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.