以C-反應蛋白作為目標抗原發展生物辨識和生物感測的通用平台

Abstract

單鏈抗體(scFv)由抗體重鏈可變區和輕鏈可變區經由彈性的短肽(linker)連接而成，它是抗體之中具有抗原辨認活性的單元。因其分子量較小，在大腸桿菌中較為容易表現，因此利用嗜菌體展現技術進行scFv的篩選，再經蛋白質表現出具辨識能力的scFv，將有機會取代傳統動物抗體的使用，以降低檢測成本。

本研究篩選的目標抗原為C反應蛋白(CRP)，它參與身體中的急性發炎反應，且與許多慢性發炎引起的相關疾病有所關聯，為現行檢測中一個常見的生物標記。在本研究中，首先我們將麥芽糖結合蛋白和C反應蛋白之融合蛋白(MBP-CRP)以大腸桿菌表現，再以純化之融合蛋白進行單鏈抗體的篩選。我們亦同時使用以經人類細胞表現之CRP為目標抗原進行單鏈抗體的篩選，以比較其對篩選造成的差異。所篩選出之單鍊抗體scFvCRP之後以基因工程的技術連接上組胺酸標籤(his-tag)以利後續使用鎳管柱(Ni column)純化。之後以間接酵素結合免疫吸附分析法 (indirect enzyme-linked immunosorbent assay, indirect ELISA)以及微量熱泳動 (microscale thermophoresis, MST) 分析 scFvCRP 對CRP的親和力，indirect ELISA測得Kd為105 nM，而MST測得之解離常數(Kd)為0.43 ± 0.06 nM。最後以自製之氧化鎳晶片於石英晶體微天平(quartz crystal microbalance, QCM) 平台上進行檢測，其有效檢測濃度可達100 nM (2.5 mg/L)以下。

Single chain fragment variable (scFv), consists of heavy chain and light chain of fragment variable linked with flexible peptide, is the smallest unit of antibody with antigen-binding activities. Reduced size of protein facilitates the expression in E. coli. Phage display technology enables screening of scFv to against interested antigen. With the scFv expressed in E. coli, it has the potential to replace the use of antibodies produced by animal and consequently reduces the cost of diagnosis. C-reactive protein (CRP), the target antigens of this study, is recognized as a mediator of the acute-phase response, associated with various chronic inflammatory mechanism. CRP is a common biomarker in current assays. In this study, Maltose-binding protein fused CRP (MBP-CRP) expressed in E. coli was employed for scFv screening using a phage display library. Simultaneously, human cell produced CRP (CRPcell) served as the other target antigen for scFv screening. The scFv obtained from the screening, scFvCRP, was further inserted with his-tag at the N-terminus genetically to facilitate the process of protein purification. The affinity of scFvCRP was further characterized with indirect enzyme-linked immunosorbent assay (ELISA) and microscale thermophoresis (MST). Kd value was estimated to be 105 nM by indirect ELISA, whereas the Kd is 0.43 ± 0.06 nM obtained from MST analysis. The discrepancy between the two measurements becomes an interesting issue to be discussed. A detection platform was further established with the application of a NiO-coated chip on quartz crystal microbalance (QCM). Valid detection concentration was achieved below 100 nM (2.5 mg/L) of CRP.