ELISA
The in-house ELISA was performed as described before [11] with
slight changes to the protocol to harmonise the assay with other
serological in-house assays. As antigen, UV-inactivated RIPA lysate from
VACV New York City Department of Health Laboratories (ATCC, catalogue #
VR-1536®) infected HEp2 cells was coated at a concentration of 4 µg/mL
in 100 µL of 50 mM carbonate buffer (pH 9.6) per well to the surface of
one half of a MaxiSorp™ ELISA plate (Nunc). Similarly, lysed
non-infected HEp2 cells were coated on the other half of each plate to
serve as a non-specific negative control. Coating was done at 4 °C
overnight. The next day, the plates were washed four times by using an
automated ELISA washer (Hydrospeed, Tecan) with 300 µL of washing buffer
(PBS with 0.1 % Tween 20) per well before the plates were blocked for 1
hour at room temperature using 200 µL per well of casein blocking buffer
(200 mM Tris pH 7.3, 2.5 % casein, Sigma-Aldrich, # C-5890, 0.1 %
Tween 20, 0.02 % 5-Bromo-5-Nitro-1,3-Dioxan, Bronidox). Subsequently,
divergent protocols were developed for the detection of either IgG or
IgM antibodies. For IgG detection, sera were diluted either 1:100 and
1:1000 or in a 1:4 dilution series ranging from 1:100 to 1:6400 in
casein blocking buffer and incubated for 1 h at 37 °C. For each serum
dilution, 100 µL were incubated on both VACV-infected as well as
non-infected HEp2 cell lysate. After a washing step was performed as
described before, 100 µL of HRP-labelled goat anti-human IgG antibody
(Fc-γ-specific, Jackson ImmunResearch, obtained from Dianova, #
109-035-008) diluted 1:2500 (final dilution 1:5000 due to storage in
glycerol at a 1:1 dilution) were added per well and incubated for 1 h at
37 °C. For IgM detection, sera were pre-treated with Mastsorb (Mast
Group) similar to the IFA protocol and diluted in LowCross-Buffer
(Candor Bioscience) to minimise non-specific background binding (see
supporting figure 1). Detection of bound IgM was done by incubation with
HRP-labelled goat anti-human IgM antibodies (5µ-specific, Jackson
ImmunResearch, obtained from Dianova, # 109-035-043) diluted 1:2500
(final dilution 1:5000 due to storage in glycerol at a 1:1 dilution).
After a final wash with eight washing steps, 100 µL of TMB substrate
(Seramun Slow TMB substrate, Seramun Diagnostica GmbH) were added per
well for 15 min at room temperature before the reaction was stopped by
adding 100 µL of 0.25 M H2SO4 per well.
The absorption was read with an ELISA microplate reader (Tecan Infinite
M200) at 450 nm referenced to 620 nm. For each serum and dilution,
signals for binding to non-infected HEp2 lysate were subtracted from
signals for binding to VACV-infected HEp2 lysates to account for
unspecific binding, leading to Delta results (VACV minus HEp2). On each
plate, a standard curve was included using a 1:4 dilution series of
Vaccinia Immune Globulin (obtained through BEI Resources, NIAID, NIH:
Polyclonal Anti-Vaccinia Virus (immune globulin G, Human), NR-2632)
starting at a 1:500 dilution and ranging to a 1:128,000 dilution. Delta
results were quantified by interpolation to the standard curve by using
a four-parameter sigmoidal fit over log-transformed VIG concentrations
using the statistical software R (version 4.1.2) and the drLumi package
(version 0.1.2) [21]. A mean concentration was calculated from all
dilutions that could be interpolated from the standard curve while
dilutions above or below the limit of quantification were excluded.