Radio Immunoassay of Thromboxane B2

Thromboxane B2 (TxB2) is one of the major end products of the cyclooxygenation of arachidonic acid (AA) in blood platelets, lung and certain other tissues. The immediate precursor of TxB2 is the unstable thromboxane A2 which has been reported to cause platelet aggregation and to constrict coronary arteries as well as the respiratory tree. We report here on the development of a novel radioimmunoassay for TxB2 in human plasma. Radioactive TxB2 was prepared by incubating 3H-AA3 (sp. act. = 64 Ci/mM) with a suspension of washed rabbit platelets for 10 min. at 37°C. The 3H-TxB2 in the lipid extract was subsequently purified by silicic acid column chromatography. Antibodies to TxB2 were generated by immunizing rabbits with TxB2 which had been coupled to albumin using 1,1’ – carbonyldimidazole. The binding of 3H-TxB2 to diluted plasma (final dilution – 1 /1500) from immunized rabbits was inhibited by authentic TxB2 (Nelson et al, Tetrahedron Letters No. 37, 1976) but not by prostaglandins. Thus, 1.5 picomoles of cold TxB2 caused 50% inhibition of the binding of 3H-TxB2 while no cross reactivity was observed with PGD2, PGE2 or PGF2α. Small amounts of TxB2 (10–50nM) were found in normal human plasma but large amounts (~500 nM) were found in sera obtained from normal human blood clotted at 37°C. The assay should be useful for determining plasma levels of TxB2 in various disease states. The work supported in part by Grant HL-14890 from the NIH. V.F. was funded by the U. S. Army (AR601–112).