Radioimmunoassay of blood bradykinin: purification of blood extracts to prevent cross-reaction with endogenous kininogen.
Alcoholic extracts of blood collected for measurement of circulating bradykinin were analysed for co-extracted kininogen which would artificially elevate measured bradykinin levels. The blood extract contained a glycoprotein with identical chromatographic properties to kininogen. Bradykinin immunoreactivity in the extract eluted as a single peak from an immunoaffinity column of anti-bradykinin IgG bound to Sepharose identically to both bradykinin and kininogen showing immunoidentity of these substances. Affinity chromatography on Concanavalin A-Sepharose or ion-exchange chromatography on CM-Sephadex C25 separated bradykinin and the glycoprotein which again eluted identically to kininogen. The decrease in measured values of blood bradykinin after purification of the extract on CM-Sephadex C25 was a similar amount to that calculated as cross-reactivity with the amount of co-extracted kininogen. Hence radioimmunoassay of bradykinin in ethanolic extracts of blood is inaccurate owing to the presence of co-extracted kininogen, and additional purification steps such as chromatography on CM-Sephadex C25 are mandatory for accurate assay by ligand binding techniques.
D000818:Animals; D001666:Binding Sites, Antibody; D001920:Bradykinin / Q000097:blood* / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods; D003429:Cross Reactions; D004285:Dogs; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D011489:Protein Denaturation; D011863:Radioimmunoassay
D007106:Immune Sera; D007704:Kininogens; D001920:Bradykinin