Microbial transglutaminase-mediated synthesis of hapten-protein conjugates for immunoassays

Hapten-protein conjugates are essential in many immunochemical assays, in particular, in assays employing titration or competitive assay formats. By exploitation of the catalytic properties of the microbial transglutaminase from Streptoverticillium mobarense sp. (MTGase), i.e., acyl transfer between gamma-carboxamide groups and various primary amines, new techniques for the synthesis of hapten-protein conjugates were developed. This is demonstrated by two examples. The feasibility of MTGase for hapten-protein conjugate synthesis was studied by coupling the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) to casein. Different procedures for the synthesis and the immobilization of these 2,4-D-casein conjugates were evaluated, comprising (i) a batch procedure, (ii) coupling of 2,4-D to an already immobilized layer of casein, and (iii) a method for simultaneous immobilization and conjugation. Kinetic studies revealed that conjugate formation in the batch procedure was almost complete after approx 2 h. By employing the conjugates in a competitive ELISA, detection limits as low as 0.05 microgram/L 2,4-D were reached. Using the approach with simultaneous immobilization and conjugation, the time for the whole assay could be reduced to only 2 h. Finally, to demonstrate the versatility of the enzymatic synthesis of hapten-protein conjugates, an ELISA for 2,4,6-trinitrotoluene (TNT) determination based on transglutaminase-synthesized conjugates was developed. In this assay, a detection limit as low as 0.04 microgram/l TNT was obtained.