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.