The Development and Application of Dengue Virus Specific Monoclonal Antibodies
|關鍵字:||登革熱病毒;單株抗體;基質輔助雷射脫附游離;dengue virus;monoclonal antibody;immunocapture;matrix-assisted laser desorption/ionization time-of-flight mass spectrometry|
因為抗體對抗原具有高度專一性結合的能力，因此抗體在臨床應用性相當廣泛。抱括開發免疫診斷方法、研究致病機轉及及疾病的治療。因此在本研究中，嚐試以登革熱病毒之表面蛋白(envelope)及登革熱病毒為標的，開發出對登革熱病毒具有專一性之抗體。這些抗體將被作進一步的特性分析，並應用於檢測方法的開發，以及研究病毒與宿主之間的作用力，以發展疫苗及抗病毒藥物。總共得到一個多株抗體和十五個單株抗體。經由酶聯免疫吸附試(ELISA)及點墨分析(dot blot analysis)驗證，篩選出六個具有型別專一性的單株抗體。其餘一個多株及九個單株抗體，則是可以辨識二個到四個型別的登革熱病毒。
具有型別專一性的抗體，可用來開發對型別具有專一性的檢測工具。另外，若對病毒有中和能力(neutralization)，其抗原決定部位(epitope)，可提供相關訊息，以助於疫苗的開發，以及探討病毒的致病機制。在此，兩個可辨識外膜熱白、具有型別專一性的單株抗體: 1-9-5 (DV1-E1) 和 3-46-1 (DV1-E2)，被拿來更進一步探討其對病毒之中和能力與分析其抗原決定部位。經由人工胜肽合成技術，證實這兩株單株抗體之抗原決定部位，位於病毒表面蛋白上346到360位置的胺基酸上(346TQNGRLITANPIVTD360)。這段位置在第一血清型的登革熱病毒上具有高度的保留性。另外，這兩株單株抗體，具有抑制登革熱病毒感染C6/36及Vero這兩株細胞的能力。因此可推論，這個位置的胺基酸，在型別專一性及病毒中和上扮演相當重要的角色。而這也是第一次被提出來的。
最後，我們結合型別專一性之單株抗體(3-46-1)、磁珠及基質輔助雷射脫附游離法 (MLADI-TOF MS)，開發出可由複雜的生物樣品中，快速且專一性地檢測出第一型登革熱病毒之檢測技術平台。此技術平台利用修飾上抗體的磁珠，初步分離出病毒。再以30%醋酸，將病毒從抗體上沖提下來後，再與基質混合後，針對病毒之殼蛋白(capsid protein)進行分析。偵測極限約為~105 pfu/mL (病毒含量)。|
Dengue virus (DV) is responsible for the highest rate of disease and mortality among members of the Flavivirus genus. Global epidemics of DV have occurred over the past few years. DV infects 50 to 100 million people each year, with 500,000 patients developing the more severe disease, namely, dengue hemorrhagic fever (DHF). This can lead to hospitalizations and results in approximately 22,000 deaths. In Taiwan, dengue has been a very common infectious disease since 1981, and several large epidemics of dengue/DHF have been reported. In the absence of a vaccine or any specific drug for its treatment, an early diagnosis is considered essential to prevent casualties. Because of the ability of antibodies to bind to antigens with a high degree of affinity and specificity, antibodies have a wide range of clinical applications, including development of immunodiagnostics, investigation of pathogenesis and therapy for diseases. In this thesis, the important role of the envelope protein of DV, as an antigen for the development of DV specific antibodies is investigated. These antibodies were characterized and applied to develop antibody-based diagnostic methods and to study the interaction between DV and host cells, which could then be applied in the design of new vaccine and antiviral drugs. One polyclonal antibody (pAb) and fifteen monoclonal antibodies (mAb) against DV were obtained. Based on the results of ELISA and dot blot analysis, including six serotype specific mAbs and nine cross-reactive antibodies. 1-10H reacted with DV4, 1-9-5 (DV1-E1), 3-46-1 (DV1-E2), 4-26-2, 3-18-5, and 1-46-3 reacted with DV1. DV4r (pAb), 1G4-1, and 2B1 were found to react with DV1 and DV4; 2-34, 3-14, 3B1, and 5-23 reacted with DV1 and DV2; 1-2H, 2B11-3, and 2E4-1 reacted with DV. We believe that mAbs and neutralizing epitopes will provide information that will lead to the development of serotype specific diagnostics and vaccines. Two serotype-specific mAbs, 1-9-5 (DV1-E1) and 3-46-1 (DV1-E2), against the E protein domain III of DV1 were selected and their serotype-specificity was characterized by ELISA, dot blot, immunofluorescence assay, western blot, and focus-forming analyses. The location of a linear epitope recognized by DV1 serotype-specific mAbs was identified using a peptide coated indirect ELISA, and the results revealed that amino acids 346-360 (346TQNGRLITANPIVTD360) of DV1 E protein are critical for the binding of DV1-E1 and DV1-E2. Based on sequence alignment and BLAST, the amino acid sequence of the epitope region was highly conserved in most DV1 strains. Further, DV1-E1 and DV1-E2 were able to block DV1 entry into Vero and C6/36 cells. These mAbs will be useful in the development of serologic diagnostic kits and feasible treatments for DV1 infection. Furthermore, DV1 epitope identification makes it possible to analyze antibody responses to DV and to determine the role of antibodies in the pathogenesis of DV1 infections. Finally, a facile method for the accurate detection of DV1 infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against DV1. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was ~105 pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures.
|Appears in Collections:||Thesis|