2.4 | Enzyme-linked immunosorbent assay (ELISA)
A 96-well ELISA plate was coated with 5 μg/mL purified EspB in PBS and
incubated overnight at 4°C. The plate was then blocked with 300 μL/well
of 3% [w/v] skim milk in PBS for 1 hr at 37°C and washed with PBS.
mAb-EspB-B7 in blocking solution was added to the first line of the
plate and serially diluted throughout the plate. The plate was incubated
for 1 hr at room temperature, washed, and incubated with goat anti-human
H+L HRP-conjugated secondary antibody in 0.05% PBST (Jackson
ImmunoResearch) for 1 hr at room temperature. Plates were then washed
and signal was developed using 3,3’,5,5’-tetramethylbenzidine (TMB). The
reactions were quenched by 1 M H2SO4 and
absorbance was measured at optical density (OD) of 450 nm (Epoch,
BioTek). ELISA assays to test mAb-EspB-B7 binding in various conditions
were carried out using similar protocol as described above with the
following modifications: (i) for binding under various pH conditions,
mAb-EspB-B7 was incubated in 0.1 M citric acid buffer pH 7.4, 7.0, 6.6,
5.6, and 4.6 during the binding step; (ii) for binding at various salt
concentrations, mAb-EspB-B7 was incubated in 45.6 nM, 68.5 nM, 137 nM,
274 nM, and 411 nM NaCl; and (iii) for assessment of the serum effect on
mAb-EspB-B7 binding, the antibody was incubated in 10% goat or horse
serum with 1% Tween 20 and 1% human serum during the binding step.
Competitive ELISA with peptides was carried out as follows: A 96-well
ELISA plate (I) and a 96-well inert Bradford plate (II) were used for
each of the peptides examined. The respective scrambled peptides
(carrying the same amino acid compositions in a scrambled order),
peptide #78 and full-length EspB were used as positive and negative
controls. Plate I was coated with 3 µg/ml EspB or PBS and incubated
overnight at 4°C. Blocking of plate I was performed as described above.
mAb-EspB-B7 (15 nM) was pre-incubated with serially diluted
concentrations of peptides, starting at 15 µg/mL for 1 hr at room
temperature, transferred to plate I, and incubated for 1 hr at room
temperature. The remaining steps were performed as described above for
regular ELISA.