Characterization of interactions involving anti-metatype antibodies and immune complexes.

Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity labeled with fluorescein 5-isothiocyanate into a rabbit elicited antibodies specific for the liganded conformation of 4-4-20 (termed "anti-metatype" antibodies). Reaction of liganded 4-4-20 with anti-metatype antibodies caused significant delay (up to 23-fold) in the rate of dissociation of fluorescein ligand from the active site. In this study, structural analogues of fluorescein, including fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, 5-dichlorotriazinyl aminofluorescein and 5-carboxyfluorescein, were bound by monoclonal antibody 4-4-20 and anti-metatype antibody reactivity was observed through delay in the dissociation rate of ligand from Mab 4-4-20. Significant delays (ranging from 5- to 242-fold) were observed for all structural analogues examined indicating that 4-4-20 maintained similar but not necessarily identical conformations upon binding fluorescein structural analogues. Additionally, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carrier molecules of increasing mol. wt (ranging from 225 to 14,600 D) in an attempt to sterically interfere with "metatopes" at the mouth of the active site and localize regions of anti-metatype antibody binding. These fluorescein-conjugated compounds were reacted with 4-4-20, and binding of anti-metatype antibodies delayed dissociation rates from 24- to greater than 1500-fold. These results indicated that the mechanism whereby anti-metatype antibodies delay the release of fluorescyl ligands from the active site probably does not solely involve steric hindrance of the ligand due to binding of anti-metatype antibodies at the mouth of the active site. Studies with 4-4-20 Fab fragments and a single chain derivative of 4-4-20 (consisting of the variable regions tethered by a 14 amino acid linker) indicated that anti-metatype reactivity was specific for the immunoglobulin variable region.