The development of a radioimmunoassay for 12-L-hydroxyeicosatetraenoic acid

Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [125I]Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21-80% the binding of antibodies and consequently of [125I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.