An enzyme-linked immunosorbent assay for quantitation of aromatase cytochrome P-450.

Traditionally, aromatase has been quantified as aromatase activity according to its ability to produce estrogen from androgen. We have developed a quantitative assay based on the protein mass of catalytically active aromatase cytochrome P-450. A solid phase sandwich enzyme-linked immunosorbent assay for aromatase cytochrome P-450 has been devised using mouse monoclonal antibody (MAb3-2C2) and rabbit polyclonal antiserum (PAb R-8-2). Two rabbit antisera (PAb R-8-1 and R-8-2) were raised by immunization against human placental aromatase cytochrome P-450 which had been isolated by immunoaffinity chromatography of MAb3-2C2-coupled to Sepharose 4B resin. Both antisera were capable of suppressing human placental aromatase activity with IC50 values of 0.6 and 0.8 microliter/ml incubate, respectively, and showed monospecific to aromatase cytochrome P-450 in the Western blot analyses. Solubilized human placental microsomal samples were incubated in microtiter wells precoated with MAb3-2C2. The unbound proteins were washed out, and the aromatase cytochrome P-450 bound with the MAb3-2C2 in the wells was then reacted with PAb R-8-2, the binding of which was subsequently probed with goat antirabbit immunoglobulin G antibody alkaline phosphatase conjugate. Immunoaffinity-purified aromatase cytochrome P-450 of human placental microsomes was used for the standard, with the current assay detection limit at 1 ng/ml. There was a positive correlation between aromatase activity and the immunoreactive aromatase cytochrome P-450 level in solubilized microsomal samples after preincubation at 22 and 37 C, indicating that the enzyme-linked immunosorbent assay measures the level of aromatase cytochrome P-450 that has catalytic activity. The mean level of aromatase cytochrome P-450 in solubilized human term placental microsomes was 16.4 +/- 10.3 (+/- SD) micrograms/ml, corresponding to 0.38 +/- 0.19% of the original microsomes. The mean specific activity of aromatization of the solubilized samples was 0.650 +/- 0.163 nmol estrogen formed/min.mg protein. These results indicate that aromatase in the solubilized placental microsomal fraction has catalytic ability of 5.3 +/- 1.6 min-1 based on the immunoassayable cytochrome P-450.