Supporting Figure 1: Impact of different commercial diluent solutions on signal intensities for binding of IgG and IgM to both specific (VACV) and unspecific (HEp2) antigens. Tested samples were tested at 1:100 dilution or 1:500 dilutions (VIG only) in the specified diluents. Sample P-16-056-001-04 had IFA titres of 1:5120 (IgG) and 1:1280 (IgM), sample P-20-162-001-01 had IFA titres of 1:1280 for both IgG and IgM antibodies, and VIG is highly positive for IgG but negative for IgM while sample SeCoV-0827 is negative for both IgG and IgM. Casein blocking buffer was prepared in-house as described, LowCross-Buffer was obtained from Candor, Neptuneā„¢, Plasma Sample, Protein-Free, and General Serum diluents were all obtained from ImmunoChemistry. For IgG detection, dilution in the established in-house casein blocking buffer enabled the lowest background signals with the highest specific signals, leading to the overall highest difference between specific and unspecific signals. In contrast, due to very high unspecific binding signals in casein blocking buffer, almost no signal difference remained in casein blocking buffer for IgM detection. All commercial diluents tested significantly lowered unspecific binding for IgM detection. Due the fact that unspecific binding was most reduced when using LowCross-Buffer, this buffer was used for IgM detection in the ELISA, although also specific signals on VACV lysate were slightly reduced as compared to the other diluents tested.
Supporting Figure 2: Comparison of ELISA signals (VACV-specific signals minus HEp2 signals) for native and sera inactivated with triton/tween + heat. Almost perfect agreement between measurement (R20.9921, slope 0.9661, y-intercept -0.004592 of linear regression) indicates that congruent results can be obtained from native and inactivated sera.