結合三明治免疫法及等溫型指數放大法開發快速偵檢癌症標記蛋白之分析平台

Abstract

根據衛生福利部統計，近年來肺癌的死亡率節節上升，成為癌症死因死亡率之首；而癌症指標分子(tumor marker)的檢測則已成為肺癌治療或預後評估的重要一環。RGS11是G蛋白下游訊息路徑的調節因子，先前研究指出RGS11與肺癌轉移有關，是個與肺癌相關的蛋白；然而由於其在血液中的濃度極低(pg/mL左右)，無法使用市售的酵素連結免疫分析(Enzyme-linked immunosorbent assay, ELISA)套組分析監測。 本研究以修飾有抗體之磁珠為基礎，結合三明治免疫法(sandwich immunoassay)以及核酸等溫型指數放大技術 (isothermal exponential amplification reaction)發展出一套快速偵測且具高靈敏度的蛋白質分析平台，對肺癌相關蛋白RGS11進行檢測。在實驗步驟方面，將anti-RGS11 antibody藉由EDC/NHS反應連結到磁珠上，形成抗體修飾磁珠(MB-Ab)，接著加入已知濃度的His-tag RGS11以及血清中未知濃度的RGS11，使兩者競爭MB-Ab上anti-RGS11 antibody的接合位；最後，加入接有一段核酸引子的anti-His antibody (Ab-P)，使反應溶液內生成三明治免疫複合物，而抗體上的核酸引子可在短時間內進行等溫型的指數放大，形成許多雙股DNA產物；最後加入對雙股DNA有專一性的螢光染劑SYBR® Green，即可測得螢光訊號，而螢光訊號強度與血清中RGS11的量成反比關係，可做為評估血清中RGS11的依據。本實驗的設計優點為只需少量(20 μL)的樣品體積，即可在短時間 (1.5 hr)內完成偵測，且靈敏度高，偵測極限約為150 fg/mL。另外，本實驗可偵測兩倍稀釋之血清中，額外加入的RGS11，且回復率(Recovery rate)在90%以上。從上述結果我們認為本偵測平台在肺癌檢測方面具有運用潛力。

Lung cancer accounts for the primary cause of cancer-associated mortality worldwide, making the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, a regulator of G-protein signaling, represents as a lung cancer biomarker, plays an important role in the cancer-related metastasis. However trace level of the RGS11 (~ pg/mL) in serum sample makes it a challenge for the quantification using currently available ELISA kits. It thus is our aim to develop a rapid and sensitive platform for the detection of RGS11 based on a sandwich immunoassay coupled with isothermal exponential amplification strategy. Our study was initiated with the functionalization of the magnetic beads with anti-RGS11 antibodies (MB-Ab) via EDC/NHS activation. Taking advantage of MB-Ab, it served as a sensing probe for the competitive immuno-recognitions between known concentration of His-tag RGS11, and unknown concentration of target RGS11 in serum. The reporter anti-His antibodies, modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to reaction mixture leading to formation of immunosandwich complexs. The exponentially amplified DNA duplex intercalated with SYBR green was designated as signal output for the assessment of RGS11, in an inversely proportional relationship. The sensing platform demonstrated the excellent performance on the determination of RGS11 with exceptional detection limit of 150 fg/mL, along with minimal sample volume (20 μL), and reaction time 1.5 hr required. In addition, we challenged the sensing platform for the detection of spiked-RGS11 in 2x diluted serum samples, and acceptable recovery rate (higher than 90%) was observed . It is suggested that this sensing platform holds a potential in the biomedical application for screening lung cancer.