DETERMINATION OF FOLLICULAR-FLUID ESTRADIOL LEVELS BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

A direct competitive heterologous enzyme-linked immunosorbent assay (E LISA) was developed and validated to determine follicular fluid estrad iol levels of different antral follicular sizes (small, medium, and la rge) obtained from the ovaries of heifers in the follicular phase of t he estrous cycle. Polyclonal antiestradiol antibodies were raised in N ew Zealand White rabbits using 6-keto-1,3,5(10)-estratriene-3,17beta-d iol 6-carboxymethyloxime: bovine serum albumin as immunogen and charac terized by the usual methods. Horseradish peroxidase conjugated to 1,3 ,5(10)-estratriene-3,17beta-diol 3-hemisuccinate was used as a label. The concentration range used for the standard curve was 0 to 1 ng per well. The low detection limit of the technique was 0.3 pg per well wit h a sensitivity at 50% binding of 93.62 pg per well. Intraassay and in terassay CV (%) were <5.3% and <7.0%, respectively (n = 10). The recov ery rate of the known estradiol concentrations added to follicular flu id averaged 96.1 +/- 1.3%. Compared with a radioimmunoassay (RIA), the values of ELISA were highly correlated (r +/- 0.99, P < 0.005). This assay was used to quantify follicular fluid estradiol concentrations w ithout previous extraction in three antral follicular sizes: small (<5 mm), medium (5.1 to 10 mm), and large (10.1 to 20 mm). The mean +/- S E follicular fluid estradiol concentrations were 77 +/- 5.2 ng/ml (n = 490) in small follicles, 111 +/- 19 ng/ml (n = 65) in medium follicle s, and 496 +/- 144 ng/ml (n = 45) in large follicles. These values are similar to those previously reported for the same species, age, folli cular size, and stage of cycle. We concluded that this technique is us eful for determining follicular fluid estradiol concentrations in antr al follicles of heifers. Furthermore, its application to other species is now being investigated.