Detection of SARS-CoV-2 Neutralizing Antibodies
SARS-CoV-2 nAb titers were determined in IVIG and human plasma samples that were used undiluted, or prediluted with cell culture medium 1:4, 1:5, 1:10, or 1:20 depending on sample amount available, and then serially diluted in 2-fold steps. Equal volumes of sample dilutions were mixed with virus stock at 103.0 tissue culture infectious doses 50% per milliliter (TCID50/mL) SARS-CoV-2 (strain BavPat1/2020, kindly provided by C. Drosten and V. Corman, Charité Berlin, Germany) and incubated for 150 minutes ± 15 minutes, before titration on Vero cells (Cat. no. 84113001, European Collection of Authenticated Cell Cultures, Porton Down, Salisbury, UK) in 8-fold replicates per dilution. The virus-induced cytopathic effect was determined after 5–7 days of incubation. The reciprocal sample dilution resulting in 50% virus neutralization (NT50) was determined using the Spearman-Kärber formula, and the calculated neutralization titer for 50% of the wells reported as 1:X. The detection limits were as follows: <1:0.8 for undiluted, <1:3.1 for 1:4 prediluted, <1:3.9 for 1:5 prediluted, <1:7.7 for 1:10 prediluted, and <1:15.4 for 1:20 prediluted IVIG.
The neutralization assay (µNT) included several validity criteria, that is confirmatory titration of input virus infectivity, cell viability, and neutralization testing of an internal reference standard, all of which had to comply with defined ranges.