Partial elucidation of an anti-hapten repertoire in BALB/c mice: Comparative characterization of several monoclonal anti-fluorescyl antibodies

The anti-fluorescyl repertoire in BALB/c mice was examined by producing nine hybridomas secreting antibodies with fluorescein specificity. All nine purified monoclonal anti-fluorescyl antibodies contained kappa light chains and gamma heavy chains (IgG1 and IgG2). Isoelectric focusing profiles of reduced and alkylated Ig preparations demonstrated restricted, yet relatively different spectrotypes. Collectively, the hybridomas provided a diverse range of antibody affinities as determined by several methods, including dissociation rate and ligand inhibition studies. Homogeneity of purified preparations was confirmed by dissociation rate experiments which showed that each monoclonal anti-fluorescyl preparation exhibited a single first-order off-rate. Competitive inhibition experiments with structurally related ligands indicated a high degree of fine specificity. Absorption profiles of bound fluorescein revealed distinct relative differences within the active sites of the molecules studied, and suggested the lack of any apparent correlation between affinity and wavelength maximum. Characteristics of the clones are discussed in terms of the range and prevalence of anti-fluorescyl antibody affinities and the diversity of possible binding mechanisms.