Journal of Chromatography & Separation Techniques
Open Access

Demonstration of homotropic positive cooperativity in antibody-antigen binding reactions

International Summit on Current Trends in Mass Spectrometry

July 13-15, 2015 New Orleans, USA

Robert Blake

Posters-Accepted Abstracts: J Chromatogr Sep Tech

Abstract :

We used macroion mobility spectrometry (macroIMS) to quantify the percentages of antibody complexes that contained 0, 1, and 2 protein antigens in different reaction mixtures of antibody and antigen. These measurements tested the following hypothesis: A bivalent antibody that exhibits positive cooperativity when it binds its protein antigen will have a higher ratio of doubly occupied to singly occupied antibody than that anticipated for an antibody that binds its protein antigens in separate and independent binding events. The binding of human peroxiredoxin 4 (PRDX4) to two different monoclonal antibodies was studied by kinetic exclusion assays on a KinExA flow fluorimeter. One antibody (PRDX4-1) exhibited positive cooperativity with a Hill coefficient of 1.6 when it bound PRDX4, while the other antibody (9D2) bound PRDX4 with no evidence of cooperativity whatsoever. MacroIMS measurements revealed that the ratios of Ab�?�Ag2 to Ab�?�Ag1were 0.5 and 1.4 when half of the antigen binding sites on antibodies 9D2 and PRDX4-1, respectively, were filled with antigen. The lower value was precisely the ratio expected for the distribution of antibody complexes when each of the antigen binding sites behaved separately and independently. The higher ratio that was expected for the distribution of antibody complexes when binding of the second equivalent of antigen occurred with higher affinity than that of the first equivalent. These studies independently verified the existence of homotropic positive cooperativity in an antibody-antigen binding event. To our knowledge, no other laboratory has reported the occurrence of homotropic positive cooperativity in selected antibody-antigen binding reactions.