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

我们建立了一种同时利用萤火虫荧光素酶（b-Luc）和萤火虫荧光素蛋白（Aq）的生物发光检测方法；此外，我们开发了一种高灵敏度和快速的串联生物发光免疫分析法（BLIA），涉及aq标记的Fab片段和b- luc -链亲和素复合物。Aq和b-Luc的最低检出限分别为9.4×10^<-21> mol assay^<-1>（空白+ 3s - d）和3.6×10^<-19> mol assay^<-1>（空白+ 3s - d）。两种发光蛋白的测定在4 s内用单一测定培养基完成。本研究以前列腺酸性磷酸酶（PAP）和前列腺特异性抗原（PSA）为分析物，采用串联bla法测定。PAP和PSA分别用aq标记的anti-Dig Fab片段和b-Luc-streptavidin复合物检测。PAP和PSA的可测范围分别为0.04 ~ 100和0.2 ~ 200 ngmL^<-1>。该技术也适用于PSA和α-胎蛋白（AFP）的同时测定。PSA和AFP的可测范围分别为0.2 ~ 200和1.95 ~ 1000 ngmL^<-1>。人血清中PAP和PSA或PSA和AFP的水平可以用提出的bla准确测定。从拟议的bla和商业试剂盒中获得的结果之间观察到令人满意的相关性

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