發展對B肝病毒表面抗原有專一性之單株抗體

Abstract

世界上約有3.5億慢性B型肝炎帶原者，約佔世界總人口數的三分之一。慢性的病毒感染可能導致一系列的肝臟疾病，像是肝纖維化、肝硬化以及肝癌等。在抗病毒藥物治療下的患者，其血液中的病毒DNA含量為藥物治療效果的指標。而由文獻指出，監測血液中preS含量的變化，可以確實反映出病毒的活躍狀態。因此本研究選擇發展對preS具有專一性的抗體，並將之應用於病毒活躍狀態的檢測。另外，由於血液當中小的表面抗原(HBsAg)含量若是過高會干擾preS的檢測，因此我們同時也發展對小的表面抗原具有專一性的抗體(我們稱之為HBV_a)。 在本研究中，我們嘗試以B肝病毒表面蛋白preS和HBV_a作為B肝病毒的檢測標的，首先我們利用大腸桿菌表現系統表現出這些重組蛋白以作為免疫老鼠之抗原，並藉由細胞融合(hybridoma)技術，開發出對B肝病毒表面蛋白preS和HBV_a具有專一性之抗體。總共得到九個anti-HBV_a單株抗體和四個anti-preS單株抗體。接著經由酶聯免疫吸附試驗(ELISA)及西方墨點法(Western blot)驗證，篩選出對兩個標的具有專一性且辨識能力較佳的單株抗體各一對(anti-HBV_a:A2A8B5,A1B11G7;anti-preS:1H9G7,3E6B6C5)並將這些抗體應用於間接型酵素連結免疫吸附法(Indirect ELISA assay)檢測系統中。在我們的傳統型(Indirect)檢測系統中，preS的偵測極限為20 ng/mL，HBV_a則為2 ng/mL。而在我們的三明治(sandwich)檢測系統中，preS的偵測極限為1.4mg/mL，HBV_a則為6.8 ng/mL。

There are about 350 million chronic HBV carriers in the world, accounting for about one third of the global population. Chronic viral infection may lead to a series of liver disease, such as liver fibrosis, cirrhosis, and HCC. The active viral state in the patient’s blood is as an indicator of anti-virus drugs treatment efficacy. The literatures indicate that monitoring the preS level changes in blood, which can reflect the active state of the virus. Therefore, in our study, we developed preS specific antibodies, which we applied to detect the viral active state. Furthermore, excessive amounts of small antigen (HBsAg) interferes with the detection of preS. Therefore, we also developed HBsAg specific antibody (called HBV_a). In this study, we used HBV surface protein preS and HBV_a as detection targets of HBV active state. At first, we expressed these recombinant proteins through E. coli expression system and used these proteins as antigens for the immunization of mice. After immunization, antibody-secreted hybridoma was created using a cell fusion (hybridoma) technology. We have obtained a total of nine anti-HBV_a monoclonal antibodies and four anti-preS monoclonal antibodies, respectively. Through ELISA and Western blot assay, we screened at least one pair antibody pair on two targets using specificity and sensitivity analyses (anti-HBV_a: A2A8B5, A1B11G7; anti-preS: 1H9G7, 3E6B6C5) and applied these antibodies on detection system. We developed a preS detection system with these specific antibodies based on indirect ELISA assay. The detection limits of each target in our indirect ELISA assay were 20 ng/mL for preS and 2 ng/mL for HBV_a. The detection limits in our sandwich ELISA assay were 1.4mg/mL for preS and 6.8 ng/mL for HBV_a.