A NEW HIGHLY SENSITIVE CHEMILUMINESCENT ASSAY OF ALKALINE-PHOSPHATASE USING LUCIGENIN AND ITS APPLICATION TO ENZYME-IMMUNOASSAY

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP(+) or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 x 10(-19) mol/assay and 2.5 x 10(-19) mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP(+) as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimetric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x - 0.04, r = 0.997 (n = 51) and y = 1.00x - 0.03, r = 0.999 (n = 10), respectively.