18] Bioluminescent assays of estrogens

Abstract

This chapter discusses the bioluminescent assays of estrogens. Estrone and estradiol concentrations are measured using the transhydrogenase function of human placental estradiol 17β-dehydrogenase. The hydrogen, from NADPH continuously formed by a regenerating system, is transferred to NAD through the rapid cycling enzymatic conversion of estrone and estradiol. The chapter describes a two-step assay of estrogens. The first reaction is the transhydrogenase or amplification reaction in which NADH is accumulated (15 min to 2 hr) and the second reaction is the bioluminescent assay of NADH. The assay requires three tubes for each sample and two steps. In the assay tube, the sample is incubated with all the reagent and enzymes as indicated in the table. The incubation time is dependent on the sensitivity required. The accumulated NADH is determined by adding the bioluminescent reagent. The estrogen concentrations are estimated by comparing the values obtained for the internal standard and the assay tube and assuming a linear relationship between absorbance and estrogen concentration. However, when the concentration of estrogen is too high, the rate of NADH formation is reduced, because of the accumulation of NADH. In this case, the use of the internal standard leads to an overestimation of the concentration of estrogen. For a linear relationship, the concentration of the NADH produced must be less than 5 × 10 –6 M. This is produced when 50 pg estrogen is incubated for 2 hr in 0.22 ml of transhydrogenase mixture.