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 differs from batch to batch and the possibility exists that the biological activity of the target protein may be affected by the labelling procedure. In the present study we have investigated an enzymatic approach to biotinylation using microbial transglutaminase (MTGase) from Streptoverticillium mobaraense. 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 primary amines. This was exploited for biotinylation using two amino-modified biotin derivatives, biotinamido-5-pentylamin (BIAPA) and biotinoyl-1,8-diamino-3, 6-dioxaoctane (BIDADOO) as acyl acceptors and a monoclonal IgG against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the acyl donor. Kinetic studies revealed that the MTGase-mediated reaction proceeds with low velocity and is almost complete after 34 h. Conjugation ratios ranging from 1.1 to 1.9 biotins per IgG were found by mass spectrometry. To investigate the influence of antibody conjugation on antigen binding a competitive ELISA for the determination of 2,4-D employing MTGase-biotinoylated IgGs was developed. In this assay lower limits of detection of 0.3 and 1.0 microg/l of 2,4-D were achieved with BIDADOO- and BIAPA-modified antibodies, respectively.
Josten
A
A
Institute of Chemical and Biochemical Sensor Research, Mendelstrasse Münster, Germany.
Haalck
L
L
Spener
F
F
Meusel
M
M
D015084:2,4-Dichlorophenoxyacetic Acid / Q000032:analysis / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013306:Streptomycetaceae / Q000201:enzymology; D011503:Transglutaminases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D015084:2,4-Dichlorophenoxyacetic Acid; D001710:Biotin; D011503:Transglutaminases