Use of fluorodinitrobenzene to identify monoclonal antibodies which are suitable for conjugation to periodate-oxidized horseradish peroxidase.
During the recent development of a double antibody enzyme-linked immunosorbent assay for detecting malaria sporozoites in mosquitoes it was found that a large number of potentially useful monoclonal antibodies lost their capacity for binding antigen after conjugation to periodate-oxidized horseradish peroxidase (HPO). Since HPO reacts with primary amino groups, we used a simple chemical reaction with fluorodinitrobenzene (FDNB) to determine if the loss of antigen binding was due to the requirement for unmodified primary amino groups in the binding site. FDNB-treated antibodies which reacted with antigen in an indirect fluorescent antibody assay (IFA) also yielded successful HPO-antibody conjugates. Conversely, those antibodies which did not react with antigen after treatment with FDNB failed to produce useful HPO-antibody conjugates. These data suggest that conjugation of oxidized HPO to primary amines in or near the antigen-combining site yields conjugates in which the antibody is inactive. Use of FDNB to predict the suitability of antibodies for conjugation to periodate-oxidized HPO may save a considerable amount of time over randomly selecting antibodies for conjugation and testing.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D004139:Dinitrofluorobenzene; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D009578:Nitrobenzenes; D010961:Plasmodium / Q000276:immunology
D000911:Antibodies, Monoclonal; D009578:Nitrobenzenes; D004139:Dinitrofluorobenzene