- 學位論文
用改良式的奈米金快速側向流免疫法及磁珠晶片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
Using magnetic beads-enriched array display immunoassay and the gold nanoparticles-based lateral flow immunoassay for determination of the prostate cancer biomarker-METCAM/MUC18 concentration in human serum
摘要
目前診斷前列腺癌的生物指標物為前列腺特異性抗原(Prostate specific antigen, PSA)，但其存在20-25%的誤診率，且無法區分病患有前列腺良性增生(Benign prostatic hyperplasia, BPH)、抑或是患有前列腺癌，因此需要開發一個新的前列腺癌生物指標蛋白。本研究分別使用改良型側向流薄膜試 條檢測法(Modified LFIA)以及磁珠晶片免疫法(magnetic beads-enriched array display immunoassay)檢測人類血清中之METCAM/MUC18蛋白濃度，並與前列腺特異性抗原(PSA)濃度和癌症指數Gleason Score之比較。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。 本研究使用兩種生物素化抗體，分別為Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278)和自製的Biotinylated Chicken anti-METCAM/MUC18 Ab，以及能辨識不同抗原決定位的兩種Rabbit anti- METCAM/MUC18 Ab (EPP11278 and MBS416853)與自製Chicken anti-METCAM/MUC18 Ab進行兩種實驗血清樣品中的METCAM/MUC18濃度偵測。實驗結果證實磁珠晶片免疫法可以純化血清中METCAM/MUC18濃度，獲得比側向流薄膜試條法高約7倍的濃度訊號，METCAM/MUC18與PSA些微正相關並能對應Gleason Score指數病況。而METCAM/MUC18具前列腺癌早期診斷的能力獲得數據上的驗證，可與目前的PSA檢測相輔助。
並列摘要
The current serum PSA test for diagnosis of prostate cancer has a 20-25% of false positive results, besides it is impossible to distinguish Benign Prostatic Hyperplasia (BPH) from Prostate cancer; thus, it is still necessary to develop a rapid and accurate diagnosis method for this cancer. In our previous studies we suggest that METCAM/MUC18 has a high potential to be used as a diagnostic biomarker for the detection of prostate cancer. Previously we first used the Western blot method to determine the specificity of several commercial antibodies. Then we used these antibodies for ELISA to determine the concentration of human METCAM/MUC18 in sera from normal persons and patients with BPH and prostate cancer (Lo, 2015). However, both ELISA and Western blot methods were too complex and time-consuming for a routine test by an untrained person. We then simplified the assay by using nitrocellulose membrane to develop a lateral flow immunoassay (LFIA) (Ho, 2016). However, this method was not easily reproducible by a lay person. Furthermore, after replacing the non-fat milk with BSA this traditional LFIA still could not be used to detect the presence of METCAM/MUC18 antigen in human serum, though it could differentiate the positive and negative controls of recombinant METCAM/MUC18 proteins, NM-GST and C-terminus-GST (Hsieh, 2017). The problem could be due to interference substances in the human serum. To overcome this problem, we further developed a modified LFIA by taking advantage of the binding of METCAM/MUC18 antigen with a biotinylated rabbit anti-METCAM/MUC18 antibody and strapavidin on the membrane (Hsieh, 2017). By using this method, METCAM/MUC18 antigen in human serum was not consistently detectable, though it could differentiate the positive and negative controls of recombinant METCAM/MUC18 proteins (Hsieh, 2017). However, all these three methods can not remove the interfering substances in the serum because of the complex composition of serum samples. Therefore, the magnetic beads-enriched array display immunoassay was developed to use magnetic beads and magnetis field to separate the METCAM/MUC18 from other interfering substances in the serum in order to enrich the the detection signal and improve the detection accuracy. This method successfully detected different concentrations of positive and negative recombinant protein and human serum samples, found that this has the potential for early diagnosis of prostate cancer (Wei, 2018). In this study, two biotinylated antibodies were used for the modified lateral flow immunoassay experiments, Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278) and homemade Biotinylated Chicken anti-METCAM/MUC18 Ab, plus two Rabbit anti-METCAM/MUC18 Ab (EPP11278 and MBS416853), which recognize different epitopes in the METCAM/MUC18 protein, and the homemade Chicken anti-METCAM/MUC18 Ab were used to detect the METCAM/MUC18 concentration in serum samples. Biotinylated Rabbit anti-METCAM/MUC18 Ab or Biotinylated Chicken anti-METCAM/MUC18 Ab was first incubated with the antigen and the nanogold-anti-METCAM/MUC18 antibody, and the complex was then captured by streptpavidin on the test line. Serum samples were from healthy men, patients with benign prostatic hyperplasia, and patients at different stages of prostate cancer. The experimental results indicate that the homemade Biotinylated Chicken anti-METCAM/MUC18 Ab, which recognizes that the epitopes in the M-portion (terminus) of METCAM/MUC18 (aa#212-374), was better than the Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278), which recognizes the epitopes in the N-terminal portion of the METCAM/MUC18 protein (aa# 26-350). The rabbit anti-(MBS416853), which recognizes the epitopes in the C-terminal portion (epitope) of METCAM/MUC18(aa#259-544), was better than the Rabbit anti-METCAM/MUC18 Ab (EPP11278), which recognize the epitopes in the N-terminal portion of METCAM/MUC18 (aa#26-350). The results showed that METCAM/MUC18 had a somewhat positive correlation with prostate-specific antigen (PSA), and could also roughly distinguish prostate cancers with different Gleason scores. This study also used the combinations of two biotinylated primary antibodies and two unmodified primary antibodies to operate the magnetic beads-enriched array display immunoassay. The first combination was Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278) combined with homemade Chicken anti-METCAM/MUC18 Ab. The second and the third combinations were the homemade Biotinylated Chicken anti-METCAM/MUC18 Ab paired with the two Rabbit anti-METCAM/MUC18 Ab (EPP11278 and MBS416853), respectively. All three combinations were used to differentiate the control positive and negative recombinant METCAM/MUC18 proteins and also to determine the METCAM/MUC18 concentrations in human serum samples by using a fluorescent tagged secondary antibody, which were Cy5-conjugated Goat anti-Rabbit Ab or Cy5-conjugated Goat anti-Chicken Ab. The cy5 was released from the immune complex after heat denaturation, spotted on the surface of a biochip, and the intensity of the fluorescent signal of cy5 was read by a laser scanner. The corresponding METCAM/MUC18 concentration in serum samples were obtained after comparing to the caliberating standard curve of the control proteins. Serum sources for the experiments included normal men, patients with benign prostatic hyperplasia, and patients at different stages of prostate cancer. The results showed that the homemade Biotinylated Chicken anti-METCAM/MUC18 Ab was better than Biotinylated Rabbit anti-METCAM/MUC18 Ab (EPP11278). The rabbit antibody MBS416853 was better than the rabbit antibody EPP11278. The experimental results confirmed that the magnetic beads-enriched array display immunoassay could enrich the METCAM/MUC18 from serum and obtained a signal about 7 times higher than the LFIA strip method. Furthermore, only 3ul of the serum sample and 1/5 to 1 /2 concentrated rabbit antibody MBS416853 were used in the assay. Overall, the modified sandwich (LFIA) and the magnetic beads-enriched array display immunoassay used in this study could accurately determine the concentration of METCAM/MUC18 in human serum samples. The magnetic beads-enriched array display immunoassay was superior to the modified sandwich type LFIA data. There was somewhat a good correlation between METCAM/MUC18 and PSA, and the concentration of METCAM/MUC18 in the serum of patients with prostate cancer was generally higher than that of normal people or BPH, showing a positive correlation between concentrations of METCAM/MUC18 and PSA. The Modified LFIA method obtained more obvious signal in GS=4+4 samples. The disease signal obtained a more obvious signal, while the magnetic beads-enriched array display immunoassay had the highest fluorescence signal with GS=4+5. Based on the experimental results, I concluded that METCAM/MUC18 has the ability to diagnose prostate cancer early, and can be compared with the current PSA prostate.