研發創新Protein G微米球以提升抗體於酵素免疫吸附測定法(ELISA)、西方墨點法(Western Blot)之偵測極限值

Development of a novel Protein G microparticle to improve detection limits of antibodies in ELISA and Western Blot systems.

賴怡婷(Yi-Ting Lai)

指導教授 ： 莊國祥

臺北醫學大學/藥學院/生技製藥企業經營管理產業碩士專班/碩士(2014年)

摘要

在傳統的生物醫學實驗上，常使用酵素免疫吸附測定法(ELISA)及西方墨點法(Western Blot)作為臨床醫學檢測的工具。ELISA和Western Blot皆是應用抗原與抗體專一性結合的特性，然而，目前所遭遇的困境為抗體偵測效率不足的問題。為了改善此難題，我們研發一款新型大腸桿菌(Escherichia coli : E. coli) 細胞膜上表現Protein G之細菌微米球，Protein G可以有效將偵測的抗體結合在微米球表面，形成一個抗體微米球 (簡稱為Protein G微米球) ，以提升抗體在ELISA及Western Blot的偵測敏感度。目前我們已在實驗室中研發出表現單一Protein G之細菌株(BL21/pET1G)和8個Protein G重複單元之細菌株(BL21/ pET8G)的細菌微米球。經由Western Blot證實BL21/ pET1G及BL21/ pET8G E. coli細菌皆正確表現Protein G或8重覆Protein G，且利用ELISA證實BL21/ pET8G抓取抗體的效率是BL21/ pET1G的10倍。此外 ，於ELISA實驗中證實無論是將細菌株保存於4oC或-80oC，經過40天後，仍不影響Protein G細菌微米球抓取抗體的功能。此細菌微米球的產量豐富、快速且方便，且能讓Protein G細菌微米球的生產品質均一，未來，可廣泛運用在ELISA或Western Blot及其他生物醫學相關實驗上，相信可大大提升偵測抗體的訊號量，以改善舊有實驗方式，加速實驗進行並節省實驗成本，進而成為一種強勁的免疫偵測工具。

關鍵字

酵素免疫吸附試驗(ELISA) ； 西方墨點法(Western Blot) ； Protein G微米球

並列摘要

In the traditional biomedical experiments, often using enzyme-linked immunosorbent assay (ELISA) and Western blot as a clinical tool for the detection, ELISA and Western blot applications are both antigen and antibody specific binding characteristics. However, difficulties currently facing shortage of antibody detection efficiency, in order to improve this problem, we developed a new type of membrane expression of bacterial Protein G microparticles, Protein G can be effective in the detection of antibody binding surface of microparticles, to form antibodies microparticles(Protein G microparticles ) to improve antibody in ELISA and Western blot detection sensitivity. Currently there is a single type of Protein G (BL21/pET1G) or 8 Repeat Protein G (BL21/pET8G) bacteria microparticles , first, we confirmed BL21/pET1G and BL21/pET8G are correctly expressing by Western blot, and confirmed BL21/pET8G crawl antibodies efficiency was better than BL21/pET1G 10 times by ELISA, and then we compare different bacterial microparticles preservation methods confirmed either Protein G bacteria microparticles stored at 4oC or -80oC freezer refrigerator, Protein G bacterial microparticles still retains crawl antibody function after 40 days , This bacterial production of microparticles fairly quick and easy , and allows Protein G bacterial production of microsparticles uniform quality , the future can be widely used in the ELISA or Western Blot or other biomedical experiments, greatly enhance the amount of detectable antibody signal to replace the old way of experiments , and accelerate cost savings, and thus become a strong immune detection tools .

並列關鍵字

enzyme-linked immunosorbent assay (ELISA) ； Western ； blot Protein G microparticles

參考文獻

參考文獻

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