Generation and Characterization of Monoclonal Antibodies Against Dengue Virus Type Ⅱ and Ⅳ

登革病毒引發人類嚴重疾病，包括有登革出血熱和登革休克症候群。近來，本研究室過去己確認第一型和第二型登革病毒的血清專一型B細胞抗原決定位。本研究因此利用登革第二型和第四型的登革病毒對BABL/c老鼠進行免疫，並以融合瘤技術生產對抗第二型和第四型登革病毒的單株抗體。初步結果己生產對抗第二型和第四型登革病毒的單株抗體分別有九株和十一株。這些單株抗體在酵素連結免疫吸附分析法、免疫螢光染色法以及免疫墨點法測試中對登革病毒有專一性的反應。一些單株抗體對抗病毒的套膜蛋白(E proteins)，另一些對抗病毒的非結構性蛋白(Non-structural protein 1)和前驅膜蛋白(prM proteins)。有四株單株抗體(DB23-30-7, DB24-2-6, DB13-19-1, DD11-42-12)在溶斑減少試驗法(PRNT)的測試中被證實對登革病毒的感染途徑發揮中和性效用。我們也利用Phage display的技術確認這些單株抗體其血清專一性和中和性B細胞抗原決定位。這些單株抗體以及藉由抗原決定位胜肽抗原可以幫助我們做登革病毒感染的血清學診斷和研發登革疫苗的依據。此外，相信藉由中和性抗體所確認的這些抗原決定位可以幫助我們了解登革病毒感染時，抗體所扮演的角色，以及這些抗體於初次或二次感染登革病毒的登革出血熱時在致病機轉上所扮演的角色。

Dengue virus (DEN) causes serious febrile illness in humans, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Recently, we identified the serotype-specific B-cell epitopes of DEN-1 (Wu et al., 2001) and DEN-2 (Wu et al., 2003). In this study, we generated monoclonal antibodies (MAbs) against DEN-2 and DEN-4 by fusing NS1/1-Ab4-1 (NS-1) mouse myeloma cells with lymphocytes from BALB/c mice immunized with DENs. We have generated nine and eleven MAbs against DEN-2 and DEN-4, respectively. The specificity of MAbs was identified by ELISA, immunofluorescence, and immunoblotting analysis. Several MAbs revealed to recognize envelope proteins (E proteins), non-structural protein 1 (NS1) and prM proteins. Four MAbs (DB23-30-7, DB24-2-6, DB13-19-1 and DD11-42-12) further demonstrated to neutralize DEN infection by plaque reduction neutralization test (PRNT) assay. The serotype-specific and neutralizing B-cell epitopes of MAbs were also identified by phage-displayed random peptide library. These MAbs and their epitope-based peptide antigens will be valuable for serologic diagnosis of DEN infection. The neutralizing MAbs and B-cell epitopes will be valuable for development of DEN vaccine. Furthermore, identification of these epitopes makes it feasible to dissect the antibody response and to address the role of antibodies in the pathogenesis of primary and secondary DEN infections.