Kinetics of the dissociation of indium-(para-substituted-benzyl)ethylenediaminetetraacetic acid hapten analogs from the monoclonal anti-hapten antibody CHA255

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Abstract
Half-lives were measured for the dissociation of a series of 20 indium-benzyl-EDTA derivatives from a monoclonal antibody that binds to them. Most haptens gave expected monoexponential dissociation curves with half-lives ranging from approximately 8 to approximately 100 min at 22 +/- 1 degree C. Precise (+/- approximately 2.5%) determinations were made using centrifugal ultrafiltration to separate free from bound hapten. A strong pH dependence of the dissociation half-life was found for the two haptens studied. Activation enthalpies were identical (23 +/- 1 kcal/mol) for the dissociation of four haptens, suggesting that, in contrast to individual rate constants, this parameter is insensitive to hapten modification. The dissociation half-lives provided evidence for the location of a positive charge in the binding site, but gave no clear indication of the role of hydrophobic interactions or of steric requirements in hapten binding. While variations in ionic strength had no effect on the dissociation rate, lowering surface tension with dioxane increased the rate somewhat. Three hapten-antibody complexes showed biexponential dissociation rates. It is postulated that this results from distinct conformations of the complex dissociating at different rates. The dissociation rate constant was found to be an extremely sensitive indicator of the hapten-antibody interaction that can be measured very precisely.

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