3.3 | Stability of mAb-EspB-B7 binding under various
physiochemical conditions
We evaluated the ability of mAb-EspB-B7 to bind EspB under various
conditions by using ELISA. We found that incubation of mAb-EspB-B7 in
human serum did not compromise the ability of the antibody to bind EspB
compared to its binding in a 3% milk solution (Figure 3A). Examination
of mAb-EspB-B7 binding under different pH conditions demonstrated that
the binding was essentially not altered under a wide range of pH values
(5.6–7.4), with the exception of pH 4.6, at which there was a reduction
in binding capacity (Figure 3B). Interestingly, testing mAb-EspB-B7
across a wide range of NaCl concentrations demonstrated that only
increased salt concentrations (> 400 mM) affected the
binding capacity of the antibody (Figure 3C). Finally, to assess the
thermal stability of mAb-EspB-B7, we determined its melting temperature,
both alone and in complex with purified EspB, by using nanoDSF. The
melting temperatures of 75.4°C and 82°C for mAb-EspB-B7 alone and in
complex with EspB, respectively (Figure S1), indicated high protein
stability.