title: Antigen-Induced Activation of Antibody Measured by Fluorescence Enhancement of FITC Label at Fc.
abstract: Three anti-carbohydrate antibodies of defined specificity isolated from plasma were used to demonstrate that macromolecular antigen binding caused considerable enhancement of fluorescence of FITC-labeled antibody. Mono and disaccharide antigens which could compete with the large antigens in antibody binding could not however produce any increase in fluorescence. Fluorescence enhancement in a given antibody sample increased with the size of the occupying macromolecular antigen. Conversely in antibody samples of same ligand specificity isolated from plasma of different individuals, fluorescence enhancement produced by the same antigen correlated with specific activity of the antibody sample. Removal of Fc part of antibody, confirmed by electrophoresis and Fc-specific antibody binding, caused abolition of most of the antigen-driven fluorescence increase. Since antigen binding sites of antibodies were protected during FITC labeling, the above results suggest that conformational shift in Fc produced by occupation of binding sites by large antigens resulted in the enhancement of fluorescence of FITC tags on Fc. Data provides a tool for detection and measurement of specific ligands using fluorolabeled whole antibodies.
authors.affiliation: Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, 695 011, India.
authors.forename: Genu
authors.initials: G
authors.lastname: George
authors.affiliation: Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, 695 011, India.
authors.forename: Mandagini
authors.initials: M
authors.lastname: Geetha
authors.affiliation: Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, 695 011, India. psappu@sctimst.ac.in.
authors.forename: Padinjaradath S
authors.initials: PS
authors.lastname: Appukuttan
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology*; D002241:Carbohydrates / Q000276:immunology; D002417:Cattle; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000276:immunology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D000941:Antigens; D002241:Carbohydrates; D005456:Fluorescent Dyes; D007141:Immunoglobulin Fc Fragments; D016650:Fluorescein-5-isothiocyanate
keywords: Anti-carbohydrate antibody; FITC; Fab; Fc; Fluorescence enhancement