| Co-Investigator(Kenkyū-buntansha) |
KOKADO Amane School of Pharmaceutical Sciences, Showa University, Assistant, 薬学部, 助手 (20266159)
ITO Katsutoshi School of Pharmaceutical Sciences, Showa University, Assistant Professor, 薬学部, 講師 (20223141)
ARAKAWA Hidetoshi School of Pharmaceutical Sciences, Showa University, Associate Professor, 薬学部, 助教授 (70129807)
TAJIMA Noriko School of Pharmaceutical Sciences, Showa University, Assistant, 薬学部, 助手 (30297022)
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| Research Abstract |
Recently, bioluminescence is extensively investigated by several researchers for its high sensitivity with the aim of applying several assays. And the various luciferases and photoprotein genes were cloned and expressed in E.coli or saccharomyces serevisiae. Especially, recombinant firefly luciferase is now commercially available and has been employed for enzyme immunoassay(EIA). However, the firefly luciferase tends to lose much of its activity when treated with the chemical cross linkage reagents used in the conjugation. To dissolve this problem, we have established two highly sensitive bioluminescent assays for thermostable acetate kinase (AK). After EIA using AK as label enzyme, AK activity was determined by measuring the amount of produced AT P using firefly luciferase.
Furthermore, we have introduced well known streptavidin-biotin system to EIA using biotinyted AK. Using these systems, we applied to assay for biologically substances such as hCG, mIL-6 and other clinically importan
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t substances. Especially, in the TSH assay, sensitivity was about 25 times higher than that of RIA. It will be useful for detection of the low concentration of TSH values such as hyperthyroidism. It have been applied to BLEIA in combination with the streptavidin-biotin system with biotinylated AK and appeared sensitive, simple, and convenient. Using the streptavidin-biotin system, mIL-6 in mouse tissue and plasma, and PACAP 38 in rat tissues: it could be accurately measured without the concentration of samples or any other pretreatment using the BLEIA. More recently, Kajiyama et al conducted mutational analysis of firefly luciferase and showed that they emit light of different colors due to single amino acid change. The study demonstrated multiple simultaneous antigen detection using the two kinds of biotinylated luciferase-streptavidin-bioanylated antibody complexes that were capable of emitting difference colors of light. As clinical application using this system, PGI and PGII in serum can be assayed, satisfactory. Further investigation is now ongoing to determine if multiple analyte in the same sample can be assayed by BLEIA. Less
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