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标题：Radioimmunoassay for leukotriene B4
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作者：Robert A. Lewis ; Jean-Michel Mencia-Huerta ; Roy J. Soberman 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1982
卷号：79
期号：24
页码：7904-7908
DOI：10.1073/pnas.79.24.7904
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A rabbit immunized with leukotriene B4 [LTB4; (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (Ka) for [14,15-3H]LTB4 of 3.2 x 109 M-1 at 37{degrees}C and in a concentration of 0.37 {micro}g/ml of the immune plasma. When 10 {micro}l of anti-LTB4 and 3.9 nCi of [14,15-3H]LTB4 (28 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) were incubated in a volume of 250 {micro}l, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB4 and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB4. The sulfidopeptide leukotrienes, LTC4 and LTD4, displaced the radioligand from this antibody with less than 1/100th the activity of LTB4, and the diastereoisomers of 6-trans-LTB4, 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB4 and quantitatively correlated with the physical measurement by integrated A269 in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB4 generated averaged 4.6 ng per 106 rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB4, a highly potent chemotactic factor, to unfractionated biologic fluids.
关键词：rat mast cell ; human neutrophil ; lipoxygenase ; chemotactic factor