Comparison of chemiluminescent and chromogenic substrates of alkaline phosphatase in a direct immunoassay for plasma estradiol

The colorimetric and chemiluminescent determination of alkaline phosphatase activity in a direct enzyme immunoassay of the competitive type for determination of estradiol-17β (E2) in human plasma or serum were compared. In this assay biotin-labeled E2 and E2 from standard or patient samples compete for free antibody-binding sites. Antibody-bound E2-biotin is then reacted with streptavidin-alkaline phosphatase. Enzyme activity in the bound fraction was detected either with the chromogenic substrate p-nitrophenyl phosphate (pNPP) or with the chemiluminescent substrate disodium 3-(4-methoxyspiro l,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.13,7]decan-4-yl)phenyl phosphate (CSPD). Contrary to expectations, the use of CSPD for chemiluminescent endpoint determination of alkaline phosphatase did not improve assay performance. Both the limit of detection of analyte E2 (160 vs. 73 pmol ml−1) and overall precision (C.V., %) were less favourable with CSPD than with pNPP. These results and the high cost of CSPD make this chemiluminescent substrate less suited for endpoint determination in competitive EIA for haptens.