectodomain and @SchmidtLabHMS @ragoninstitute RBD were the ideal antigens. (2/9)

Evaluating sensitivity & specificity of spike + RBD double positive, focusing on specificity, showed 0/100 negative controls came back “#seropositive” (Spike+RBD+) for IgG/IgM/IgA. We have since shown on robot w 0/300 for IgG (100% specificity @ 95% CI 98.78% to 100%) (3/9)

We tested this assay on a *small-scale* test cohort in a high-exposure community with 68 individuals that reported symptoms. 22 of these had a positive PCR test, we found 59 with IgG antibodies. This is a test & not indicative of broad community. (4/9)

We saw that RBD OD values trend lower than Spike, maybe some interesting stuff there regarding polyclonal responses? ... we have some more interesting data comparing these in patients for a future manuscript. (5/9)

We also found from another 6 donors that reported no symptoms, but with known exposure to #SARSCoV2, 4 that tested seropositive for IgG (two of which were IgM+), so using these assays we can detect people that had the virus but no symptoms (6/9)

We also looked at potential issues with cross-reactivity from different coronaviruses and found no correlation between signal intensity of #SARSCoV2 with seasonal (OC43, HKU1) and epidemic (MERS, SARS1) coronaviruses, or the flu (H1N1). (7/9)

Our statisticians from @NIAIDNews looked forward, determining the # of controls we need to be certain in these serology tests for large-scale data sets. #Specificity matters much more than sensitivity; you need at least 300-1000 negative controls, even w/ 100% specificity. (8/9)

We also include our protocols for our at-home blood sampling and how that compares to fresh serum! (9/9)

Again, massive collaborative effort towards serology in our group at the @NIH. With @NIBIBgov, @ncats_nih_gov, @FredNatLab, and @NIAIDNews. Huge congrats to first authors Carleen Klumpp-Thomas @hanta716 (NCATS) and Heather Kalish (NIBIB) who really pushed these ELISAs forward!