PMC:7650537 / 6141-9621 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7650537","sourcedb":"PMC","sourceid":"7650537","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7650537","text":"2.1. Reference Samples and Tests Chosen for Evaluation of Immunoassay Performance\nThirty-seven serum samples were obtained from 26 outpatient patients with a PCR-confirmed SARS-CoV-2 infection. Blood was drawn between four and 60 days (median 19 days) after a positive real-time RT-PCR result in which parts of the SARS-CoV-2 E or N genes [18] were detected in oropharyngeal swabs with a cycle threshold ≤35 (median 26.8). Serum was obtained from the blood and stored at −20 °C until tested. These sera are likely to contain SARS-CoV-2 IgG/total antibodies and, therefore, were considered to determine the serological assay sensitivities (as previously suggested by [19]). This variant of the sensitivity calculation only took into account whether a SARS-CoV-2-infected patient was reactive at all, regardless of when this reactivity was developed (reference method 1).\nA plaque reduction neutralization assay (PRNT) using a SARS-CoV-2 isolate designated as Kiel M16502/2020 and Vero cells (order no. 605372, CLS Cell Lines Service GmbH, Eppelheim, Germany) was included as a further reference for the calculation of the sensitivities. All sera classified as reactive in the PRNT were considered. This also covered follow-up sera (reference method 2). Testing was done under biosafety level 3 conditions and was in accordance to previous reports [20,21] with minor modifications. One to two days before infection, 1.0 × 105 cells were seeded into each well of 48-well plates. These were then incubated under standard conditions until the cells became confluent. Directly prior to the PRNT, patient sera were heat-inactivated at 56 °C for 30 min and then diluted from 1:10 to 1:1280 in cell culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Bio\u0026SELL GmbH, Feucht, Germany) supplemented with 3.7 g/L NaHCO3, 4.5 g/L glucose, 2 mM L-glutamine, and 1% (v/v) Penicillin/Streptomycin/Fungi-Mix (Bio\u0026SELL). A serum dilution series was made by mixing 50 µL of each dilution step with 50 µL virus suspension containing 100 plaque-forming units, followed by an incubation for 1 h at 37 °C. The cells were washed with phosphate-buffered saline (Bio\u0026SELL), inoculated with 100 μL of these virus serum dilutions, and incubated for one hour at room temperature on a rocking shaker. Then, 100 µL of the cell culture medium supplemented with 20% (v/v) fetal calf serum (FCS) was added to each well to achieve a FCS concentration of 10% (v/v). After four to five days, cells were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline and stained with an aqueous solution of 0.1% (w/v) crystal violet and 20% (v/v) methanol. The stained plates were photo-documented. All dilution steps were tested in quadruplicates, and plaque formation was compared to an untreated cell control and a virus control. A serum dilution \u003e1:10, which prevented plaque formation in at least 50% of the wells compared to the virus control, was classified as reactive and probably as protective. The reciprocal titers were used to visualize the kinetics. If an exact titer could not be given, a geometric mean value was calculated from the two adjacent titers.\nFor the calculation of assay specificities, 100 archived sera collected during the summer 2018 (N = 50) and during winter 2018/2019 (N = 50) were used. None of these sera were expected to contain SARS-CoV-2 antibodies. The inclusion of such pre-pandemic sera for the demonstration of specificity was previously suggested 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