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 competitiveassay formats. By exploitation of the catalytic properties of the microbial transglutaminase from
Streptoverticillium mobarense
species (MTGase), that is, acyl transfer between γ-carboxamide groups and various primary amines, new techniques for theenzymatic modification of proteins were developed. One example of bioconjugation is the biotinylation of antibodies for immunochemicalapplications using two species of activated biotin. In this case, the activated biotin acts as the acyl acceptor and is coupledto the glutamine residues of a monoclonal antibody. Because of the substrate specificity of the MTGase with regard to thelimited number of glutamine residues and the surrounding microenvironment, only a limited number of binding sites on the targetprotein 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 procedureand two
in situ
procedures, are described.