STUDY OF ANTIGEN-BINDING PROPERTIES OF BISPECIFIC ANTIBODIES

Antigen-binding properties of bispecific antibodies (bAbs) produced by mouse hybrid hybridomas were studied. One of the bAbs held binding si tes for two different antigens with relatively high molecular mass: hu man IgG (M(r) similar to 160,000) and horseradish peroxidase (HRP, M(r ) similar to 40,000). Another bAbs showed specificity to antigens diff ering in molecular mass by more than an order of magnitude: peptide cr -endorphin (END, M(r) similar to 1600) and HRP (M(r) similar to 40,000 ). The studied antibodies also contained different immunoglobulin chai ns. Both heavy chains of the anti-IgG/HRP bAbs molecule were of mouse subclass IgG1. Anti-END/HRP bAbs was formed by a combination of heavy chains which belong to two subclasses of IgG: IgG2a and IgG1. bAbs wer e purified from ascitic fluid by a two-step affinity chromatography on columns with Sepharose-4B conjugated with the corresponding antigen. Radioimmune and immunoenzyme assays were used to analyze antigen-antib ody binding and equilibrium constants of association (K-a) for each pa rental antibody and bAbs were determined by the Scatchard method. No s ignificant changes in the affinity of bAbs antigen-binding sites were observed as compared to the corresponding parental antibodies. It was also shown that bAbs interaction with an excess of one of the antigens did not affect binding of the other antigen to the second bAbs site. Two-dimensional gel electrophoresis was used to analyze the compositio n of bAbs light and heavy chains specific to END/HRP. This analysis co rroborated that bAbs molecules contained light and heavy chains from b oth parental hybridomas. Hence, it was demonstrated that the hybridoma fusion method can provide bispecific IgG molecules fully preserving a ntigen binding properties of the parental antibodies.