Xiao L, Yang C, Nelson C O, Holloway B P, Udhayakumar V, Lal A A
Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Atlanta, GA 30333, USA.
J Immunol Methods. 1996 Dec 15;199(2):139-47. doi: 10.1016/s0022-1759(96)00174-3.
We described here a bioluminescence-based immunoassay for the quantitation of RT-PCR amplified cytokine mRNA. This technique uses a standard RT-PCR procedure, with the following modifications. The forward primer in the PCR reaction is labeled with a 5' biotin molecule. Following PCR, a digoxigenin-conjugated oligonucleotide probe is hybridized to the target biotin-labeled DNA template. The hybridized duplex is captured onto a streptavidin-coated microtiter plate. The bound product is quantitated by adding digoxigenin-specific antibodies conjugated with the photoprotein aequorin. The amount of specific DNA captured onto the plate is quantitated by triggering the bioluminescence reaction through the addition of calcium ions. This technique detected as low as 40 amol of amplified cytokine products, or 500 copies of templates when 27 PCR cycles were used. The high sensitivity of this technique enables the quantitation of target DNA during the exponential phase of the PCR reaction. The aequorin-bioluminescence assay is an alterative non-radioactive method for the quantitation of PCR products.
我们在此描述了一种基于生物发光的免疫测定法，用于定量逆转录聚合酶链反应（RT-PCR）扩增的细胞因子信使核糖核酸（mRNA）。该技术采用标准的RT-PCR程序，并作如下修改。PCR反应中的正向引物用5'生物素分子标记。PCR之后，将地高辛配体缀合的寡核苷酸探针与目标生物素标记的DNA模板杂交。杂交双链体被捕获到包被有链霉抗生物素蛋白的微量滴定板上。通过加入与光蛋白水母发光蛋白缀合的地高辛特异性抗体来定量结合产物。通过加入钙离子触发生物发光反应来定量捕获到板上的特异性DNA的量。该技术能够检测低至40阿托摩尔的扩增细胞因子产物，或在使用27个PCR循环时检测到500个模板拷贝。该技术的高灵敏度使得能够在PCR反应的指数期对目标DNA进行定量。水母发光蛋白-生物发光测定法是一种用于定量PCR产物的非放射性替代方法。
