title: Study of antigen-binding properties of bispecific antibodies.
abstract: Antigen-binding properties of bispecfic antibodies (bAbs) produced by mouse hybrid hybridomas were studied. One of the bAbs held binding sites for two different antigens with relatively high molecular mass: human IgG (M(r) approximately 160,000) and horseradish peroxidase (HRP, M(r) approximately 40,000). Another bAbs showed specificity to antigens differing in molecular mass by more than an order of magnitude: peptide alpha-endorphin (END, M(r) approximately 1600) and HRP (M(r) approximately 40,000). The studied antibodies also contained different immunoglobulin chains. 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 subclass of IgG: IgG2a and IgG1. bAbs were 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-antibody binding and equilibrium constants of association (Ka) for each parental antibody and bAbs were determined by the Scatchard method. No significant 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 composition of bAbs light and heavy chains specific to END/HRP. This analysis corroborated that bAbs molecules contained light and heavy chains from both parental hybridomas. Hence, it was demonstrated that the hybridoma fusion method can provide bispecific IgG molecules fully preserving antigen binding properties of the parental antibodies.
authors.affiliation: Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: M B
authors.initials: MB
authors.lastname: Smirnova
authors.forename: N N
authors.initials: NN
authors.lastname: Dergunova
authors.forename: E A
authors.initials: EA
authors.lastname: Kizim
authors.lastname: Massino YuS
authors.forename: V A
authors.initials: VA
authors.lastname: Nikulina
authors.forename: O L
authors.initials: OL
authors.lastname: Segal
authors.forename: E B
authors.initials: EB
authors.lastname: Tereshkina
authors.forename: G I
authors.initials: GI
authors.lastname: Kolyaskina
authors.forename: A D
authors.initials: AD
authors.lastname: Dmitriev
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D018822:alpha-Endorphin / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D000941:Antigens; D007074:Immunoglobulin G; D018822:alpha-Endorphin; D006735:Horseradish Peroxidase