We report a novel antigen heterologous enzyme linked immunosorbent assay for the direct estimation of estrone-3-glucuronide (E1-3-G) in diluted human urine. The differential behavior of antibody towards the labeled and unlabelled analyte in heterologous systems forms the basis of the present assay. Antiserum was raised in New Zealand white rabbits using estrone-3-glucuronide-bovine serum albumin (E1-3-G-BSA) as immunogen and nandrolone-17-HS (N-17-HS) coupled to horseradish peroxidase (HRP) with and without urea bridge ((N-17-HS-Urea-HRP/N-17-HS-HRP) as an enzyme labeled reagent. To the E1-3-G antibody coated wells, 50 microL standard or appropriately diluted (1:20) female urine samples were added along with nandrolone-17-HS-HRP/N-17-HS-Urea-HRP conjugate (100 microL) and were incubated at room temperature (RT) for 1 hour. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H(2)O(2)) as substrate. The cross-reaction of E1-3-G antiserum with C(18), C(19), C(21), C(27) steroids was less than 0.1% in both assays. Incorporation of urea bridge in the enzyme conjugate has decreased the effective displacement dose, i.e., ED(50) from 20 ng/ml to as low as 2 ng/mL. The sensitivity of the assay using N-17-HS-HRP and N-17-HS-Urea-HRP was 0.6 ng/mL and 0.4 ng/mL, respectively. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 4.7-9.0% and 5.1-6.4%, respectively. The estrone glucuronide level was also determined in female urine samples of 26 and 28 days cycle depicting a prominent peak corresponding to the preovulatory phase. The urinary E1-3-G values correlated well with those obtained by heat denaturation of urine samples r=0.94 (n=27).