Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis.
A rabbit immunized with a conjugate of leukotriene D4 (LTD4) and bovine albumin via the icosanoid carboxyl produced antibodies with comparable affinities for leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4) and their 11-trans stereoisomers. The antibodies bound 3H-labeled 11-trans-LTC4 and 11-trans-LTC4 with the same average association constant (Ka) of 2.8 x 10(9) M-1 at 37 degrees C and were present at a concentration of 0.32 microgram/ml of the immune rabbit plasma. When 9.5 microliter of anti-LTD4 and 108 pmol of 11-trans-[3H]LTC4 (40 Ci/mmol) were incubated in a volume of 300 microliter with LTC4, LTD4, LTE4, or their 11-trans stereoisomers, 50% inhibition of 11-trans-[3H]LTC4 binding was achieved at levels varying between 0.3 and 0.7 ng. As assessed with synthetic analogs of the natural leukotrienes, the antibodies recognized neither those changes within the 6-sulfidopeptide unit of LTD4 produced by deamination or modest peptide lengthening nor the specific stereochemistry of the delta 14-cis double bond. However, the antibodies did recognize the triene lipid domain and the position and spatial orientation of the glutathione or cysteinylglycine function. Binding of 11-trans-[3H]LTC4 by anti-LTD4 was not inhibited by glutathione, cystinylbisglycine, arachidonic acid, or 5-hydroxy-6,8,11,14-icosatetraenoic acid, and leukotriene B4 (LTB4) was about 1/1000th as active as LTC4, LTD4, or LTE4. Mouse lymphoma (WEHI-5) and rat basophil leukemia (RBL-1) cells, when stimulated with calcium ionophore A23187, each produced immunoreactive leukotrienes; and LTC4, LTD4, and LTE4 from RBL-1 cells were individually quantitated by radioimmunoassay after resolution by high-performance liquid chromatography.
D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D007942:Leukemia, Experimental / Q000378:metabolism; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D011863:Radioimmunoassay; D013189:SRS-A / Q000276:immunology* / Q000378:metabolism
D013189:SRS-A