Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase

使用水母发光蛋白标记的 Fab 片段和生物素化的萤火虫荧光素酶进行高灵敏度和快速串联生物发光免疫分析

• 批准号: 17590039
• 负责人: ITO Katsutoshi
• 金额: $ 2.05万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590039/
• 关键词: Bioluminescent assay; Biotinylated firefly luciferase; Aequorin; Sensitive ＆ simultaneous assay; Bioluminescent immunoassay; Prostate specific antigen; Prostatic acid phosphatase; α-Fetoprotein; 抗前立腺酸性ホスファターゼ; ハイスループットスクリーニング; カルシウムイオン; ルシフェリン

We established a simultaneous bioluminescent assay utilizing aequorin (Aq) and biotinylated firefly luciferase (b-Luc) ; furthermore, we developed a highly sensitive and rapid tandem bioluminescent immunoassay (BLIA) involving the Aq-labeled Fab fragment and b-Luc-streptavidin complex. Minimum detection limits of Aq and b-Luc were 9.4×10^<-21> mol assay^<-1> (blank + 3S.D.) and 3.6×10^<-19> mol assay^<-1> (blank + 3S.D.), respectively. Measurements of two luminescent proteins were completed in 4 s with a single assay medium. In this study, prostatic acid phosphatase (PAP) and prostate specific antigen (PSA), which served as analytes, were measured in the tandem BLIA. PAP and PSA were detected by the Aq-labeled anti-Dig Fab fragment and b-Luc-streptavidin complex, respectively. The measurable ranges of PAP and PSA were 0.04-100 and 0.2-200 ngmL^<-1>, respectively. This technique was also applied to the simultaneous measurement of PSA and α-fetoprotein (AFP). Measurable ranges of PSA and AFP were 0.2-200 and 1.95-1000 ngmL^<-1>, respectively. Levels of PAP and PSA or PSA and AFP in human serum could be accurately determined with the proposed BLIA. Satisfactory correlations were observed between results obtained from the proposed BLIA and those derived from commercial kits

我们利用水母发光蛋白（Aq）和生物素化萤火虫荧光素酶（b-Luc）建立了一种同时生物发光检测方法;此外，我们还开发了一种涉及Aq标记的Fab片段和b-Luc-链霉亲和素复合物的高度灵敏和快速的串联生物发光免疫分析（BLIA）。Aq和b-Luc的最低检测限分别为9.4×10 ~（-1）<-21>mol/L<-1>（空白+3S. D.）和3.6×10^<-19>mol含量测定^<-1>（空白+3S.D.），分别两种发光蛋白质的测量用单一测定介质在4秒内完成。在这项研究中，前列腺酸性磷酸酶（PAP）和前列腺特异性抗原（PSA），作为分析物，在串联BLIA测量。PAP和PSA分别用Aq标记的抗Dig Fab片段和b-Luc-链霉亲和素复合物检测。PAP和PSA的可测量范围分别为0.04-100和0.2-200 ng/mL<-1>。该技术还应用于同时测量PSA和甲胎蛋白（AFP）。PSA和AFP的测量范围分别为0.2-200和1.95-1000 ng/mL<-1>。该方法可准确测定人血清中PAP和PSA或PSA和AFP的水平。从拟议的BLIA和商业试剂盒获得的结果之间观察到令人满意的相关性