Radioimmunoassay for leukotriene B4

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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 × 109 M-1 at 37°C and in a concentration of 0.37 μg/ml of the immune plasma. When 10 μl of anti-LTB4 and 3.9 nCi of [14,15-3H]LTB4 (28 Ci/mmol; 1 Ci = 3.7 × 1010 becquerels) were incubated in a volume of 250 μl, 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.

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