Synthesis of hapten-protein conjugates using microbial transglutaminase

Hapten-protein conjugates are essential in many immunochemical assays and in particular in assays using titration or competitive assay formats. By exploitation of the catalytic properties of the microbial transglutaminase from Streptoverticillium mobarense species (MTGase), that is, acyl transfer between gamma-carboxamide groups and various primary amines, new techniques for the enzymatic modification of proteins were developed. One example of bioconjugation is the biotinylation of antibodies for immunochemical applications using two species of activated biotin. In this case, the activated biotin acts as the acyl acceptor and is coupled to the glutamine residues of a monoclonal antibody. Because of the substrate specificity of the MTGase with regard to the limited number of glutamine residues and the surrounding microenvironment, only a limited number of binding sites on the target protein are available; the proposed method is thus particularly suitable when only a few biotin molecules need to be attached. Another example for the modification of proteins is the synthesis of hapten-protein conjugates used in competitive-type immunoassays. Methods for the synthesis of 2,4-D-casein conjugates (2,4-diclorophenoxyacetic acid, a herbicide) are presented. Various approaches, including a batch procedure and two in situ procedures, are described.