The effect of fluorescein labels on the affinity of antisera to small haptens

Small haptens such as methylamphetamine (MW 149) cannot, on their own, induce an immune response. It is also unlikely that they fill the binding site of any antibody that recognises them. Under such circumstances any attached label might be expected to enter the area of the binding site and exert an influence on overall binding. To investigate the possible influence of the label on binding, a range of fluorescein-labelled derivatives, differing in bridge length, were prepared. Antiserum binding of these labelled derivatives was then compared to that of the unlabelled drug. Evidence is presented which suggests that, with small haptens, the closeness of the fluorescein molecule can markedly influence antibody binding. Significant differences were found in titre, sensitivity, and assay kinetics. These overall effects appear to be brought about by the change in affinity of the antibody for the labelled hapten.