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.