Forchlorfenuron-mimicking haptens: from immunogen design to antibody characterization by hierarchical clustering analysis

Abstract

To obtain highly-specific and selective forchlorfenuron binders, a collection of functionalized derivatives with different spacer arm locations and lengths was prepared. By immunization with target-mimicking haptens, a large battery of monoclonal and polyclonal antibodies against this synthetic cell regulator was produced and exhaustively characterized in two immunoassay formats using homologous and heterologous conjugates. Antibodies with IC₅₀ values lower than 0.3 nM were successfully raised from the prepared immunogens, thus evidencing the efficacy of the explored strategies. In order to identify significant epitopes in the antibody–antigen interaction, a series of new chemical forchlorfenuron analogues, with slight modifications at both rings of the target molecule, were synthesized and evaluated in competitive assays. As a novel approach in hapten recognition studies, data processing was performed by computational classification methods based on hierarchical clustering. This strategy was shown to be highly valuable for a straightforward profiling of antibodies according to analogue recognition patterns. A relationship could be established between the antigen binding properties of antibodies and the structure of the immunogen. Whereas antibodies with equivalent affinities had been obtained from all of the derivatives, their specificity was found to be largely influenced by the differential exposition of the molecule to the immune system.