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Development of a radioimmunoassay for [DES-ARG9]-bradykinin
Antisera to [des-Arg9]-bradykinin were elicited in rabbits immunized with the peptide conjugated to thyroglobulin and/or ovalbumin. Sera were screened for the presence of antibody with three radioactive antigens, mono-125I-labeled derivatives of [Tyr1, des-Arg]-kallidin, [Tyr, des-Arg]-bradykinin, and [Tyr, des-Arg]-bradykinin that were prepared by treating mono-125I-labeled [Tyr]-kallidin, [Tyr]-bradykinin, and [Tyr]-bradykinin with carboxypeptidase B. Of the six animals immunized, five produced antibodies to [des-Arg]-bradykinin as evidenced by the ability of their sera to bind at least 33% of the added radioactivity at a final dilution of 1:500. Sensitivity and specificity studies were performed with each labeled antigen and a dilution of antiserum that bound 30-50% of the radioactivity. The best labeled antigen-antibody combination, with respect to titer, sensitivity, and specificity was obtained with [mono-125I-Tyr, des Arg]-bradykinin and serum from a rabbit immunized with [des-Arg]-bradykinin conjugated to ovalbumin with toluene diisocyanate. The lowest concentration of [des-Arg]-bradykinin inhibiting 50% of this radioactive antigen binding was 0.23 ng/ml and the lowest concentration which could be distinguished from no [des-Arg]-bradykinin added was 67 pg/ml. This antiserum cross-reacts with bradykinin and lysyl-bradykinin about 9% but not with methionyl-lysyl-bradykinin.
Antisera to [des-Arg9]-bradykinin were elicited in rabbits immunized with the peptide conjugated to thyroglobulin and/or ovalbumin. Sera were screened for the presence of antibody with three radioactive antigens, mono-125I-labeled derivatives of [Tyr1, des-Arg]-kallidin, [Tyr, des-Arg]-bradykinin, and [Tyr, des-Arg]-bradykinin that were prepared by treating mono-125I-labeled [Tyr]-kallidin, [Tyr]-bradykinin, and [Tyr]-bradykinin with carboxypeptidase B. Of the six animals immunized, five produced antibodies to [des-Arg]-bradykinin as evidenced by the ability of their sera to bind at least 33% of the added radioactivity at a final dilution of 1:500. Sensitivity and specificity studies were performed with each labeled antigen and a dilution of antiserum that bound 30-50% of the radioactivity. The best labeled antigen-antibody combination, with respect to titer, sensitivity, and specificity was obtained with [mono-125I-Tyr, des Arg]-bradykinin and serum from a rabbit immunized with [des-Arg]-bradykinin conjugated to ovalbumin with toluene diisocyanate. The lowest concentration of [des-Arg]-bradykinin inhibiting 50% of this radioactive antigen binding was 0.23 ng/ml and the lowest concentration which could be distinguished from no [des-Arg]-bradykinin added was 67 pg/ml. This antiserum cross-reacts with bradykinin and lysyl-bradykinin about 9% but not with methionyl-lysyl-bradykinin.