Use of microbial transglutaminase for the enzymatic biotinylation of antibodies

Nowadays many reagents are available for the biotinylation of proteins. As most of them bind to amino groups of the protein the degree of labelling di ffers from batch to batch and the possibility exists that the biological ac tivity of the target protein may be affected by the labelling procedure. In the present study we have investigated an enzymatic approach to biotinylat ion using microbial transglutaminase (MTGase) from Streproverticillium moba raense. The proposed method is particularly suitable when only a few biotin molecules need to be attached to the target proteins. The enzyme catalyses the acyl transfer reaction between gamma-carboxyamide groups and various p rimary amines. This was exploited for biotinylation using two amino-modifie d biotin derivatives, biotinamido-5-pentylamin (BIAPA) and biotinoyl-1,8-di amino-3,6-dioxaoctane (BIDADOO) as acyl acceptors and a monoclonal IgG agai nst the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the acyl donor. Kinetic studies revealed that the MTGase-mediated reaction proceeds with l ow velocity and is almost complete after 34 h. Conjugation ratios ranging f rom 1.1 to 1.9 biotins per IgG were found by mass spectrometry. To investig ate the influence of antibody conjugation on antigen binding a competitive ELISA for the determination of 2,4-D employing MTGase-biotinoylated IgGs wa s developed. In this assay lower limits of detection of 0.3 and 1.0 mu g/l of 2,4-D were achieved with BIDADOO- and BIAPA-modified antibodies, respect ively. (C) 2000 Elsevier Science B.V. All rights reserved.