研究第二型登革病毒血清型專一性抗體的B細胞抗原決定位及評估其為偵檢試劑之可行性

Abstract

本計畫主要是要研究登革病毒的B 細 胞抗原決定位。經由ELISA, immunofluorescence 和immunoblotting 方 法，我們找出具專一性的第二型登革病毒 單株抗體(Ab4)。此抗體之抗原決定位的確 認，有助於病毒感染機制的暸解，疫苗及 偵檢試劑的研發。在此研究中，我們以噬 菌體顯現法來研究第二型登革病毒的抗原 決定位。我們也已經篩選出多株的噬菌體 可以專一性的與第二型登革病毒抗體結 合。這些專一性噬菌體所攜帶的外來 peptide，都含有His-Arg/Lys-Leu/Ile motif。 以synthetic peptide 代替噬菌體所攜帶的外 來peptide，可與第二型登革病毒抗體明顯 反應。第二型登革病毒感染之老鼠及兔子 血清，可以用此epitope-based peptide 來確 認。另外，此epitope-based peptide 可有效 的分辨登革病毒及日本腦炎病毒感染之老 鼠血清，這些抗原決定位的確認與研究， 將有助於epitope-based peptide antigen 之研 發，及了解出血性登革之病理機制。

Serotype-specific monoclonal antibody (MAb), D2 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. This MAb recognized non-structural protein 1 (NS1) of DEN-2; its specificity was determined by ELISA, immunofluorescence and immunoblotting analysis. Serotype-specific B-cell epitope of Ab4 was further identified from a random phage-displayed peptide library. Selected phage clones specifically reacted with Ab4 and did not react with other MAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile. Synthetic peptide corresponding to phage-borne peptide specifically bound to antibody. His and Arg in this epitope were crucial for peptide binding to Ab4: binding activity dramatically decreased when His or Arg was changed to Leu. Epitope-based synthetic peptide identified serum samples from DEN-2-immunized mice and rabbits by ELISA assay. The peptide clearly differentiated between serum samples from DEN-2- and Japanese encephalitis virus (JEV)-immunized mice. This monoclonal antibody and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect the antibody response and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.