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 bound3H-labeled 11-trans-LTC4and 11-trans-LTC4with the same average association constant (Ka) of 2.8× 109M-1at 37 degrees C and were present at a concentration of 0.32 μ g/ml of the immune rabbit plasma. When 9.5 μ l of anti-LTD4and 108 pmol of 11-trans-[3H]LTC4(40 Ci/mmol) were incubated in a volume of 300 μ l with LTC4, LTD4, LTE4, or their 11-trans stereoisomers, 50% inhibition of 11-trans-[3H]LTC4binding 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 LTD4produced by deamination or modest peptide lengthening nor the specific stereochemistry of the Δ14-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]LTC4by anti-LTD4was 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 LTE4from RBL-1 cells were individually quantitated by radioimmunoassay after resolution by high-performance liquid chromatography.