Enzyme-Linked Neutralization Assay (ELNA):
VeroB4 cells (ACC-33, DSMZ) were seeded in flat-bottom 96 well plates (Sarstedt, Germany) with Medium199 (Thermo Scientific Gibco, USA) and 10% fetal calf serum (Thermo Scientific Gibco, USA) at a density of about 106 cells/ml to receive a confluent monolayer. Next day, an infectivity titration was carried out to determine 100 tissue culture infectious dose 50% (100 TCID50).
Inactivated serum samples were titrated in duplicate in twofold dilution steps, starting at a dilution of 1:4 in Medium199 containing 3% fetal calf serum. Equal volumes of virus (1 x 105TCID50) and serum dilutions in Medium199 were mixed and subsequently incubated for 1 to 1.5 hours at room temperature in U-bottom 96 well plates (Thermo Scientific Thermo Scientific Nunc, USA). After incubation, a pre-seeded flat-bottom 96 well plate with confluent VeroB4 cells was used, medium was discarded, the incubated mixture of patient´s serum and defined virus solution was transferred to each corresponding well of the flat-bottom plate and the plate was incubated for 24 hours at room temperature. Incubation was stopped by discarding supernatant, cells were washed in PBS twice, fixed with ice-cold acetone-methanol (1:1) and frozen for at least 15 minutes. All steps were performed under strict observation and in compliance with biosafety level 3. The analysis was carried out like an enzyme-linked immunosorbent assay using a BEP III (Siemens, Germany) according to the following steps: blocking (45 minutes, 37° C, Candor Biosciences, Germany), washing 3 x (wash pod, Siemens, Germany), anti-SARS-CoV-2 nucleocapsid protein IgG (Bioss bsm-41413M, dilution 1:5000) for 30 min. at 37° C), washing 3 x, adding of horseradish-peroxidase-conjugated goat anti mouse IgG (ABIN376241, dilution 1:5000) for 30 min. at 37° C), washing 3 x, adding substrate tetramethylbenzidine (TMB) and stop solution (Siemens, Germany). 24 hours post infection the reading of the results was done.
The cut-off titer was set by titrating defined negative human sera from volunteers out of healthy Tyrolean blood donors from the year 2009 and was set at 1:4 in combination with the viral dose of 1x105 TCID50 and calculated as median optic density minus the standard deviation. A sample was considered positive when the given optic density was higher than the cut-off titer.
Titers of single 1:4 in ELNA were valued as borderline, titers of 1:4 in duplicate as weak neutralization ability, 1:8 to 1:16 as moderate/good and > 1:16 as strong neutralizing ability.