COMPARISON OF CHEMILUMINESCENT AND CHROMOGENIC SUBSTRATES OF ALKALINE-PHOSPHATASE IN A DIRECT IMMUNOASSAY FOR PLASMA ESTRADIOL

The colorimetric and chemiluminescent determination of alkaline phosph atase activity in a direct enzyme immunoassay of the competitive type for determination of estradiol-17 beta (E(2)) in human plasma or serum were compared. In this assay biotin-labeled E(2) and E(2) from standa rd or patient samples compete for free antibody-binding sites. Antibod y-bound E(2)-biotin is then reacted with streptavidin-alkaline phospha tase. Enzyme activity in the bound fraction was detected either with t he chromogenic substrate p-nitrophenyl phosphate (pNPP) or with the ch emiluminescent substrate disodium '-chloro)-tricyclo[3.3.1.1(3,7)]deca n)-4-yl)phenyl phosphate (CSPD). Contrary to expectations, the use of CSPD for chemiluminescent endpoint determination of alkaline phosphata se did not improve assay performance. Both the limit of detection of a nalyte E(2) (160 vs. 73 pmol ml(-1)) and overall precision (C.V., %) w ere less favourable with CSPD than with pNPP. These results and the hi gh cost of CSPD make this chemiluminescent substrate less suited for e ndpoint determination in competitive EIA for haptens.