Each plate also contained two positive control wells (CR302218,19, an anti-SARS-CoV-1 monoclonal antibody that reacts to the SARS-CoV-2 RBD, at 0.5 g/mL) and two negative PF-05180999 control wells (pooled human sera taken from 2017C2018 (Gemini Biosciences, 100C110, lot H86W03J, pooled from 75 donors). of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of 1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children had neutralizing activity, including one that neutralized at a dilution 1:18,000. Therefore, an PF-05180999 increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests. One of the first cases of community transmission of SARS-CoV-2 in the United States was identified in the greater Seattle area in late February, 20208,9. By late March, thousands of cases had been identified in Washington state by viral RT-PCR testing, mostly among adults PF-05180999 (https://www.doh.wa.gov/Emergencies/Coronavirus). Schools closed statewide on March 17, and a statewide stay-at-home order was issued the next week. March and April of 2020 are therefore critical months for understanding the early dynamics of the SARS-CoV-2 pandemic in the Seattle area. Because SARS-CoV-2-infected children often experience little or no disease1,4C6, we sought to identify infections using an approach independent of symptom-based viral testing. Serological assays, which detect antibodies induced by infection, provide such an approach. When interpreting these assays in a temporal context, note that individuals do not become seropositive until 1 to 2 2 weeks post symptom onset10C14, while PCR-based testing generally only detects viral RNA during the first few weeks after symptom onset11,12. We serologically screened 1, 775 residual serum samples from Seattle Childrens Hospital that were collected between March 3 and April 24, 2020 following approval from the Human Subjects Institutional Review Board. These samples were collected from 1,076 unique children who visited the hospital and received blood draws for any reason, including respiratory illnesses, surgery, or ongoing medical care. Demographics and reason for medical admission are presented below with results of our serological testing. The generalizability of this study population to all children in Seattle is unknown, particularly because hospital visitors were primarily those with urgent medical needs during the statewide stay-at-home order. We used a multi-assay serological testing approach based on an enzyme-linked immunosorbent assay (ELISA) protocol that recently received emergency use authorization from New York State and the FDA15,16, although we increased stringency by adding a second validation ELISA and confirming putative seropositives with the Abbott SARS-CoV-2 IgG chemiluminescent microparticle immunoassay (CMIA), which identifies IgG antibodies to the nucleocapsid protein and has been shown to have 99.9% specificity and 100% sensitivity for samples taken greater than 17 days post symptom onset17. Furthermore, as described below, we confirmed that seropositive samples had activity in pseudovirus neutralization assays. We NF2 first screened all sera at a 1:50 dilution in an ELISA for IgG binding to the SARS-CoV-2 spike receptor binding domain (RBD) and compared results to a negative control consisting of a pool of sera collected in 2017 and 2018 (Figure 1a). We identified 102 of 1 1,775 samples with PF-05180999 PF-05180999 readings that exceeded the average of the negative controls by 5 standard deviations. These preliminary hits were further assessed with IgG ELISAs using serial dilutions of sera against two antigens: RBD and pre-fusion stabilized spike ectodomain trimer (Figure 1b). As negative controls, we included twelve serum samples and two serum pools collected before 2020; we also tested some pediatric samples that were negative in the initial RBD screen. We summarized the ELISAs by calculating the area under the curve (AUC), and called samples as putatively seropositive if the AUC exceeded the average of the negative controls by 5 standard deviations for both RBD and spike (Figure 1b). We then performed a final validation by testing with the Abbott CMIA all putative seropositives from the ELISAs as well as most other samples with sufficient volume that passed the initial RBD screen. The AUCs for the RBD and spike ELISAs.