Preparation of Biotinylated Cypridina Luciferase and Its Use in Bioluminescent Enzyme Immunoassay
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- 作者：Chun Wu ; Kosei Kawasaki ; Yoko Ogawa ; Yasukazu Yoshida ; Satoru Ohgiya ; and ; Yoshihiro Ohmiya
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：February 15, 2007
- 年：2007
- 卷：79
- 期：4
- 页码：1634 - 1638
- 全文大小：271K
- 年卷期：v.79,no.4(February 15, 2007)
- ISSN：1520-6882
文摘
Cypridina luciferase, a well-known secretory enzymeinvolved in the bioluminescence of marine ostracod, isused to monitor gene expression in mammalian cells.Here we report the preparation of biotinylated Cypridinaluciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase wasexpressed in yeast and successfully purified to nearhomogeneity. The luciferase was biotinylated with conventional biotinylation reagents, and the biotinylatedlysine sites were determined by liquid chromatography-tandem mass spectrometry. The biotinylated luciferasewas stable when stored at 4 
C. The stability of syntheticS-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a modelstudy of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/mL interferon-alpha. Our results show that Cypridinaluciferase is a very sensitive and versatile bioluminescentreporter.
C. The stability of syntheticS-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a modelstudy of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/mL interferon-alpha. Our results show that Cypridinaluciferase is a very sensitive and versatile bioluminescentreporter.