用改良式奈米金快速側向流免疫法來檢測以高親和力鍵結之磁珠純化過的血清中前列腺癌惡化之生物指標-METCAM/MUC18之濃度

Using a gold nanoparticles-based lateral flow immunoassay to determine the prostate cancer biomarker METCAM/MUC18 concentration in human serum that was purified by magnetic bead via a high affinity bond

傅彥誠(Yan-Cheng Fu)

指導教授 ： 吳瑞璋

共同指導教授 ： 吳光哲(Guang-Jer Wu)

中原大學/工學院/化學工程研究所/碩士(2022年)

https://doi.org/10.6840/cycu202200007

摘要

目前臨床上使用前列腺特異性抗原（Prostate specific antigen, PSA）作為診斷前列腺癌的生物指標物，但PSA並非前列腺癌特異性之生物指標物，因PSA值可能因其它因素而升高，是以PSA指數常導致20-25%之誤診率，使其無法正確預診列腺腫瘤之癌前病變(惡化)之可能性。而研究顯示METCAM/MUC18有可能為良好之前列腺癌標誌物。
本研究結合磁珠篩選法，使用改良型側向流薄膜試條檢測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，配合經純化的血清樣品，調整所有一級抗體用量以增加其靈敏度。兩種生物素化抗體Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278) (47μg/mL)、Biotinylated Chicken anti-METCAM/MUC18 Ab(20μg/mL)，每次分別使用8μL和6μL與純化之目標樣品做預混合；接金標記抗體Chicken anti-METCAM/MUC18 Ab(100μg/mL)每次使用量為4μL，而辨識不同抗原決定位的兩種Rabbit anti- METCAM/MUC18 Ab(40μg/mL)，EPP11278與MBS416853皆每次使用8μL以對純化血清樣品中的METCAM/MUC18濃度執行檢測。
將純化血清之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。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。以未處理血清之實驗結果做比對，數據顯示磁選純化後之血清其METCAM/MUC18濃度與前者差距2.6倍(High PIN樣品)，檢測訊號提高約160%；另外在銀增強劑的測試中，檢測訊號增加約120%，獲得比原先提升一至兩倍訊號值且可視度有明顯改善。
實驗結果證實此法可成功檢測純化血清中METCAM/MUC18濃度，而且METCAM/MUC18之濃度在前列腺腫瘤之癌前病變(high PIN)之血清中最高，在不同Gleason Score的前列腺癌之血清中次高，它們都高於有良性前列腺增生(BPH)、正常健康者及治療過之前列腺癌血清中之METCAM/MUC18濃度，並進一步證實血清中METCAM/MUC18濃度可以做為前列腺腫瘤有癌前病變(惡化)之指標，是以METCAM/MUC18可以作為目前PSA檢測無法達到的前列腺癌之早期診斷指標。

關鍵字

側向流免疫試條 ； 快速檢測 ； 膠體金 ； 磁珠 ； 磁珠分離

並列摘要

Prostate specific antigen (PSA) is currently used clinically as a biomarker for the diagnosis of prostate cancer. However, the PSA level often leads to a false diagnosis rate of 20-25%; thus, it cannot correctly predict the malignant potential of prostate cancer. Therefore, it is necessary to use a new biomarker for developing the diagnosis of prostate cancer.
From our previous research results, METCAM/MUC18 has been suggested to be a possible good prostate cancer biomarker. To further validate this possibility, this study developed a new approach to combine the magnetic-bead screening method with the modified lateral flow immunoassay (Modified LFIA) to determine the METCAM/MUC18 protein concentrations in human serum samples. All working antibodies were optimized for their optimal quantities to obtain the best performance. Two conjugate antibodies, biotinylated rabbit anti-METCAM/MUC18 Ab (47μg/mL) and biotinylated chicken anti-METCAM/MUC18 Ab(20μg/mL), were optimized to 8μL and 6μL per strip, respectively, when mixed with target analytes. The gold-labeled reporting antibody, chicken anti-METCAM/MUC18 Ab (100μg/mL), was optimized to 4μL per strip. The reporting antibodies, rabbit anti-METCAM/MUC18 Ab (40μg/mL) EPP11278 and MBS416853, were both optimized to 8μL per strip.
The correlation of METCAM/MUC18 with PSA concentrations and the pathological index of prostate cancer (Gleason Score) were also investigated. Comparing with the tests of untreated serum, the signals of METCAM/MUC18 protein were increased to about 160% by the magnetic-bead screening serum. By using the additional silver enhancement, the detection signals were further increased about 120% and hence the visibility was also significantly improved.
The experiment results indicated that our approach was able to detect the concentrations of METCAM/MUC18 in serum samples, and the METCAM/MUC18 concentrations were found to be at the highest level in patients with a high PIN and at the second highest level in patients with prostate cancer with various Gleason Scores. They were both higher than the patients with benign prostatic hyperplasia (BPH), normal healthy individuals, and treated prostate cancer patients.
From this study it concludes that the measurement of METCAM/MUC18 concentration in serum sample can be used as an early diagnosis of the malignant potential of prostate cancer that cannot be achieved by the PSA detection. METCAM/MUC18 is conclusive to be a newly emerging alternative biomarker for clinical detection of malignant prostate cancer.

並列關鍵字

lateral flow assay ； rapid detection ； colloidal gold ； magnetic bead ； magnetic separation

參考文獻

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