Development of immuno PCR method and its biomedical application

免疫PCR方法的发展及其生物医学应用

• 批准号: 07807211
• 负责人: ENDO Hiroshi
• 金额: $ 0.96万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07807211/
• 关键词: immuno PCR; immunoassay; C- reactive protein; イムノ PCR

A high sensitive assay, which was termed immuno PCR,for C- reactive protein as model molecule was developed. PCR (polymerase chain reaction) was employed in detection system of this assay. C- reactive protein was immobilized on microplate through APPC (p-aminophenylphosphorylcholine) - BSA,based upon its Ca^<2+>-dependent binding property to phosphorylcholine. After blocking, 1st Ab, biotinylated 2nd Ab, avidin and biotinylated template DNA were added in turn. In final, PCR was performed with SP6 promoter primer and T7 promoter primer. As assessed by image analyzer after electrophoresis followed by EtBr staining, the amounts of PCR product to be amplified (497 bp) was shown to be dependent on the amounts of initially added C- reactive protein. In addition, it varied with some parameters, including PCR cycles, temperature and period for annealing. C- reactive protein as little as 2.5 ng/ml could be detected by his immono PCR,while not by sandwich- type conventional enzyme immunoassay.

一种高敏感测定，称为Model分子的C-反应性蛋白，称为免疫PCR。 PCR（聚合酶链反应）用于该测定的检测系统。 C-反应性蛋白通过AppC（P-氨基磷酸胆碱）-BSA固定在微板上，基于其CA^<2+> - 依赖性的结合特性与磷酸胆碱的依赖性结合。阻塞后，又添加了第1 AB，生物素化的第二AB，Avidin和生物素化模板DNA。在最终，使用SP6启动子底漆和T7启动子底漆进行PCR。正如电泳后通过图像分析仪评估的，然后进行ETBR染色，显示要扩增的PCR产物的量（497 bp）被证明取决于最初添加的C-反应性蛋白的量。另外，它随一些参数而变化，包括PCR周期，温度和退火期。 c-反应性蛋白只能通过其Immono PCR检测到2.5 ng/ml，而不是通过三明治常规酶免疫测定法。

项目成果

森秀治: "疎水性クロマトグラフィに伴う耐熱性ホスファターゼ活性の変動" 岡山大学医療技術短期大学部 紀要. 7・1. 17-21 (1996)

森秀晴：“与疏水色谱相关的耐热磷酸酶活性的变化”冈山大学医学技术学院通报7·1（1996）。

Motoi Okamoto: "A enzyme-linked immunosorbent assay for heparan sulfate proteoglycans" Life Sciences. (印刷中). (1997)

Motoi Okamoto：“硫酸乙酰肝素蛋白聚糖的酶联免疫吸附测定”生命科学（1997 年出版）。

Motoi Okamoto, et al.: "A enzyme- linked immunosorbent assay for heparan sulfate proteoglycans" Life Sciences. (in press). (1997)

Motoi Okamoto 等人：“硫酸乙酰肝素蛋白聚糖的酶联免疫吸附测定”生命科学。

Motoi Okamoto: "A enzyme-linked immunosorbent assay for heparan sulfate proteoglycan." Life Sciences. (発表予定). (1997)

Motoi Okamoto：“硫酸乙酰肝素蛋白聚糖的酶联免疫吸附测定”（即将出版）。

Shuji Mori, et al.: "Alternation of thermostable phosphatase activity after hydrophobic chromatography" Bull.Sch.Health Sci.Okayama Uiv.7. 17-21 (1996)

Shuji Mori 等人：“疏水层析后热稳定磷酸酶活性的改变”Bull.Sch.Health Sci.Okayama Uiv.7。