Development of digoxigenin-labeled peptide: application to chemiluminoenzyme immunoassay of bradykinin in inflamed tissues.
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 ED50 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.
Décarie
A
A
Département de Physiologie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Drapeau
G
G
Closset
J
J
Couture
R
R
Adam
A
A
D015097:3-Mercaptopropionic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000208:Acute Disease; D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000032:analysis*; D002216:Captopril / Q000494:pharmacology; D004076:Digoxigenin; D004195:Disease Models, Animal; D007124:Immunoenzyme Techniques; D007249:Inflammation / Q000378:metabolism*; D008163:Luminescent Measurements; D001121:Lysine Carboxypeptidase / Q000037:antagonists & inhibitors; D008297:Male; D008969:Molecular Sequence Data; D015394:Molecular Structure; D051381:Rats; D017208:Rats, Wistar
C029729:2-mercaptomethyl-3-guanidinoethylthiopropionic acid; D002216:Captopril; D015097:3-Mercaptopropionic Acid; D001121:Lysine Carboxypeptidase; D004076:Digoxigenin; D001920:Bradykinin