Development of dual-enzyme-based simultaneous immunoassay for measurement of progesterone and human chorionic gonadotropin

The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin (hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) and alkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells, 50 μL of different concentrations of combined standards or serum samples was added in duplicate and then 100 μL of combined conjugate reagent, composed of 17-α-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1 h at 37 °C. After incubation, the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 μL alkaline phosphatase substrate along with streptavidin–horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 °C. After incubation, the developed color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of the wells were decanted and washed. In the next step, 100 μL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 min of incubation, 100 μL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage provides the result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- and inter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3 for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and for hCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnancies such as miscarriages and ectopic pregnancies.