- 學位論文
用免疫法來檢測血液中 METCAM/MUC18之濃度以應用於預診前列腺癌肇生及惡化之傾向
Using immunological methods to determine human serum METCAM/MUC18 concentration for prediction of the possible malignant potential of prostate cancer.
摘要
前列腺癌患者約有10%是罹患具侵略性的轉移性腫瘤會在診斷後的一年內死亡。目前最主要的診斷依據就是患者的前列腺癌特異性抗原(prostate specific antigen, PSA)水平。然而這個測試至少有20~25%的結果是錯誤的，PSA的測試不能預測前列腺癌的病理分級或目前的轉移性疾病狀況。大部分具有潛力的前列腺癌診斷標誌物仍需要驗證，不能精確的判斷怠惰性腫瘤和侵略性腫瘤的差別。因此，我們仍迫切的需要對前列腺癌轉移潛力早期診斷的標誌物。之前的研究證實了METCAM/MUC18抗原的表達對惡性的前列腺癌有正相關。而且它又是一個轉移性基因，它能夠引發和驅動非轉移性前列腺癌細胞轉移至多個器官；因此它對於非侵略性前列腺癌轉移成侵略性前列腺癌扮演一個很重要的角色，它也可能是人類前列腺癌的早期診斷的一個新的生物標誌。
本研究希望發展一個診斷的方式，能利用抗體針對怠惰性和侵略性的前列腺腫瘤進行區分，以取代現有PSA測試。
實驗先利用西方墨點法，來證實抗體和抗原間有高的敏感度和親和性，能夠辨認METCAM/MUC18抗原，接著發展ELISA對METCAM/MUC18之定量分析。之後建立標準曲線以定量正常的、良性前列腺增生的和前列腺癌患者的血清之抗原濃度。
四種待測抗體中以ELISA實驗證實自製的chicken antibody anti-middle portion (aa#212-375) 為最佳。最佳一級抗體與二級抗體濃度分別為50 μg/ml與0.4 μg/ml。以此最佳一級抗體濃度進一步測定18個人類血清樣品中METCAM/MUC18的濃度，包含正常的男性、前列腺增生的病患與罹患前列腺癌不同階段之患者的血清。結果發現ELISA和西方點墨法皆可檢測血清中METCAM/MUC18抗原的存在。當PSA<7 ng/ml 和PSA< 25 ng/ml時，血清中METCAM/MUC18與PSA之濃度趨近於正相關。然而，當25 ng/ml
並列摘要
Prostate cancer in 10% of prostate cancer patients are aggressive and metastatic, killing the patients within one year of the initial diagnosis. The current dominant diagnosis for the cancer is to test an elevated serum PSA level. However the diagnosis test has at least 20-25% false results; the elevated level of PSA in the serum is not always predictive of the pathologic stage of the prostate cancer or the presence of a metastatic disease. Many potential diagnostic markers for prostate cancer progression have been validated; however, most of them cannot accurately distinguish between indolent and aggressive cancers. As such, there is still an urgent need to search for a better marker for the early detection of the metastatic potential of prostate carcinomas. Previous research suggested that METCAM/MUC18 may be used as a novel diagnostic biomarker for early detection of the metastatic potential of human prostate cancer and for distinguishing the aggressive cancers from indolent ones. The purpose of my research is to test the possibility of using METCAM/MUC18 as a diagnostic biomarker for developing a reliable, cost-effective test for predicting the malignant progression of prostate cancer. We have used immunological methods, both ELISA (enzyme-linked immunosorbent assay) and Western blot (WB) analysis, to establish a standard curve by using recombinant METCAM/MUC18 proteins and then used the tests to determine METCAM/MUC18 concentrations in human serum samples. To initiate the research, we used Western blot (WB) analysis to identify an antibody to have the highest sensitivity and specificity to recognize the METCAM/MUC18 antigen and using the antibody to develop a 96-well-ELISA for quantitative analysis of the METCAM/MUC18 antigen. Four antibodies were used for the tests: our home- made chicken antibody anti-middle portion (aa#220-375), MyBioscource MBS275688 rabbit antibody against METCAM/MUC18 aa#14-234, Santa Cruz SC-28667 rabbit antibody against the C-terminus aa#586-646 of METCAM/MUC18, and Santa Cruz SC-18940 goat antibody against METCAM/MUC18 unknown internal epitopes. We found that the home-made chicken antibody anti-middle portion (aa#212-375) was the best and MyBioscource MBS275688 rabbit antibody against METCAM/MUC18 aa#14-234 the second best for ELISA. Second, we determined the best concentrations of the primary antibodies and secondary antibodies for obtaining optimal ELISA signals. Third, we established a protein standard curve for ELISA by using five recombinant proteins (antigen #1: M, antigen#2:M-GST, antigen#4: N-M-GST, antigen#5:C-terminal portion, and GST control). Finally, we used the two best primary antibodies for ELISA to determine and quantify the serum METCAMMCUC18 concentrations in 18 human serum samples obtained from normal individuals, BPH patients, and patients at different stages of prostate cancer in comparison with the protein standard curve. We found that it was possible to detect the presence of METCAM/MUC18 antigens in the clinical serum specimens by Western blot analysis and ELISA. We also found that the serum METCAM/MUC18 concentrations were linearly proportional to most of the PSA concentrations when serum PSA concentrations were at <7 ng/ml and < 25 ng.ml. However, the serum METCAM/MUC18 concentrations remained statistically similar versus most PSA concentrations when serum PSA concentrations were at between >25 and <1500 ng/ml. We also compared the results of ELISA with those of WB analysis and found that the results were similar to ELISA results except the serum METCAM/MUC18 concentrations determined by WB were higher than those by ELISA, which we did not know the exact reason. One likely reason was that the resolution of Image J, which we used for quantitative analysis, was not high enough for a more precise quantitation. We also found that serum METCAM/MUC18 concentrations appeared to be higher in prostate cancer patients than normal individuals and BPH patients. Since the survey cohort was not large enough to obtain statistically significant results, we still could not differentiate malignant prostate cancers from indolent ones and also to monitor treatment outcome of the patients. In conclusion, the preliminary results by using immunological test to determine serum METCAM/MUC18 concentrations were promising, so that in the near future we should expand our survey cohort in Taiwanese males and to simplify and increase sensitivity of our tests by exploring the use of 2D-MBLF and 3D-aerogel bio-chip. Furthermore, it may also be possible to detect the presence of METCAM/MUC18 in urine samples, because the presence of METCAM/MUC18 antigen in serum was detectable.