比較用磁珠晶片免疫法及兩種奈米金快速側向流免疫法以偵測血清中前列腺癌惡化之生物指標 -METCAM/MUC18之濃度

Comparing magnetic beads-enriched array display immunoassay with the two gold nanoparticles-based lateral flow immunoassay for determination of the prostate cancer biomarker-METCAM/MUC18 concentration in human serum

魏瑜均(Yu-Chun Wei)

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(2018年)

https://doi.org/10.6840/cycu201800376

摘要

目前診斷前列腺癌的生物指標物為前列腺特異性抗原(Prostate specific antigen, PSA)，但其存在20-25%的誤診率，且無法區分病患有前列腺良性增生(Benign prostatic hyperplasia, BPH)、抑或是患有前列腺癌，因此需要開發一個新的前列腺癌生物指標蛋白。過去二十幾年的研究當中暗示METCAM/MUC18具有高度潛力成為檢驗前列腺癌的生物指標物。先前的方法是以西方墨點法(Western Blot)檢測所使用的抗體與抗原-METCAM/MUC18之間的親和性，再以酵素連結免疫吸附法(Enzyme-Linked Immunosorbent Assay, ELISA)進行血清中METCAM/MUC18的定量分析，血清來源包含健康人血清、BPH血清以及前列腺癌病人血清(羅杏雯, 2015)。但由於Western Blot以及ELISA檢測法步驟繁複、且時間較長，加上消耗抗體與抗原量較多，因而開發側向流薄膜檢測法(Lateral Flow Immunoassay, LFIA)進行METCAM/MUC18檢測，希望藉由此法的應用，進行快速、方便的檢測血清樣品中的METCAM/MUC18濃度(何仲堃, 2016)。但由於此檢測法再現性不易，且試條存在高背景值及偽陽性等問題，因此進行LFIA的實驗最適化，實驗結果發現使用脫脂牛奶會使得試條產生偽陽性，因此改以BSA取代脫脂牛奶作為添加。實驗結果顯示，此法可以順利檢測不同濃度的正負控重組蛋白，但在檢測人類血清樣品時，仍是受血清中的雜質干擾造成測試線訊號低落。為了改善試條測試線訊號，開發了改良型LFIA，以Streptavidin與Biotin進行訊號優化，可初步檢測出血清樣品的METCAM/MUC18濃度。(謝承祐, 2017)
本研究使用兩種生物素化抗體操作磁珠篩選實驗，分別為Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278)以及自製的Biotinylated Chicken anti-METCAM/MUC18 Ab，以及使用兩種Rabbit anti- METCAM/MUC18 Ab (EPP11278 and MBS416853)進行血清樣品中的METCAM/MUC18 Ab篩選，並透過標記螢光物質的二級抗體Goat anti-Rabbit Cy5 conjugated Ab或是Goat anti-Chicken Cy5 conjugated Ab呈現訊號，最終透過加熱變性的方式，將抗體抗原的免疫結構破壞，再點樣至生物晶片表面上，並以晶片雷射掃描儀檢測Cy5訊號強度。透過一連串操作條件最適化實驗，本方法已可檢測正負控重組抗原，並建立校正曲線，更可進一步應用於測定人類血清樣品中的METCAM/MUC18濃度。血清來源包含正常男性、前列腺良性增生的病患與罹患前列腺癌不同階段之患者。實驗結果證實磁珠篩選法可移除血清中的雜質，提高檢測的準確性，且發現METCAM/MUC18於病人血清中的濃度普遍高於正常人或是BPH的情況，且PSA與METCAM/MUC18呈現一定程度的相關性。未來繼續優化此法的實驗條件後，相信此法相當具有前列腺癌早期診斷的潛力。
本研究嘗試用側向流薄膜試條檢測法(Lateral Flow Immunoassay, LFIA)可分為二種類型：傳統三明治型以及改良三明治型。為了改善此二種LFIA的背景值，嘗試使用BSA以及BSA Fraction V取代緩衝溶液中的Non-fat milk。根據傳統三明治型LFIA的實驗結果顯示，所使用的接金兔抗Rabbit anti-METCAM/MUC18 Ab (MBS416853)會辨識METCAM/MUC18之C端的抗原決定位(aa#259-544)，其檢測效果會優於辨識METCAM/MUC18之N端的抗原決定位(aa#26-350)的二株抗體：Rabbit anti-METCAM/MUC18 Ab (EPP11278)及Mouse anti-METCAM/MUC18 Ab (EPM12190)。檢測血清樣品的實驗結果亦顯示PSA與METCAM/MUC18存在些許相關性，且前列腺癌病人之METCAM/MUC18濃度也普遍高於正常人或是BPH的情況。
改良三明治型利用測試線上的Streptavidin與一級抗體Biotinylated Rabbit anti- METCAM/MUC18 Ab或Biotinylated Chicken anti-METCAM/MUC18 Ab結合，得到較傳統三明治型更佳的訊號強度。實驗結果指出使用生物素化雞抗體(home-made)的檢測效果優於生物素化兔抗體(EPP11278)，且發現使用生物素化雞抗體所搭配的二種接金之兔抗體：辨識METCAM/MUC18之C端抗原決定位(aa#259-544)之兔抗(MBS416853)表現優於辨識METCAM/MUC18之N端抗原決定位(aa#26-350)之兔抗(EPP11278)。且血清樣品的實驗結果亦顯示PSA與METCAM/MUC18存在些許相關性，且前列腺癌病人之METCAM/MUC18濃度也普遍高於正常人或是BPH的情況。
總體而言，本研究所使用的傳統三明治型LFIA、改良三明治型LFIA以及磁珠篩選法皆可檢測人類血清樣品中的METCAM/MUC18濃度，實驗結果也都顯示在METCAM/MUC18與PSA存在著些許相關性，且前列腺癌患者血清中的METCAM/MUC18濃度普遍高於正常人或是BPH。根據實驗結果，可以初步認為METCAM/MUC18具有前列腺癌早期診斷的能力。相信所使用的這三種方法經過進一步的優化後，可在不久的將來成為一個檢測前列腺癌的實用工具。

關鍵字

側向流免疫層析法 ； 膠體金 ； 磁性篩選 ； 前列腺癌 ； 血清

並列摘要

The current serum PSA test for diagnosis of prostate cancer has a 20-25% of false positive results, besides it is impossible to distinguish Benign Prostatic Hyperplasia (BPH) from Prostate cancer; thus, it is still necessary to develop a rapid and accurate diagnosis method for this cancer. In our previous studies we suggest that METCAM/MUC18 has a high potential to be used as a diagnostic biomarker for the detection of prostate cancer. Previously we first used the Western blot method to determine the specificity of several commercial antibodies. Then we used these antibodies for ELISA to determine the concentration of human METCAM/MUC18 in sera from normal persons and patients with BPH and prostate cancer (Lo, 2015). However, both ELISA and Western blot methods were too complex and time-consuming for a routine test by an untrained person. We then simplified the assay by using nitrocellulose membrane to develop a lateral flow immunoassay (LFIA) (Ho, 2016). However, this method was not easily reproducible by a lay person. Furthermore, after replacing the non-fat milk with BSA this traditional LFIA still could not be used to detect the presence of METCAM/MUC18 antigen in human serum, though it could differentiate the positive and negative controls of recombinant METCAM/MUC18 proteins, NM-GST and C-terminus-GST (Hsieh, 2017). The problem could be due to interference substances in the human serum. To overcome this problem, we further developed a modified LFIA by taking advantage of the binding of METCAM/MUC18 antigen with a biotinylated rabbit anti-METCAM/MUC18 antibody and strapavidin on the membrane (Hsieh, 2017). By using this method, METCAM/MUC18 antigen in human serum was not consistently detectable, though it could differentiate the positive and negative controls of recombinant METCAM/MUC18 proteins (Hsieh, 2017).
In the present studies, we further improved the detection method by using two kinds of biotinylated antibodies (biotinylated rabbit anti-METCAM/MUC18 antibody (EPP11278) and home-made biotinylated chicken anti-METCAM/MUC18 antibody) and two rabbit anti-METCAM/MUC18 antibodies and by using the streptavidin-coated magnetic beads to enrich the METCAM/MUC18 antigen from human serum (the magnetic beads-enriched array display immunoassay). The signal was amplified by attaching the cy5-secondary goat antibody against chicken or rabbit anti-METCAM/MUC18 antibody, which was added to the formed complex of streptavidin-coated magnetic beads-biotinylated chicken or rabbit primary antibody-the METCAM/MUC18 antigen. After heat denaturation, the final cy5-containing extract was spotted on a glass slide coated with Anhydride and the intensity of the fluorescence spot was detected by Microarray confocal scanner. After optimization of the conditions by using different temperatures (25o C > 4o C > 0o C), different antibodies at different concentrations, I could differentiate the two control recombinant METCAM/MUC18 proteins, and establish the caliberation curve for quantitating the protein in human serum. I found that the METCAM/MUC18 concentrations were proportional to PSA concentrations and the METCAM/MUC18 concentrations were higher in sera from patients with prostate cancer than in sera from BPH patients and normal persons. This magnetic beads-enriched array display immunoassay method has the high potential to be used for diagnosis after further improvement.
Since both the traditional LFIA and the modified LFIA methods have the merits of rapidity and simplicity than the magnetic beads-enriched array display immunoassay, I also futher improved both methods by replacing the non-fat milk in the LF buffer and in DB with BSA-FV and BSA, respectively, and by using different antibodies. By using the improved traditional LFIA method, now I could visualize and detect the signal of METCAM/MUC18 antigen from human serum on the membrane. I found that the gold-conjugated rabbit anti-METCAM/MUC18 MBS416853, which recognizes the METCAM/MUC18 epitopes at the C-terminal end (aa#259-544), was much better than the rabbit anti-METCAM/MUC18 antibody (EPP11278) and the mouse monoclonal anti-METCAM/MUC18 antibody (EPM12190), both of which recognize the METCAM/MUC18 epitopes at the N-terminal end (aa#26-350). I also found that the concentrations of METCAM/MUC18 in huma serum were somewhat proportional to lower serum PSA concentrations and the serum METCAM/MUC18 concentrations were higher in prostate cancer patients than in BPH patients and in normal persons.
By using the further improved modified LFIA, I could also visualize and detect the signal of METCAM/MUC18 antigen from human serum on the membrane. Furthermore, the signal of serum METCAM/MUC18 antigen detected on the membrane by this further improved modified LFIA method was much higher than that by the improved traditional LFIA method. I found that the combination of biotinylated chicken antibody and gold-conjugated rabbit anti-METCAM/MUC18 antibodies was better than the combination of biotinylated rabbit antibody (EPP11278) and gold-conjugated chicken antibody. Futhermore, the combination of biotinylated chicken antibody and gold-conjugated rabbit anti-METCAM/MUC18 antibody MBS416853, which recognizes the METCAM/MUC18 epitopes at the C-terminal end (aa#259-544), was much better than the combination of biotinylated chicken antibody and the gold-conjugated rabbit anti-METCAM/MUC18 antibody EPP11278, which recognize the METCAM/MUC18 epitopes at the N-terminal end (aa#26-350). I found that the concentrations of METCAM/MUC18 in huma serum were proportional to lower serum PSA concentrations and the serum METCAM/MUC18 concentrations were higher in prostate cancer patients than in BPH patients and in normal persons.
In summary, I could use the improved traditional LFIA, the improved modified LFIA, and magnetic beads-enriched array display immunoassay to quantitate the serum concentrations of METCAM/MUC18 antigen and showed that the serum concentrations of METCAM/MUC18 antigen were somewhat proportional to lower serum PSA concentrations. Since the serum concentrations of METCAM/MUC18 antigen from prostate cancer patients were higher than those in BPH patietns and normal persons, I suggest that METCAM/MUC18 may be a biomarker for predicting the early stage of prostate cancer. We expect that after further improvement the three methods have a high potential in the near future to become useful diagnostic assays.

並列關鍵字

Lateral flow immunoassay ； colloidal gold ； magnetic separation ； prostate cancer ； METCAM/MUC18 ； serum

參考文獻

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