Ts. Jokiranta et S. Meri, BIOTINYLATION OF MONOCLONAL-ANTIBODIES PREVENTS THEIR ABILITY TO ACTIVATE THE CLASSICAL PATHWAY OF COMPLEMENT, The Journal of immunology, 151(4), 1993, pp. 2124-2131
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.