3.5 | mAb-EspB-B7 epitope mapping
To identify the epitope of mAb-EspB-B7 within the EspB protein, we
designed a peptide array of 78 cyclic peptides that covers the full
sequence of EspB (321 residues long). Each peptide was 15 residues long,
with an overlap of 11 residues between the peptides. Recombinant EspB
(full-length) served as a positive control, while BSA served as a
negative control. Incubation of mAb-EspB-B7 with the peptide array
revealed that mAb-EspB-B7 bound mostly to two cyclic peptides within the
array, namely, to peptides #49 (positions 193-207) and #50 (positions
197-211), which have the sequences TSAQKASQVAEEAAD and KASQVAEEAADAAQE
of the EspB protein, respectively (Figure 5A). To confirm that this
epitope is indeed recognized by mAb-EspB-B7, we synthesized the
following peptides: peptide #49; peptide #50; a peptide that comprises
the combined sequences of peptides #49 and #50 (TSAQKASQVAEEAADAAQE);
peptide #78, which was not detected by the mAb-EspB-B7 and was
therefore suitable as a negative control; and two peptides with
scrambled sequences of peptides #49 and #50. Competitive ELISA between
full-length EspB and the cyclic peptides revealed that pre-incubation of
peptides #49, #50 or #49+#50 (1 µg/ml) with mAb-EspB-B7 completely
abolished the ability of the antibody to bind full-length EspB (Figure
5B-D). To determine whether the competitive effect is derived directly
from the binding of mAb-EspB-B7 to the peptides, we assessed the ability
of mAb-EspB-B7 to recognize and bind these peptides. We observed that
mAb-EspB-B7 can bind peptides #49, #50 and the combined peptide
(#49+#50), while no binding was detected for the scrambled peptides or
for peptide #78 (Figure S2A-C). These results confirm that the main
mAb-EspB-B7 epitope is the KASQVAEEAAD sequence of the EspB protein
(peptide sequences are presented in Figure S2D).