以奈米金微粒為主的側向流免疫法偵測血清中前列腺癌惡化之生物指標 -METCAM/MUC18之濃度

Gold nanoparticles-based lateral flow immunoassay detection of METCAM/MUC18 concentration in human serum for its validation as a biomarker to predict the malignant progression of prostate cancer.

何仲堃(Chung-Kun Ho)

指導教授 ： 吳瑞璋 ； 吳光哲

中原大學/工學院/化學工程研究所/碩士(2016年)

https://doi.org/10.6840/cycu201600534

摘要

前列腺癌據統計於過去五年（2007-2011）年為全世界成年男性罹癌比率的最大宗，於美國是過去 20 幾年（1975-2011）間排名第一名，而在台灣為 2013 年之成年男性十大癌症發生率排名第五名，另外目前檢測前列腺癌的方法－ PSA 測試（PSA test），其精確度不能完全檢測前列腺癌是否存在，因此需要針對前列腺癌早期病發，能夠快速且精確度高的檢測方法。

本研究發展以奈米金粒子為檢測媒介的側向流免疫層析試紙檢測法，來測定人類血清中之 Human METCAM/MUC18 相對強度。經過多次蛋白質檢測抗原實驗，除了得到實驗的最佳化條件，也確定一級抗體 Chicken anti-HuMETCAM/MUC18 Ab 在薄膜試條上的顯色效果，比一級抗體 Rabbit anti-HuMETCAM/MUC18 Ab 還要佳，而且兩個一級抗體認定的位置不同，因此將顯色效果較好的前者噴印於測試線上，後者則作為鍵結膠體金之抗體。而一級抗體與二級抗體使用量之最佳化結果，以一級抗體 Chicken anti-HuMETCAM/MUC18 Ab 的稀釋濃度為 600 倍，而另一個一級抗體 Rabbit anti-HuMETCAM/MUC18 Ab 的稀釋濃度為 100 倍，二級抗體 Goat anti-Rabbit IgG 的稀釋濃度也為 100 倍為最佳。

以最佳條件經過多次實驗後，建立人類重組抗原的標準曲線圖，不但能分辨正控制（NM-GST）與負控制（C-terminus-GST）之人類重組抗原，兩者差別有五倍之多（P= 0.021），且能觀察到 Human METCAM/MUC18 之相對強度和不同稀釋濃度人類重組抗原之呈正相關關係，正控制之值在扣去負控制之值之後，就可以做為標準曲線以計算未知之濃度。進一步的實驗結果顯示受試者血清中的 PSA 濃度與其血清中的 Human METCAM/MUC18 之強度亦成正相關之關係，以標準曲線為準，由強度可以計算出血清中的 Human METCAM/MUC18 之濃度，雖然我們只有四個來自前列腺癌病患之血清之 METCAM/MUC18 之數據及五個正常人之數據，但發現前列腺癌患者之血清中的 Human METCAM/MUC18 濃度比正常人的濃度明顯的高，此數據在統計兩者之統計分析結果是 P = 0.01, 證明此偵測方法比 ELISA 及 PSA 所得的數據（P = 0.09-0.1）更能分辨有無前列腺癌，此結論雖仍有待驗證更多個受試者血清之結果而成定論。

基於本研究目前之成果，LFIA 在經過進一步的研發和改良之後，具有高潛力成為一個快速、簡便及精確度高的診斷前列腺癌的方法，且能用於檢測正常人和前列腺癌病患之血清中 METCAM/MUC18 的不同的濃度。盼望這簡單的檢測方法未來能用於診所或醫院中，且能精確地診斷及辨別惰性的前列腺癌與具轉移性的前列腺癌。

關鍵字

奈米金 ； 膠體金 ； 側向流薄膜免疫法 ； 前列腺癌檢測 ； 血清 ； 生物標記

並列摘要

Prostate cancer is the major prevalent cancer of men in the world from 2007 to 2012. It has the 1st cancer incidence rate of men in the USA over the past from 1975 to 2011. And it also has been ranked as the top 6th of the ten cancer incidence rate of men in Taiwan in 2013. Therefore, it is necessary to develop a rapid and high accurate method to detect the early stage of prostate cancer since the current PSA test was not accurate enough to detect the presence of prostate cancer.

In this study, a gold nanoparticles-based lateral-flow immunoassay（LFIA）was developed to detect the human METCAM/MUC18 antigen in human serums. The results indicated that the primary antibody of chicken anti-HuMETCAM/MUC18 Ab had a better performance than that of rabbit anti-HuMETCAM/MUC18 Ab in LFIA. Thus the former was used for printing in the test line, the latter for conjugation with gold nanoparticles, since the two primary antibodies recognize different epitopes. The optimal conditions of primary antibodies and secondary antibody were in 1/600 dilution of chicken anti-HuMETCAM/MUC18, 1/100 dilution of rabbit anti-HuMETCAM/MUC18 Ab, and 1/100 dilution of goat anti-Rabbit IgG, respectively.

Under the above optimal conditions, the standard curve of human recombinant antigens was established and the positive control（NM-GST）and negative control（C-terminus-GST）could be clearly differentiated with a significant five-fold difference（P = 0.021）. Furthermore, the color intensity of the human METCAM/MUC18 antigens had a positive correlation with the human recombinant antigens. Thus after correcting the values of the positive control by substracting the values of the negative control, the standard curve of the positive control can be established for calculating the concentrations of the unknowns. Moreover, the color intensity of human METCAM/MUC18 antigens in human serum specimens also had a positive correlation with the serum PSA concentration Serum concentrations of METCAM/MUC18 were calculated from the standard curve. The average serum METCAM/MUC18 concentration from prostate cancer patients was statistically significantlty higher than that in normal individuals, though we had only five serum specimens from prostate cancer patients and from normal individuals, respectively（P = 0.01）. Therefore, this method of detection was significantly much better than the PSA test and our previous ELISA test（P = 0.09-0.1）of the same set of serum specimens for diagnosis of the presence of prostate cancer in an individual.This conclusion still waits to be further scrutinized by testing more human serum specimens.

Based on our evidence, LFIA had a high potential to be further improved to become an accurate, simple, and rapid diagnostic method for comparing the concentrations of the human METCAM/MUC18 antigens in serums from normal human individuals with those from prostate cancer patients. Hopefully, this simplified diagnostic assay can be used for accurate diagnosis of prostate cancer and for differentiation of indolent prostate cancers from aggressive ones in clinics.

並列關鍵字

Colloidal gold ； Gold nanoparticles ； lateral flow immunoassay ； METCAM/MUC18 ； serum ； prostate cancer detection ； biomarker

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