Investigations of polyclonal catalytic antibodies

A key feature of catalytic antibodies is that recognition can be programmed into the catalyst by varying hapten design. Understanding the limitations of this programmability and recognition is of great importance to the production of more efficient antibody catalysts. Polyclonal catalytic antibody studies offer the opportunity to establish a framework from which haptens can be designed to produce the best catalysts. The work described will probe the limitations of the polyclonal catalytic immune response by investigating two systems designed to elicit catalysts for carboxylic ester hydrolysis and four systems designed to elicit catalysts for carbonate ester hydrolysis. Five out of these six systems failed to elicit significant catalytic activity. Studies of the binding properties using ELISA and competition ELISA of the non-catalytic polyclonal samples were performed and suggest that in some cases affinity for the hapten can be driven too high, precluding binding of the substrate in a manner conducive for catalysis. In other cases, it appears that ground state stabilization of the substrate was too great. Quenched dynamics molecular modeling of some of the haptens and their substrates suggest that conformational flexibility of the hapten and substrate can influence the production of polyclonal catalysts. As an interesting control experiment, one set of animals that did not produce polyclonal catalytic antibodies over the course of an immunization regimen was used for a second immunization regimen with a phenyl phosphate immunogen that was known to elicit polycional catalytic antibodies. These rabbits produced polyclonal antibodies that catalyzed the hydrolysis of a phenyl carbonate substrate with {dollar}rm ksb{lcub}cat{rcub}/ksb{lcub}uncat{rcub}{dollar} values of 1400 for one sample and 4700 for the other. This is the first demonstrated re-use of an animal in polyclonal catalytic antibody experiments. Another set of animals was immunized repeatedly with this same phosphate hapten to investigate the influence that immunization number has on the magnitude of catalytic activity when using a transition state analog hapten. It was found that although hapten affinity continued to increase throughout the immunization regimen, catalytic activity peaked after four to five immunizations and then decreased.