CHEMILUMINESCENT AND BIOLUMINESCENT ASSAYS FOR POLYADENYLIC-ACID AND APPLICATIONS TO DNA PROBE ASSAY AND IMMUNOASSAY

We developed a chemiluminescent (CL) assay for polyadenylic acid (poly A) based on the enzymatic cycling reaction following the conversion to ATP by pyruvate kinase/polynucleotide phosphoryrase enzyme reaction. Poly A was also measured with the bioluminescent (BL) assay using luciferase from firefly Photinus pyralis. The detection limits of poly A by CL and BL assay were 10 amol and 2 amol, respectively. We prepared the biotinylated poly A using biotin hydrazide and glutaraldehyde as a bifunctional coupling reagent to apply the poly A assay to a general avidin-biotin reaction system. When the CL assay and the BL assay of biotinylated poly A are applied to the DNA probe assay of lambda-phage DNA and the immunoassay of thyroid-stimulating hormone (TSH) using avidin-biotin reaction as detection system, 500 pg assay-1 well of lambda-phage DNA and 3-mu-U ml-1 of TSH were determined.