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Abstract
We developed a competitive chemiluminescent (CL) enzyme immunoassay (CLEIA) for rapid, sensitive analysis of 19‐nortestosterone (19β‐NT) in aquaculture tissues. Anti‐19‐NT polyclonal antibodies raised against protein conjugates of two haptenic derivatives of 19‐NT were utilized in immobilized, antibody‐based, competitive immunoassays. With homologous and heterologous ELISA systems, most antiserum titers fell in the range of 1:3000–1:100,000. Assay conditions, including concentrations of antisera and enzyme tracer, were optimized. The effect of pH and various blocking agents was also investigated. The optimized CLEIA system allowed 19‐NT determination in the range of 0.07–30.5 ng/mL with apparent IC50 values of 1.2–2.8 ng/mL. Cross‐reactivies with a number of compounds were determined, and the assay proved highly selective for 19‐NT. The good recoveries achieved with different aquaculture tissue samples indicate that CLEIA is suitable for high‐throughput screening of 19‐NT in this kind of sample.