最佳化奈米金微粒為主的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

目前針對前列腺癌的檢測所使用的抗原為前列腺特異性抗原(Prostate specific-antigen, PSA)，然而其精確度不夠，其誤診率可達20-25%．是以仍急需用別的抗原，開發一個既快速且又高精確度的檢測方法，以便正確地檢測早期發病之前列腺癌。過去二十幾年之研究，已暗示METCAM/MUC18有高度潛力成為檢驗前列腺癌之生物指標。為達此目標，過去幾年用此抗原及自製的雞抗體發展了ELISA檢驗法(羅杏雯, 2015)，發現血清之METCAM/MUC18濃度與大都之PSA濃度成正比，而且其濃度在前列腺癌患者的血清中高於正常人，可是此檢驗法之手續太繁複，不易為一般人所採用。之後為了要發展一個既快速又簡便的檢驗法，又進一步開發一個用側向流層析法做檢驗(何仲堃, 2016)，此法亦證實：血清之METCAM/MUC18濃度與大多之PSA濃度成正比，而且其濃度在前列腺癌患者的血清中高於正常人，美中不足的是：此檢驗法之再顯性不易，也不為一般人所使用。本研究的目的乃是進一步簡化及最佳化此檢驗法　使其成為一個容易操作、高再顯性、及正確診斷早期之前列腺癌之方法。本研究先使用西方墨點法證實抗體和抗原間有高的敏感度和親和性，確定了抗體辨認METCAM/MUC18抗原之位置，使之可應用於側向流薄膜試條檢測METCAM/MUC18抗原。試條實驗中使用不同物種的抗體分別為自製抗體Chicken polyclonal antibody anti-middle portion of METCAM/MUC18抗原，Rabbit polyclonal antibody against METCAM/MUC18 與 Mouse monoclonal antibody against METCAM/MUC18。利用側向流訊號欲對兩種不同的重組蛋白(antigen #1(正控)：N-M-GST, antigen #2(負控)：C-terminus-GST) 建立檢測標準曲線，以便應用在側向流實驗中測定人類血清中METCAM/MUC18的濃度。血清來源包含正常男性、前列腺增生的病患與罹患前列腺癌不同階段之患者。試著用三種方式來發展側向流薄膜試條檢測: 競爭型、螢光三明治型、及三明治型。在競爭型側向流實驗中發現，使用脫脂牛奶所配製的稀釋溶液和緩衝溶液，因不易流動會出現膠體金殘留在膜上，且用其緩衝溶液進行跑膜時會導致背景值過高。即使保持脫脂牛奶所配製的稀釋溶液稀釋抗體並噴印在膜上，仍出現偽陽性，其實BSA也會帶來較弱的偽陽性(因其濃度較低之故)，代表脫脂牛奶及BSA所配製的稀釋溶液會使各種抗體對C-terminus-GST 及GST抗原產生具有非專一性鍵結，是以放棄使用競爭型做進一步之測試。也放棄使用螢光三明治型，因其亦有類似的問題, 此法仍有待改進。在三明治型側向流實驗中，為了最適化試紙系統，將上述所發生之情況依序一一排除，使用以BSA作為配製溶液之材料而獲得改善，改用BSA作為配製溶液並沒有太明顯之偽訊號，以此最佳條件之下，使用三明治型非但能分辨正及負控之重組蛋白，兔抗體及雞抗體之組合比鼠抗體及雞抗體之組合好很多，其標準曲線有可能用於定量血清中METCAM/MUC18之濃度。然而在此最佳條件下，血清樣品仍無法顯示夠強的訊息足以定量，懷疑血清中之METCAM/MUC18受到其他成分干擾，不易被抗體捕捉到，導致test line 上之訊息太弱。為了突破此困難，新開發了一個的新的三明治型側向流薄膜試條檢測法，用biotin-rabbit anti-METCAM/MUC18 Ab及streptavidin來放大test line上之訊息: 先把抗原與接金之雞或鼠抗體及接biotin之兔抗體預先在LFB2中混合，之後使抗原與抗體在冰浴或在室溫作用30或 60分鐘，然後點在樣品墊上，用LFB2跑膜，膜上已有噴印好之streptavidin 在test line及 goat anti-mouse or anti-chicken Ab在control line，用此新開發之三明治型做側向流薄膜試條檢測，發現不但可分辨正及負控之重組蛋白，亦使血清在test line顯示較強之訊息，相信在進一步改進實驗條件至最佳情況之後，以此來定量血清中之METCAM/MUC18濃度，應當指日可待。最後此新開發之三明治型側向流薄膜試條檢測可能發展成為一個快速且精確度高的方法來檢測早期病發之前列腺癌。

關鍵字

奈米金側向流薄膜免疫法前列腺癌檢測血清生物標誌物

並列摘要

The current serum PSA test for diagnosis of prostate cancer has a 20-25% of false results; thus, it is still necessary to develop a fast, accurate diagnosis method for this purpose. In our previous studies we suggest that METCAM/MUC18 has the high potential to be used as a biomarker for the detection of prostate cancer. To reach this goal, in the previous research we have used our unique chicken anti-METCAM/MUC18 antibody to develop an ELISA for serum test, in which the serum concentration of METCAM/MUC18 appears to be proportional to that of PSA (Lo, 2015). However, the procedure of ELISA has a limitation for routine use since the procedure is too tedious and difficult to be reproducibly performed by an un-trained person. To further develop a fast and easy method, we used nitrocellulose membrane for developing a lateral flow immunoassay (LFIA). We found that the detected serum METCAM/MUC18 concentrations were also proportional to most PSA concentrations and the serum concentration of METCAM/MUC18 is higher in patients with a high-grade prostate cancer than normal individuals (Ho, 2016). However, the method was not easily reproducible by a lay person. The purpose of this research is to further simplify and optimize the method for an early and accurate detection of prostate cancer. In this study, we first used the Western blot (WB) analysis of our home-made chicken anti-METCAM/MUC18 antibody and several commercial antibodies to identify an antibody for having the highest sensitivity and specificity to recognize the METCAM/MUC18 antigen. We found that all these antibodies were complementary to each other to be used for organizing different combinatory sets for a gold nano-particles based lateral-flow immunoassay (LFIA). We explored three different LFIA methods: the competition method, the fluorescence-sandwich method, and a sandwich method for the purpose. We found that addition of non-fat milk or BSA to a dilution buffer in the LFIA appeared to create false positive signals in which non-fat milk were worse than BSA. Furthermore, addition of non-fat milk also created a higher background than BSA.Thus, we decided to use 0.5% BSA to decrease non-specific interactions of an antibody with the antigen. When the competition method was used, all the antibodies could not differentiate the positive control (the recombinant N-M-GST METCAM/MUC18 protein) from the negative control (the recombinant C-terminus-GST METCAM/MUC18 protein); thus, this method was abandoned for further use. The fluorescence-sandwich method was also abandoned for the time-being till further improvements, since it also had similar problems. Taken all the parameters into consideration, we used regular sandwich method for further optimization. We found that this method was successfully demonstrated to be capable of differentiating between the positive control recombinant protein from the negative control a when the combination of rabbit antibody and chicken antibody was used for the assay; thus, a standard curve could be established. The combination of mouse antibody and chicken antibody could yield a similar positive result; however, differences were not as great as the combination of rabbit & chicken antibodies. Unfortunately, both combinations were incapable of yielding any strong signals for serum samples. To further improve the method, we decided to amplify the signals by including the biotinylated rabbit antibody and streptavidin in the assay. To be brief, the gold nano-particles-conjugated chicken antibody or mouse antibody was first mixed with antigens and the biotinylated rabbit antibody in the LFB2 (namely, LF buffer added 0.5% BSA instead of 5% non-fat milk) and the mixture was incubated in an ice bath or in an water bath at room temperature for 30 or 60 minutes to allow ample time for the interaction of antigens with the antibodies before it was applied to the sample pad. The LFIA was developed in the presence of LFB2. With this improvement, the positive control could be well differentiated from the negative control when gold nano-particles-conjugated chicken antibody was used. Furthermore, strong signals were obtained when serum samples were used. A similar result was obtained when the gold nano-particles-conjugated mouse antibody was used; however, the result was not as dramatic as when the chicken antibody was used. However, this improved method still required further improvements before it can be successfully used for quantitation of the METCAM/MUC18 antigens in the serum. We predict that this further improved method has a high potential in the near future to become an useful diagnostic assay.

並列關鍵字

Gold nanoparticles lateral flow immunoassay METCAM/MUC18 serum prostate cancer detection biomarker

參考文獻

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for rapid nucleic acid detection based on miniaturized lateral flow chromatography. Nucleic Acids Res, 35(10), e74.

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最佳化奈米金微粒為主的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

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