IgG-anti-SARS-CoV-2 Avidity
IgG avidity was used as a surrogate marker for synergistic
antibody-antigen-binding and IgG maturation during clonal hypermutation
of specific memory B cells over time. The IgG-anti-SARS-CoV-2 avidity
was determined in IgG positive samples by adaptation of the agile
IgG-SARS-CoV-2 ELISA (Virion/Serion) evaluating the remaining bound IgG
after incubation with 1M ammonium thiocyanate (NH4SCN)
and using PBS as reference (Almanzar, Ottensmeier et al. 2013) and was
performed in duplicates. The relative avidity index (RAI) was determined
in serum samples at an O.D. equivalent for 100U/mL and calculated as
follows: the IgG-anti-SARS-CoV-2 concentrations after
NH4SCN / the IgG-anti-SARS-CoV-2 concentrations after
PBS multiplied by 100. RAI values were considered as: RAI
>60% high avidity, 40% ≤ RAI ≤ 60% as moderate, and RAI
< 40% as low avidity according to experience with
varicella-specific avidities (Kneitz, Schubert et al. 2004).
IFN γELISPOT
SARS-CoV-2-specific T cell response was determined by using an IFNγ
ELISPOT assay after stimulation of 4x105 PBMCs in the
presence of 10µg/mL SARS antigen (Ag) (SARS-CoV-2 Spike Ectodomain
(S1-S2), Virion/Serion) and 2µg/mL measles virus lysate (Virion/Serion)
as a specific positive control for 18h at 37° C and was performed in
triplicates. One hundred ng/mL phytohemagglutinin A (PHA,
Sigma, Taufkirchen, Germany) and
X-VIVOTM-20 medium (Lonza, Verviers, Belgium) were
used as positive and negative controls, respectively. Briefly, a
preactivated 96-well plate (Milipore, Merck, Darmstadt, Germany) plate
was coated with 5µg/mL anti-IFNγ (clone 1-D1K, Mabtech, Nacka Strand,
Sweden) in PBS O.N. at 4°C. Unspecific binding sites were blocked with
5% BSA/PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) for 1h at 37°
C. Detection of IFNγ was performed after incubation with 2µg/mL of
biotinylated anti-IFNγ (clone 7-b6-1, Mabtech) in 0.5% BSA/PBS for 1h
at room temperature (RT). Spot development was done after incubation
with BCIP/NBT plus (Mabtech). Spot forming units (SFU) were quantified
by C.T.L. ELISPOT reader software (Bonn, Germany) and expressed as
SFU/106 cells. An arbitrary limit of <20
SFU/106 cells was set as negative, as determined by
pilot experiments with defined positive samples. Data were normalized by
calculating the stimulation index (SI) as follows: the number of
SFU/106 antigen divided SFU/106negative control. Positive T cell reactivity to SARS-CoV-2 was
considered for SI values above 1.04 as determined by pilot experiments
with defined negative samples.