Biotinylation of monoclonal antibodies prevents their ability to activate the classical pathway of complement.
Biotinylation of mAb has become a standard procedure for a variety of applications that exploit the specific high affinity interaction between biotin and avidin. In the present study, we investigated how biotinylation of mAb affects their ability to sensitize target cells to C-dependent lysis in vitro. mAb were biotinylated by cross-linking biotin covalently with an N-succinimidyl ester to the epsilon-amino groups of lysine residues. Human RBC were treated with two rat mAb, either alone or together: one against glycophorin A (YTH89.1), another against CD59 (protectin; YTH53.1), an inhibitor of the membrane attack complex of C. Melanoma cells (G361) were attacked by a mouse mAb (27A) against an O-acetylated GD3 ganglioside. As compared with the nonbiotinylated mAb, the biotinylated forms of all the investigated mAb were much weaker in causing classical C pathway-mediated lysis of the target cells. Biotinylation did not reduce the ability of the mAb to bind to their Ag, nor of the anti-CD59 mAb to neutralize the C lysis-restrictive effect of CD59. In binding assays using 125I-labeled C1q, significantly less C1q bound to the biotinylated anti-glycophorin-A and anti-CD59 mAb than to the nonbiotinylated mAb. These data show that biotinylated antibodies do not activate the classical C pathway because binding of C1q to the antibody Fc-regions is blocked.
Jokiranta
T S
TS
Department of Bacteriology and Immunology, University of Helsinki, Finland.
Meri
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000276:immunology; D001710:Biotin / Q000737:chemistry*; D018957:CD59 Antigens; D015922:Complement C1q / Q000378:metabolism*; D003171:Complement Pathway, Classical; D066298:In Vitro Techniques; D007518:Isoantibodies / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D051381:Rats
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018957:CD59 Antigens; D007518:Isoantibodies; D008562:Membrane Glycoproteins; D001710:Biotin; D015922:Complement C1q