Restriction in IgM expression--V. Fine structure analysis in the anti-lactose system.
A methodology for the analysis of the fine specificity of monoclonal anti-lactose IgM and IgG antibodies is described using structural variants of the homologous lactoside epitope. These variants are used as inhibitors of the binding of a reference ligand N-(5-dimethylaminonaphthalene-1-sulfonyl)-p-aminophenyl-beta-lactoside. Excitation of the antibody with bound ligand at 295 nm leads to resonance energy transfer to and fluorescence emission by the ligand. Titration of the antibody-ligand mixture with the inhibitor and measurement of the emission at 550 nm provide the data for the calculation of the binding constants of the inhibitors. A comparison of two IgM and two IgG antibodies showed that the higher affinity of the IgG antibodies arises from their specific interaction with both hexosides of the lactoside in contrast to IgM antibodies which do not engage the non-terminal hexoside as effectively. The quantitative significance of this difference is a differential free energy contribution of about -3 kcal/mole to the binding of lactoside by IgG. A finer discrimination between homologous and several cross-reactive molecules is evident with IgG antibody compared to IgM. The former exhibits about 100-fold greater difference in their binding constants than does IgM. These differences applied to biologically relevant multivalent interactions, where functional affinity governs complex formation, suggest a possible explanation for the IgM to IgG conversion characteristic of the humoral immune response.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D003619:Dansyl Compounds / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology*; D007785:Lactose / Q000276:immunology*; D008024:Ligands; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D003619:Dansyl Compounds; D000939:Epitopes; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008024:Ligands; C043238:N-(5-dimethylaminonaphthalene-1-sulfonyl)-4-aminophenyl-beta-lactoside; D007785:Lactose