ATTACHMENT OF BIOTINYLATED ANTIBODY TO RED-BLOOD-CELLS - ANTIGEN-BINDING CAPACITY OF IMMUNOERYTHROCYTES AND THEIR SUSCEPTIBILITY TO LYSIS BY COMPLEMENT

A biotinylated monoclonal antibody (mAb) to human IgM (b-anti-IgM) has been attached to human red blood cells (RBC) by two different approac hes. The first method is performed with biotinylated RBC (b-RBC) and i nvolves stepwise binding of streptavidin (SA) to b-RBC followed by add ition and binding of specific b-anti-IgM or b-IgG. b-RBC were prepared with differing input levels of biotin N-hydroxysuccinimide ester (BNH S). At moderate BNHS levels (100 mu M) the resulting b-RBC (designated b4-RBC) bound 50,000 molecules of b-IgG after treatment with SA. Howe ver, at high BNHS levels (>1000 mu M) the resulting b-RBC bound b-IgG poorly, presumably due to multivalent binding of each SA to several bi otins in close proximity on the RBC. b-RBC prepared at high BNHS input s (but not b4-RBC) were lysed by serum plus SA. Stepwise attachment of b-anti-IgM to SA-coated b4-RBC allows binding of up to 6 X 10(4) mole cules of b-anti-IgM/RBC. The second method is based on attachment of b -anti-IgM to RBC via CR1, the primate RBC complement receptor. The SA- biotin system is used to prepare bispecific mAb complexes (heteropolym ers) in which a biotinylated mAb to CR1 is cross-linked with b-anti-Ig M via SA. Binding of these heteropolymers to RBC via CR1 is specific a nd saturable and can facilitate binding of up to 2500 molecules of b-a nti-IgM/RBC. RBC containing b-anti-human IgM prepared by either method bound human IgM stably and with high avidity; heteropolymer mediated binding reached saturation at 400 molecules of IgM bound per RBC, and b4-RBC plus SA and b-anti-IgM bound up to 4000 molecules of IgM per RB C. Both types of RBC were stable in serum after antigen binding. The h eteropolymer-RBC system may be useful for CR1-mediated clearance of an tigens from the bloodstream, while the SA-b-RBC system may be appropri ate for targeting of encapsulated drugs to intravascular antigens acce ssible in circulation. (C) 1994 Academic Press,Inc.