ANTIVASOPRESSIN ANTIBODY . CHARACTERIZATION OF HIGH-AFFINITY RABBIT ANTIBODY WITH LIMITED ASSOCIATION CONSTANT HETEROGENEITY

High-affinity antivasopressin antibody with limited association constant heterogeneity has been induced in rabbits by immunization with vasopressin coupled by carbodiimide-initiated amide linkage to equine γGab-globulins, and also by immunization with unmodified vasopressin. The antivasopressin antibodies of hyperimmune antisera have been shown to be 7S γG-globulins by radioimmunoelectrophoresis and sucrose density gradient ultracentrifugation. Radioimmunoprecipitation vasopressin binding curves have been constructed with radioiodinated vasopressin, employing sheep antirabbit γG-globulin serum to precipitate the complexes of rabbit antivasopressin antibody and radioiodinated vasopressin. The average intrinsic association constants, K0, of the antivasopressin antibodies did not vary substantially during the immunization schedules, or between animals immunized either with unmodified vasopressin or with vasopressin covalently linked to equine γGab-globulins (K0 = 1.6-4.5 × 109 M-1). The r/c vs. r plots (r = moles of hapten bound per mole of antibody; c = free hapten concentration) were linear over a wide range of values for r (r = 0.1- 1.9). Analysis of the binding data by use of the Sips distribution function (log (r/(n-r)) = a log c + a log K0) furnished values for the index of heterogeneity, a, which were near unity, again indicating that there was no substantial heterogeneity of the association constants of the antibodies of individual sera. The ability of analogs of vasopressin and chemically modified vasopressin to displace radioiodinated vasopressin from antivasopressin antibody has been evaluated by constructing radioimmunoassay inhibition curves. 8- Lys-vasopressin and 8-Arg-vasopressin displayed equal capacities to displace radioiodinated vasopressin from rabbit antivasopressin antibody. Oxytocin (3-Ile-8-Leu-vasopressin) required a concentration ten times greater than 8-Lysvasopressin or 8-Arg-vasopressin to effect a similar result. Performic acid oxidized vasopressin required a concentration 104 times greater than native vasopressin to displace an equivalent amount of radioiodinated vasopressin from antibody, indicating that disruption of the disulfide bond of the nonapeptide and conversion of the half-cystinyl residues at positions 1 and 6 into cysteic acid residues resulted in a profound alteration in the antigenic structure of the hormone. Conversion of the half-cystinyl residues into neutral S-carbamidomethylcysteinyl residues by reduction with 2-mercaptoethanol and alkylation with iodoacetamide also led to a marked alteration in the antigenic structure of the nonapeptide. Space-filling molecular models of vasopressin and of chemically modified vasopressin have been constructed and used in the analysis of the experimental findings. © 1969, American Chemical Society. All rights reserved.