奈米金快速側向流免疫法及磁珠晶片免疫法以高親和力之鍵結來檢測血清中前列腺癌惡化之生物指標-METCAM/MUC18之濃度

目前臨床上以前列腺特異性抗原（Prostate specific antigen, PSA）為前列腺癌的生物指標物，但PSA不是前列腺癌特異性的生物指標物，因為很多因素會使PSA值升高，例如前列腺良性增生(Benign prostatic hyperplasia, BPH)，然而以PSA指數常導致20-25%之誤診率，是以它無法正確預診列腺腫瘤之癌前病變(惡化)之可能性。因此需要開發一個新的前列腺癌生物指標物。吳光哲教授先前的研究顯示METCAM/MUC18有可能為良好之前列腺癌標誌物[18, 20]。本研究分別使用改良型側向流薄膜試條檢測法以及磁珠晶片免疫法來檢測人類血清中之METCAM/MUC18蛋白濃度，並進一步優化實驗條件，以降低背景，並提高靈敏度。對於改良型側向流薄膜試條檢測法，不是使用以前的冰浴環境（0℃）[50, 51]將抗體與膠體金結合，而是使用室溫環境（25℃）以降低背景值，並優化抗體濃度以增加其靈敏度。對於磁珠晶片免疫法，利用電漿技術改質疏水晶片，作為生物檢測平台，並且將斷鍵溫度從先前使用的80℃一分鐘[50, 51]提高至95℃一分鐘，使最後的斷鍵步驟更完全。並將血清之METCAM/MUC18濃度與前列腺特異性抗原(PSA)濃度和癌症指數Gleason Score比較。本研究使用兩種生物素化抗體，分別為Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278)、吳光哲教授自製的Biotinylated Chicken anti-METCAM/MUC18 Ab、以及兩種Rabbit anti- METCAM/MUC18 Ab (EPP11278 and MBS416853)、與吳光哲教授自製Chicken anti-METCAM/MUC18 Ab，進行血清樣品中的METCAM/MUC18濃度檢測。實驗結果證實兩種方法皆可成功檢測血清中METCAM/MUC18濃度，而且METCAM/MUC18之濃度在有前列腺腫瘤之癌前病變(PIN)之血清中最高，在有不同Gleason Score之前列腺癌之血清中次高，它們都高於有良性前列腺增生(BPH)及正常健康的人及治療過之前列腺癌血清中之METCAM/MUC18濃度，本研究數據上的驗證結果，證實與以前之結果一致[50, 51]。本研究最重要的供獻是，進一步又証實血清中METCAMMUC18濃度之上升可以做為前列腺腫瘤有癌前病變(惡化)之指標，是以METCAMMUC18可以做為前列腺癌之早期診斷之指標，此結果是目前的PSA檢測無法達到的。

關鍵字

奈米金；側向流免疫法；磁珠；高親和力；前列腺癌；METCAM/MUC18

並列摘要

Prostate-specific antigen (PSA) is currently used as a clinical biomarker for the malignant propensity of prostate cancer. However, PSA is not a prostate cancer-specific biomarker, since other physiological factors, for example BPH, may increase the serum PSA level. Furthermore, an elevated PSA concentration often leads to 20-25% of false diagnosis because it cannot be used to predict the malignant potential of prostate cancer. Therefore, it is necessary to develop a new biomarker for the diagnosis of prostate cancer. Previous studies by Professor Guang-Jer Wu have indicated the possibility of using METCAM/MUC18 for this purpose [18, 20]. In this study, I used the modified lateral flow immunoassay (Modified LFIA) and magnetic beads-enriched array display immunoassay to determine the concentration of METCAM / MUC18 protein in human serum, and also further optimized the experimental conditions to decrease the background and increase the sensitivity. For the modified lateral flow immunoassay detection method, instead of using ice bath temperature (0o C) as previously used for the conjugation of antibody with colloidal gold [50, 51] I used room temperature to decrease the background value and also optimized the antibody concentrations to increase sensitivity. For the magnetic beads-enriched array display immunoassay, I used the plasma technology to modify the hydrophobic wafer as a biological detection platform, and also made complete the final bond breaking step by increasing the bond breaking temperature from 80℃, which was previously used [50, 51], to 95℃ for one minute. The resulting serum METCAM/MUC18 concentrations were compared with the PSA and correlated with the pathological index of prostate cancer, Gleason Score. Two biotinylated antibodies were used, such as biotinylated rabbit anti-METCAM / MUC18 antibody (EPP11278) and biotinylated chicken anti-METCAM / MUC18 antibody home-made by Professor Guang-Jer Wu, as well as two rabbit anti-METCAM / MUC18 antibodies (EPP11278 and MBS416853) and home-made chicken anti-METCAM / MUC18 antibody to detect the METCAM / MUC18 concentration in human serum samples. The experimental results show that both methods were able to detect the serum concentration of METCAM / MUC18. Furthermore, the serum concentration of METCAM / MUC18 was found to be at the highest level in PIN and higher in prostate cancer patients with various Gleason scores than normal individuals and those with BPH and the treated patients. In consistent with the previous findings (50, 51), the most significant contribution of this study is that the elevated serum METCAM / MUC18 concentration may be used for early prediction of the malignant potential of prostate cancer, which cannot be achieved by the current PSA testing.