Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin.

We compared a chemiluminescent assay and a colorimetric endpoint assay for measuring an alkaline phosphatase (EC 3.1.3.1) label in an enzyme immunoassay of thyrotropin (TSH). The substrate in the chemiluminescent assay is a derivative of adamantyl 1,2-dioxetane phosphate. On dephosphorylation, catalyzed by alkaline phosphatase, the 1,2-dioxetane decomposes further and emits a glow of light (lambda max 470 nm). We modified the Hybritech Tandem-E TSH High Sensitivity assay for chemiluminescent detection of bound alkaline phosphatase label by using this substrate (with 20-, 40-, and 60-min incubations). Detection limits (mean +2 SD of zero standard) were 6.0, 5.2, and 4.5 micro-int. units/L for these incubation periods, respectively, vs 20 micro-int. units/L for the conventional colorimetric version of the assay. Comparison of results for 44 clinical specimens assayed by the chemiluminescent (20-min incubation, y) and colorimetric (60-min endpoint, x) TSH immunoassays gave statistical values of: slope = 1.17, intercept = -0.22, and r = 0.98. Hemoglobin, but not bilirubin, lipids, or protein, interfered; but these interferents were removed by the washing steps in the enzyme immunoassay.