Attachment of Biotinylated Antibody to Red Blood Cells: Antigen-Binding Capacity of Immunoerythrocytes and Their Susceptibility to Lysis by Complement
✍ Scribed by V.R. Muzykantov; R.P. Taylor
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 614 KB
- Volume
- 223
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A biotinylated monoclonal antibody ( (\mathrm{mAb})) to human IgM (b-anti-IgM) has been attached to human red blood cells (RBC) by two different approaches. The first method is performed with biotinylated RBC (b-RBC) and involves stepwise binding of streptavidin (SA) to bRBC followed by addition and binding of specific (b) anti-IgM or b-IgG. b-RBC were prepared with differing input levels of biotin (\boldsymbol{N})-hydroxysuccinimide ester (BNHS). At moderate BNHS levels ((100 \mu \mathrm{M})) the resulting b-RBC (designated b4-RBC) bound 50,000 molecules of b-IgG after treatment with SA. However, at high BNHS levels ((>1000 \mu \mathrm{m})) the resulting b-RBC bound b-IgG poorly, presumably due to multivalent binding of each SA to several biotins in close proximity on the RBC. b-RBC prepared at high BNHS inputs (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 \times 10^{4}) molecules of (\mathrm{b})-anti-IgM/RBC. The second method is based on attachment of (b)-antiIgM to RBC via CR1, the primate RBC complement receptor. The SA-biotin system is used to prepare bispecific mAb complexes (heteropolymers) in which a biotinylated mAb to CR 1 is cross-linked with b-anti-IgM via SA. Binding of these heteropolymers to (R B C) via CR1 is specific and saturable and can facilitate binding of up to 2500 molecules of (b)-anti-IgM/RBC. (R B C) 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 (\operatorname{IgM}) bound per (\mathrm{RBC}), and b4-RBC plus SA and b-anti-IgM bound up to 4000 molecules of IgM per RBC. Both types of (R B C) were stable in serum after antigen binding. The heteropolymer-RBC system may be