Preparation of Anti-Nortestosterone Antibodies and Development of an Indirect Heterologous Competitive Enzyme-Linked Immunosorbent Assay to Detect Nortestosterone Residues in Animal Urine

Abstract

The steroid 19-Nortestosterone (NT) has been illegally used in horse racing, dog games to boost physical performance, and also in animal husbandry to accelerate weight gain. This paper reports an indirect heterologous competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody for rapid, sensitive analysis of NT in animal urines. After derivation, NT haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method and mixed-anhydride technology respectively. Two antisera raised in rabbits and two coating antigens synthesized were screened and optimized using homologous and heterologous ELISA formats. The most sensitive heterologous icELISA (antisera generated from NT-17-BSA, with NT-3-OVA as coating antigen) gave an IC₅₀ value of 27 ng/mL, with a dynamic range from 4 to 198 ng/mL, and the limit of detection (LOD) of 1.9 ng/mL. Except for cross-reactivity (CR) toward 17α-Nortestosterone (67%) and β-Boldenone (16.4%), no significant CR was observed for other chemical analogues tested. When applied to the authentic animal urines, the intra- and inter-assay precision values were below 8.4%, while the recoveries were in the range of 95–110%. The correlation coefficient between the established icELISA and LC-MS/MS method was greater than 0.98.