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

我们建立了一种利用木犀草素(AQ)和生物素化的萤火虫荧光素酶(b-Luc)同时进行生物发光检测的方法；此外，我们还建立了AQ标记的Fab片段和b-Luc-链霉亲和素复合体的高灵敏、快速的串联生物发光免疫分析(BLIA)。AQ和b-Luc的最低检出限为9.4×10~(-1)~(-21)。和3.6×10~(-19)~(-1)~(-1)(空白3S.D.)。用单一的测定介质完成了S 4号的两个发光蛋白质的测定。在本研究中，前列腺酸性磷酸酶(PAP)和前列腺特异性抗原(PSA)作为分析物，在串联的BLIA中进行了测定。分别用AQ标记的抗地高辛Fab片段和b-Luc-链霉亲和素复合体检测PAP和PSA。PAP和PSA的测量范围分别为0.04~100和0.2~200ngmL^~(-1)；该技术还可用于PSA和甲胎蛋白(α)的同时检测。PSA和AFP的测量范围分别为0.2~200和1.95~1000ngmL^~(-1)。该方法可准确测定人血清中PAP和PSA或PSA和AFP水平。从拟议的BLIA获得的结果与从商业试剂盒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