Chemical synthesis of bispecific monoclonal antibodies: potential advantages in immunoassay systems

To date, the applications of bispecific antibodies in immunoassay and immunocytochemical procedures have been directed at uniting two different biomolecules through the binding of epitopes on each respective substance. In this study, bispecific antibodies were constructed in which both binding sites were directed to two different epitopes of the same target molecule. Two types of bispecific antibody were constructed; a bivalent bispecific monoclonal antibody and multivalent bispecific polymers. The binding characteristics of each were investigated for changes in specificity and binding strength relative to 1:1 mixtures of parent antibodies. A bivalent bispecific antibody (BBA) was synthesised by the method of Glennie et al. (1985) from monoclonal antibodies recognising the 'M' or 'B' units of creatine kinase (CKMB). The BBA had enhanced specificity for CKMB with diminished recognition of CKMM and CKBB. A less tedious method of producing bispecific antibody involving heterobifunctional cross-linkage was used to produce multivalent bispecific antibodies (MBAs). Certain MBAs constructed to bind 'M' and 'B' units of CKMB demonstrated enhanced specificity and affinity for CKMB. MBAs were also produced to opposite ends of the 39 amino acid peptide adrenocorticotrophic hormone (ACTH). One of these demonstrated an enhanced affinity of 41-fold. We conclude that while conventional synthesis of bispecific bivalent antibodies is not a practical proposition for immunoassay development, antibodies with similar advantages can be produced with a simple method using the heterobifunctional cross-linker. The production of certain bispecific antibody combinations appears to enhance the formation of antibody-antigen matrices conferring higher binding affinities than can be achieved with an antibody mixture alone.