Bioluminescent PCR-RFLP Enzyme-Linked Immunosorbent Assay for Analysis of Vitamin D Receptor Gene Polymorphism

Abstract

Osteoporosis is a major public health problem which affects quality of life and raises costs for health care providers. Although an osteoporotic fracture is caused by multiple factors, the risk of osteoporotic fracture in later life is determined by the peak bone density achieved in early adulthood in relation to age and menopause-related bone loss. The known determinants of peak bone density are body mass index (BMD), calcium intake, exercise, and genetic factors. It is reported that common allelic variation in the vitamin D receptor locus (VDR) can be used to predict bone turnover and bone mineral density.1-3 Japanese premenopausal women, homozygous subjects in whom the ApaI in intron 8 and TaqI site in exon 9 is present (AA, tt) were found to have lower BMD than those with the (aa, TT) genotype respectively. 3 Analysis of VDR gene polymorphism is generally done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single strand DNA confirmational polymorphism. 4 These techniques are not suitable for routine clinical analysis because of problems of rapidity, reproducibility and use of electrophoresis gel separation (time consuming). Recently, enzyme linked immunosorbent assays (ELISA) combined with RFLP using restriction enzymes and hybridization using a DNA probe are used for analysis of PCR products. [5][6][7] These methods have enabled the fast detection and quantification of PCR products. At present, a more sensitive and simpler method for PCR-ELISA is required. The sensitivity of ELISA depends remarkably on the enzyme used as a marker and on the detection method used. Highly sensitive chemiluminescent (CL) methods 8 and bioluminescent methods 9 have become available for detecting alkaline phosphatase and β-D-galactosidase labels used generally in ELISA. We have already developed a sensitive bioluminescent binding assay 10 and EIA using firefly luciferase and acetate kinase (AK) as a label enzyme 11,12 , respectively. In the research reported here, we developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system. FITC-labeled sense primer and biotin-labeled anti-sense primer were used for PCR amplification of vitamin D receptor gene. After PCR, the products were digested with Taq I or Apa I enzyme, and the reaction products were analyzed by ELISA using an anti FITC antibody coated plate and an avidin/biotinylated AK system determined by bioluminescence reaction. DNA polymorphism types of VDR could be clearly determined by measuring the bioluminescent intensity or by using photon imaging with a CCD camera. This assay system is suitable for determining simultaneously DNA polymorphisms of a large number of samples. Materials and Methods InstrumentsHamamatsu ARGUS-50 (Hamamatsu Photonics K. Medicine, Bunkyo, Tokyo, Japan We developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system. AK used as a label enzyme could sensitively be detected by bioluminescent assay u...

Simultaneous Analysis of Two-Site Gene Polymorphisms Using Time-Resolved Fluorescence Immunoassay

“…8 The method enabled a fast detection and sensitive assay of PCR products. However, a more useful method, such as the simultaneous analysis of two-site gene polymorphism is also required in the clinical field.…”