Detection strategies for catalytic antibodies

Abstract

This paper discusses the detection of antibody catalysis using soluble test substrates. Antibodies raised against a transition state analog of a chemical reaction typically show dissociation constants for the antibody–hapten complexes in the range of 10 −9–10 −7 M. If hapten binding is transferred to catalysis as measured by the transition-state dissociation constant K TS= K M/( k cat/ k uncat)= k uncat/( k cat/ K M), this corresponds to the concentration of antibody necessary to double the apparent rate of the reaction. This sets a lower limit for the detection of catalysis if no additional effects are present to induce catalysis. The concentration of antibodies in hybridoma cell culture supernatants (5–50 μg/ml) meets this requirement. The use of high-throughput screening (HTS) for catalysis together with ELISA to select hybridomas leads to the isolation of not only one, but also many catalytic antibodies. As examples, the application of HTS for catalysis using fluorogenic reactions to isolate retro-Diels-Alderase and pivalase catalytic antibodies useful for prodrug activation chemistry are discussed.