結合高親和力鍵結之免疫反應、磁珠純化程序、與放大側向流奈米金訊號以檢測血清中前列腺癌惡化之生物指標-METCAM/MUC18之濃度

Combination of the High-Affinitive-Bonding Immunoassay and the Magnetic-Bead Purification with the Silver-Enhanced Gold-Nanoparticle Signals of the Lateral-Flow Detection on the Concentrations of the Prostate Cancer Biomarker METCAM/MUC18 in Human Serum.

劉慶安(Liu Ching-An)

指導教授 ： 吳瑞璋 ； 吳光哲

中原大學/工學院/化學工程學系/碩士(2022年)

https://doi.org/10.6840/cycu202201562

摘要

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不只是前列腺癌特異性的生物指標物，很多其他因素例如前列腺良性增生(Benign prostatic hyperplasia, BPH)也會造成其異常升高，因此使用PSA指數當作診斷依據常導致20-25%之誤診率，進而無法正確預診前列腺腫瘤之癌前病變(惡化)之可能性。而根據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。
本研究使用改良型側向流薄膜試條檢測法以及結合磁珠篩選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，以降低背景值及增強訊號，獲取更佳最終判讀訊號。血清來源包括健康男性、前列腺良性增生的病患與罹患前列腺癌階段之患者。
本研究使用生物素化抗體為購買的Biotinylated rabbit anti-METCAM/MUC18 Ab (EPP11278)，另外參與免疫反應的抗體有辨別不同抗原決定位的自製Chicken anti-METCAM/MUC18 Ab （接金），可進行血清樣品中的METCAM/MUC18濃度檢測。本研究不僅證實此點，更可進一步加入銀試劑放大訊號，尤其是上面兩種抗體之組合 (即是此第一組合比起其他兩組合) 效果最明顯， (即是: 增強在改良型側向流薄膜試條之C-及T-bands之訊號, 使其肉眼可見度明顯升高)。通過28個血清樣品檢測實驗顯示，稀釋DB後血清樣品比原始未稀釋樣品整體檢測訊號提高約270%；使用銀試劑放大訊號後，整體也有效提升約70%。其中PSA較易誤診的前列腺癌前病變（High PIN）血清(編號9)，在稀釋實驗中訊號辨別度提高約323 %，加入銀試劑後也提高約65%，兩者皆具有診斷能力。
本研究發現METCAM/MUC18之濃度與癌指數Gleason score成正比。但是，METCAM/MUC18之濃度與大部分的PSA濃度無正相關，除非PSA在 12 ng/ml以下。又發現METCAM/MUC18之濃度在前列腺腫瘤之癌前病變(high PIN)患者之血清中最高，在不同Gleason Score的前列腺癌患者之血清中次高，它們都高於有良性前列腺增生(BPH) 患者、正常健康者及治療過之前列腺癌患者血清中之METCAM/MUC18濃度，進一步證實METCAM/ MUC18濃度相較於PSA檢測可作為檢驗前列腺癌癌前病變(惡化)指標，是以METCAM/MUC18在臨床上可以作為目前PSA檢測無法達到的前列腺癌之早期診斷指標。

關鍵字

磁珠純化 ； 側向流 ； 奈米金訊號 ； 前列腺癌

並列摘要

Prostate specific antigen (PSA) is currently used clinically as the popular biomarker of the prostate cancer. However, using the PSA index as the diagnostic basis often results in a misdiagnosis rate of 20-25%. Furthermore, it cannot correctly predict the possible presence of precancerous lesions (malignant) of prostate tumors. According to previous researches, METCAM/MUC18 was indicated to be a good biomarker for the prostate cancer. This study further applied the improved version of the Lateral-Flow Immunoassay (LFIA) combined with a magnetic-bead purification technology to detect the METCAM/MUC18 concentrations in human serum.

This study utilizes a combination of the Biotinylated rabbit anti-METCAM/MUC18 Ab (EPP11278) and the nano-gold-conjugated self-made Chicken anti-METCAM/MUC18 Ab to undergo the immunoassay. After optimizing the experiment conditions to reduce the signal background for better detection of the difference in the signals. The immunoassay was confirmed to successfully detect METCAM/MUC18 in serum samples.
This study also conducted a trial to amplify the detection signals by the addition of a silver reduction reagent. The amplified signals were prominently visible in the modified LFIA ananlysis by using the above combination of antibodies. After amplifying the signal, the overall detection signal was also effectively increased by about 70%, though the signal amplification accompanied an elevated background at the early stage of trial, which was later resolved by diluting the diluting buffer. The signals of all serum samples were increased by about 270% in comparison with the original samples. Especially, the singal of the serum from a patient with prostate precancerous lesion (High PIN) (No.9) was increased about 65% and the signal discrimination further improved about 323%.
By this immunoassay, the elevated serum METCAM/MUC18 concentrations were found to be correlated with the cancer index of Gleason Score; however, they were not directly correlated with the majority of the PSA concentrations except at very low PSA concentrations (< 12 ng/ml). Furthermore, 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 clinically treated prostate cancer patients.
In summary, this study demonstrated that the silver-reduction reagent was able to enhance the signals for facilitating the visible detection of the elevated serum METCAM/MUC18 concentrations in the prostate cancer patient. The elevated METCAMMUC18 concentrations in serum not only can be used to detect the presence of prostate cancer, but also the precancerous (pre-malignant) lesions, suggesting that METCAM/MUC18 can be used as a diagnostic biomarker or biosensor for predicting prostate cancer, especially at the early premalignant stage. Therefore, METCAM/MUC18 is conclusively proven to be a newly emerging alternative biomarker for clinical detection of malignant prostate cancer at the early clinical stage. The clinical significance of this finding surpasses that of the PSA detection.

並列關鍵字

Magnetic-Bead Purification ； Silver-Enhanced ； Lateral-Flow Detection ； Gold-Nanoparticle Signals ； METCAM/MUC18

參考文獻

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4. Catalona, W.J., et al., Comparison of digital rectal examination and serum prostate specific antigen in the early detection of prostate cancer: results of a multicenter clinical trial of 6,630 men. The Journal of urology, 1994. 151(5): p. 1283-1290.

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