Development of a polyclonal antibody based icELISA for detection of testosterone levels in sheep

An indirect competitive ELISA (icELISA) has been developed for the determination of testosterone (TES) residue in urine. For this reason, EDC method was employed to synthesize the artificial antigen of TES-BSA, and New Zealand white rabbits were used to prepare the polyclonal antibody. Based on the square matrix titration, an icELISA method was developed. The dynamic range was from 0.096 to 92.2 ng/mL, with LOD and IC₅₀ value of 0.035 ng/mL and 2.8 ng/mL, respectively. Except for a slight cross-reactivity (22.3%) to 19-nortestosterone, negligible cross-reactivity to other compounds was observed. After optimization, 20-fold dilution in sheep urine gave an inhibition curve almost the same as that in PBS buffer. The regression equation was y = 0.9186 × + 1.4569, with a correlation coefficient of 0.9852. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of TES residue in urine.