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 betwe en biotin and avidin. In the present study, we investigated how biotin ylation of mAb affects their ability to sensitize target cells to C-de pendent 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 alon e or together: one against glycophorin A (YTH89.1), another against CD 59 (protectin; YTH53.1), an inhibitor of the membrane attack complex o f 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 weak er in causing classical C pathway-mediated lysis of the target cells. Biotinylation did not reduce the ability of the mAb to bind to their A g, nor of the anti-CD59 mAb to neutralize the C lysis-restrictive effe ct of CD59. In binding assays using I-125-labeled C1q, significantly l ess C1q bound to the biotinylated anti-glycophorin-A and anti-CD59 mAb than to the nonbiotinylated mAb. These data show that biotinylated an tibodies do not activate the classical C pathway because binding of C1 q to the antibody Fc-regions is blocked.