Development of ELISA and sensor-based assays for the detection of ethynyl estradiol in bile

A polyclonal antibody, EEAb1, was produced against the synthetic steroid hormone, ethynyl estradiol (EE). EEAb1 demonstrated low levels of cross-reactivity with related compounds. Competitive and inhibitive ELISA assays were developed along with a biosensor-based assay using a BIAcore(R) 3000 for the detection of EE. For EEAb1 the detection ranges were 6-100 000 ng ml(-1) in the competitive ELISA, 3-50 000 ng ml(-1) in the inhibitive ELISA and 12-100 000 ng ml(-1) in the biosensor-based assay. The competitive ELISA data corresponded more closely to the 4-parameter curve model used and also required less time to perform than the inhibitive ELISA. The assays were optimized for use with bile. The bile was diluted 1/2 with buffer (phosphate-buffered salin or Hepes-buffered saline) prior to analysis. The assay formats described in this paper offer quick and simple methods for the detection of ethynyl estradiol in bile samples from cattle.