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).