Homologous ELISA for detection of 17α-methyltestosterone in serum

Abstract

17 α Methyl testosterone is a synthetic steroid hormone having androgenic properties. It is used in aquaculture, medical treatments, in sports as doping drugs, in improving food efficacy but its residue in meat and their products can cause risk to the human, animals and environment. In this article, we have focused on developing homologous ELISA using 17α Methyl testosterone-3-Carboxymethyloxime-Bovine serum albumin antiserum and 17α Methyl testosterone-3-Carboxymethyloxime-Horseradish peroxidase enzyme conjugate. Immunogen and enzyme conjugate both were prepared by N-hydroxysuccinimide mediated carbodiimide reaction method. The sensitivity, affinity and ED50 of the assay were found to be 0.11 ng/ml, 0.02x10-8 L/mol and 5.78 ng/ml respectively. The cross reactivity for this assay combination was seen with eight steroids out of 59 structurally related steroids. The assay was then studied for analytical variables like recovery (94.8%-111.6%), precision (Inter and Intra-assay coefficient of variation less than 10%), correlation (R2= 0.96) by comparing with commercially available kit and further validated by measuring levels of 17α methyltestosterone in rat serum after injecting rats intramuscularly.