Study design, population, and serum samples. This was an observational study of SARS-CoV-2 serial seroprevalence in New York City, Westchester County, and the Lower Hudson Valley from February 24 to March 29, 2020. The targeted population included patients accessing the Emergency Department at CUIMC in northern Manhattan and CareMount Medical, a network of ambulatory care clinics in Westchester County and the Lower Hudson Valley (Fig. 1b). Clinical specimens collected from patients were designated as residual when they were no longer required for the diagnostic purpose for which they were originally collected. CUIMC and CareMount retained diagnostic samples for 5 and 7 days post collection, respectively. Anonymized serum samples about to be discarded were requested from each clinical laboratory. A total of 814 serum samples from CUIMC-ED was randomly retrieved between February 24 and March 29, and each chosen vial of serum was de-identified with a label onto which was marked a study-specific identifier. The clinical laboratory retained a link between the study-specific identifier and the patient’s medical record number. Slightly later but in parallel, a total of 1,841 serum samples to be discarded was obtained from CareMount in three discrete batches between March 13 and March 28. Duplicate specimens for the same individual were identified and excluded before de-identification labels were applied. Specimens from one CareMount site in New York City were also excluded.
Assay validation. For the purpose of defining the sensitivity and specificity of our SARS-CoV-2 antibody tests, we also utilized 106 serum samples obtained from distinct healthy blood donors from New York City between 2015 and 2018, 146 serum samples from distinct patients with PCR-positive SARS-CoV-2 infection at variable time points after onset of symptoms, and 48 serum samples from contemporaneous patients with respiratory illnesses who tested negative for SARS-CoV-2 by PCR, most of whom were confirmed to be infected by another viral pathogen, although prior infection with SARS-CoV-2 cannot be absolutely ensured.
SARS-CoV-2 antigens and ELISA. SARS-CoV-2 antigens and ELISA. The ectodomain of the SARS-CoV-2 spike trimer24 was cloned into mammalian expression vector pCAGGS (Addgene, Watertown, MA), with a foldon tag followed by 6xHis tag and Strep tag II at the C-terminal. This expression vector was transiently transfected into Expi293 or HEK293F cells and the spike trimer was purified from the supernatant 3days post transfection using either Strep-Tactin XT Resin (IBA Life Sciences) followed by size exclusion chromatography on a Superose 6 Increase 10/300 GL column (GE Healthcare). SARS-CoV-2 nucleocapsid protein (NP) was cloned into pET28a(+) vector (Millipore-Sigma, Burlington, MA) with an AAALE linker and 6xHis tag at the C-terminus. The NP construct was then used to transform into E. coli Rosetta 2 (DE3) cells and the target protein was produced and purified from the bacterial lysate using PEI precipitation, affinity purification with Ni-NTA agarose beads (Thermo Fisher Scientific, Carlsbad, CA), chromatography on a HiTrap Heparin-HP column, followed by size-exclusion chromatography on a Superdex 200 10/300 GL column (Garg et al, bio-protocol 2020). SARS-CoV-2 receptor-binding domain (RBD)24 was cloned into vector pCAGGS (Addgene, Watertown, MA), with an HRV-3C protease cleavage site and monoFc tag followed by a 6xHis tag at the C-terminus. This expression vector was transiently transfected into Expi293 cells and the protein was purified 5 days post transfection using protein A agarose (Thermo Fisher Scientific, Carlsbad, CA). The monoFc and 6xHis tags were subsequently cleaved with HRV-3C protease (Millipore-Sigma, Burlington, MA) and counter-selected with protein A agarose (Thermo Fisher Scientific, Carlsbad, CA) as indicated by the manufacturer.
SARS-CoV-2 spike trimer and NP were coated on 96-well ELISA plate at a concentration of 200 ng/well and 50 ng/well, respectively at 4℃ overnight. The unbound proteins were then removed by two washes with 300 μl PBST (0.5% Tween-20 in PBS) per well. Afterwards, the ELISA plates were blocked with 300 μl 1% BSA in PBS at 37℃ for 2 hours, before the plates were washed 4 times with PBST. Serum samples were diluted 400-fold (or serially diluted for the quantitative ELISA) with dilution buffer (1% bovine serum albumin and 20% bovine calf serum in PBS) and tested using 100 μl per well of each diluted serum. The ELISA plates were then incubated at 37℃ for 1 hour, followed by washing 6 times with PBST. Later, 100 μl of 10000-fold diluted peroxidase affiniPure goat anti-human IgG (H+L) antibody or anti-human IgM antibody (Jackson Immune Research, New Grove, PA), or both, were added into each well and incubated for 1 hour at 37℃. After washing 6 times with PBST, the TMB substrate (Sigma, St. Louis) was added and the reaction was stopped using 1M sulfuric acid. Absorbance was measured at 450 nm and expressed as an optical density, or OD450 value. What constitutes a positive antibody test is discussed below.
Western Blot. SARS-CoV-2 S trimer, NP and RBD (1.5 μg each) were separated on a 4%-12% NuPage gel (Invitrogen, Carlsbad, CA) and stained with SimplyBlue™ SafeStain (Invitrogen, Carlsbad, CA). For the western blot, 200 ng of each protein were mixed and separated on a 4%-12% NuPage, and then electrophoretically transferred to a PVDF membrane (Millipore-Sigma, Burlington, MA). The membrane was then blocked with 10% Blotting-Grade Blocker (Bio-Rad, Hercules, CA) in PBST for 30 min at room temperature and then incubated with 1:400 dilution of serum overnight at 4℃. After washing in PBST, the membrane was incubated with a mixture of 10,000-fold diluted Peroxidase AffiniPure goat anti-human IgG (H+L) antibody and anti-human IgM antibody (Jackson Immune Research, New Grove, PA) for 1 hr at room temperature. Finally, it was washed, treated with chemiluminescent substrate, and imaged via iBright 1500 (Thermo Fisher Scientific, Carlsbad, CA).
Clinical information. Basic demographic data, such as age, sex, zip code, and date of onset of symptoms, were collected from the CUIMC-ED cases through the medical record number linked to the study-specific identifier log retained by the clinical pathology laboratory. For a small number of seropositive cases, the date of sample collection, the reason for visiting the ED, and additional medical history were extracted from the medical record. In parallel, the number of daily new SARS-CoV-2 cases were tracked from the websites of New York City and the counties with CareMount clinics (Fig. 1b), as well as from internal reports of CUIMC and CareMount.
Statistical analysis. We estimated the prevalence of SARS-CoV-2 infection at each clinical site by dividing the number of positive tests by the total number of samples tested for each time period, and reported 95% confidence intervals. We calculated posterior distribution of prevalence in the ED population using overall sensitivity and specificity of our S-IgG assay by the methodology described in Larrimore et al through their calculator (https://larremorelab.github.io/covid-calculator2)25. We extrapolated from the seroprevalence observed in the first week of sampling at CUIMC-ED to the total number of SARS-CoV-2-positive cases for that period based on the known number of ED visits.
Protection of human subjects and confidentiality. The study was approved by the Institutional Review Board (IRB) of CUIMC through an honest broker protocol (E. Hod, Principal Investigator) that allows the clinical laboratory to code specimens while retaining a linked key to medical record numbers from which the demographic and clinical information could be obtained. As all testing for the CareMount discarded serum specimens was done on samples that were irrevocably deidentified, the IRB determined that this component of the study did not require ethical approval.