Abstract
A new ultrasensitive chemiluminoenzyme immunoassay (CLEIA) using digoxigenin-labeled bradykinin (BK) as a tracer is proposed to quantify kinins in tissue samples. Rabbit polyclonal IgGs anti-BK directed against the C- terminal end were used for the immunoconcentration step along with dioxetane derivative for the revelation step. The sensitivity of the assay for BK was 0.1 fmol/ml with ED 50 of 0.78 pmol/ml. This method was applied on extracts of normal and carrageenan-inflamed tissues. The edema produced by the injection of carrageenan in rat hindpaws was associated with a sevenfold increase of immunoreactive kinins in the inflamed paw extract ( from 0.021 ± 0.007 to 0.141 ± 0.021 pmol/g tissue ; p < 0.01), the immunoreactivity corresponded to BK, kallidin, and T-kinin after HPLC separation. When a mixture of inhibitors of kininase I (mergepta) and kininase II (captopril) was coinjected with carrageenan, the carrageenan-induced edema was unaffected but the kinin tissue content was significantly enhanced ( 0.207 ± 0.003 pmol/g tissue ; p < 0.01). However, the kinin tissue content and the edema response were unaltered by inhibitors given separately. Hence, this highly sensitive assay provides a biochemical evidence that kinins may act as proinflammatory mediators, and highlights a compensatory increase of kininase I and II activities in inflamed tissues.
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