| Co-Investigator(Kenkyū-buntansha) |
ARAKAWA Hidetoshi Showa University, Sch.of Pharm.Sci., Assistant Professor, 薬学部, 助教授 (70129807)
ITO Katsutoshi Showa University, Sch.of Pharm.Sci., Lecture, 薬学部, 講師 (20223141)
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| Research Abstract |
We have developed the highly sensitive simultaneous bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) using a firefly luciferase-luciferin reaction, and applied this assay to a tandem bioluminescent enzyme immunoassay (BL-EIA) for Insulin and C-peptide, Angiotensin and endothelin. Recently, Nelson et al. have reported that neonatal blood concentration of VIP, CGRP, BDNF, NT 4/5 were higher in autistic spectrum than in control children. Therefore, measurements of these four factors in neonatal blood are made possible to diagnose and care for autism in early stage. In this study, we established highly sensitive tandem BL-EIA for BDNF and NT 4/5. The measurable ranges of BDNF and NT 4/5 were 4.9 - 40000 and 31.25 - 2000 pg/mL, the detection limits (at blank + 3 SD) of BDNF and NT 4/5 were 1.2 and 11.4 pg/mL, respectively. The intra-assay coefficients of variation of BDNF and NT 4/5 with each standard point were 1.8 - 9.8 % (n=8) and 2.3 - 6.4 % (n=8), resp
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ectively.
We also have developed the highly sensitive simultaneous bioluminescent assay of firefly luciferase and aequorin, and applied this assay to a tandem bioluminescent enzyme immunoassay (BL-EIA). Luciferin-luciferase reaction is specific and sensitive for the determination of ATP, therefore, this reaction has been used e.g. hygiene monitoring. Aequorin bounds specifically to Ca^<2+> and then emits blue light, thus aequorin is a valuable prove to study intercellular Ca^<2+>. We developed simultaneous assay of aequorin and luciferase by these different bioluminescence reaction. In the proposed assay, each of aequorin and luciferase solution were added to a microtiter plate, and Ca^<2+> solution was added, and then the bioluminescent intensity was integrated for 1 sec, immediately. Then, the reagent solution containing ATP, luciferin, Mg^<2+> was added to the same wells. The bioluminescent intensity obtained luciferin-luciferase reaction was integrated for 1 sec after a delay of 2 sec. The detection limits (at blank + 3SD) of aequorin and luciferase were 7.6x10^<-20> and 3.16x10^<-18> mol per assay, respectively. Therefore, this proposed assay is high throughput and sensitive detection. Furthermore, we have applied this assay to tandem immunoassay for prostate specific antigen and prostatic acid phosphatase Less
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