Chemiluminescent detection integrated with microdialysis sampling for label-free measuring the affinity of...

A novel label-free protocol was developed for measuring the affinity between ractopamine and its monoclonal antibody (McAb) based on microdialysis (MD) on-line sampling integrated with flow injection chemiluminescent detection. In this study, unbound ractopamine was sampled by MD probe from homogeneous immunoreaction equilibrious systems, and then real-time quantified using flow injection chemiluminescent detection. The quantified concentrations of unbound ractopamine in the immunoreaction equilibrious systems were treated with Scatchard analysis and Klotz analysis to obtain the affinity constant. The mean recovery of MD probe for sampling ractopamine was found to be 24.2%. The affinity constants calculated by Scatchard analysis and Klotz analysis both were 1.0 × 10⁶ M^-1, indicating that the investigated ractopamine mouse McAb was a medium-affinity antibody. The result showed good agreement with that obtained from thiocyanate elution test. This protocol for measuring antibody affinity is free of protein conjugation of hapten and enzyme labeling of McAb. Therefore it avoids affinity decrease resulting from steric hindrance, occupancy of the antigenic determinants, and deactivation of antibody, which has been frequently encountered in the reported conventional approaches. It opens up a new pathway for direct measurement of antibody affinity with a facile, rapid, accurate and low-cost approach.