Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry.
Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.
D000818:Animals; D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm / Q000032:analysis*; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex / Q000032:analysis; D001667:Binding, Competitive; D000939:Epitopes; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D012516:Osteosarcoma / Q000276:immunology; D012513:Sarcoma, Experimental / Q000276:immunology
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000939:Epitopes