Novel bioluminescent assay of alkaline phosphatase using adenosine-3'-phosphate-5'-phosphosulfate as substrate and the luciferin-luciferase reaction and its application.
This paper describes a novel bioluminescent assay of alkaline phosphatase (ALP) utilizing ATP-sulfurylase and the luciferin-luciferase reaction. The principle governing the assay is as follows. Adenosine-3'-phosphate-5'-phosphosulfate, which serves as the substrate for ALP, is hydrolyzed enzymatically to produce adenosine-5'-phosphosulfate (APS). APS is converted into ATP by ATP-sulfurylase in the presence of pyrophosphate. The ATP produced is detected by the luciferin-luciferase reaction. The measurable range was 1 zmol to 100 fmol/assay and the detection limit at blank+3 SD was 10 zmol/assay. The coefficient of variation (CV, n=5) was examined at each point of the standard curve; the mean CV percentage was 4.47% (n=6). This assay system was applied to enzyme immunoassay of human chorionic gonadotropin and allele-specific PCR enzyme-linked immunosorbent assay of verotoxin gene using ALP as the label enzyme; 10(-2) mIU/mL hCG in urine and 5 pg of Escherichia coli O157 DNA could be assayed directly and with high sensitivity by the proposed method.
Arakawa
H
H
School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan. arakawa@pharm.showa-u.ac.jp
Shiokawa
M
M
Imamura
O
O
Maeda
M
M
D000250:Adenosine Phosphosulfate / Q000031:analogs & derivatives / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D004269:DNA, Bacterial / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D049454:Firefly Luciferin / Q000378:metabolism*; D006531:HEPES / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques / Q000379:methods / Q000592:standards; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D015636:Magnesium Chloride / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013379:Substrate Specificity; D013430:Sulfate Adenylyltransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D004269:DNA, Bacterial; D015636:Magnesium Chloride; D012710:Serum Albumin, Bovine; D010724:Phosphoadenosine Phosphosulfate; D000250:Adenosine Phosphosulfate; D049454:Firefly Luciferin; D008156:Luciferases; D013430:Sulfate Adenylyltransferase; D000469:Alkaline Phosphatase; D006531:HEPES