Development and application of 5-HETE radioimmunoassay (RIA)

5-hydroxyeicosatetraenoic acid (5-HETE) lactone was converted to omega-aminopropylamide and coupled to BSA using 1,5-difluoro-2,4-dinitrobenzene as a coupling agent. Antibodies were developed by immunizing rabbits with 5-HETE-BSA conjugate for 11 months with monthly boosters. The antiplasma at 1 to 800 dilution bound 35% of the total radioactivity of the tritiated 5-HETE (150 Ci/mmole). The detection limit of the RIA (one SE of the buffer control) was 19 picogram. The antiplasma showed the greatest cross reactivity (0.24%) with LTB4 among compounds tested (arachidonic acid, diarachidonyl phosphatidyl choline, linoleic acid, dilinoeyl phosphatidyl choline, linolenic acid, LTB4, 5,12-diHETE, 5,15-diHETE, 15-HETE, 12-HETE, TXB2 and other major prostaglandins). When the ethanol extract of rat macrophages (peritoneal), stimulated by Calcium ionophore, was injected into HPLC and each fraction was assayed for 5-HETE by RIA, there was only one immunoreactive peak which appeared at fractions corresponding to the elution time of 5-HETE standard. RIA for LTB4 showed that the sample also contained LTB4 and 12-HETE. Thus, this antiplasma can be used to monitor 5-HETE, which is a relatively stable relic of arachidonic acid metabolism via 5-lipoxygenase pathway, synthesized from the endogenous precursor in various biological samples