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.