Quantitative determination of kinins released by trypsin using enzyme-linked immunosorbent assay (ELISA) and...

Rapid ELISA and HPLC procedures were developed for quantitation and identification of various natural kinins. Antibradykinin mouse monoclonal antibodies were used to determine kinin levels in the range of 20-200 ng. Bradykinin coupled to bovine serum albumin was used to coat the plates in a 3- to 4-hr ELISA. Synthetic kinin standards isoleucine-seryl-bradykinin (Ileu-Ser-BK), methionyl-lysyl-bradykinin (Met-Lys-BK), tyrosine-bradykinin (Tyr-BK) and bradykinin (BK) yielded almost identical curves with a mixture of A5 and D9 monoclonal antibodies. [Tyr5]-BK, [Tyr8]-BK and des-arginine9 bradykinin (des-Arg9-BK) showed negligible amounts of cross-reactivity. ELISA-compatible trypsin digestion developed for release of kinins from plasma of normal humans, rats and turpentine-treated rats gave values of 3.2, 6.9 and 70 micrograms/ml plasma, respectively. High performance liquid chromatography methods were developed for complete resolution of kinins on a C-18 reversed phase mu-Bondapak column before and after derivatization with phenyl isothiocyanate (PITC). The simple PITC derivatization procedure yielded good quantitation above 20 pmol. The ELISA and HPLC methods were used in a complementary fashion to assay and identify kinins in biological fluids as well as during the course of kininogen purification.