Preparation and characterization of monoclonal antibodies against envelope protein of Japanese encephalitis virus
Author(s): NI Hui, WANG Yu, MAO Yan, CHEN Puyan, YANG Mei, CAO Ruibing, College of Veterinary Medicine, Nanjing Agricultural University
Pages: 1-9
Year: 2016 Issue: 7
Journal: Animal Husbandry & Veterinary Medicine
Keyword: Japanese encephalitis virus; envelope protein; monoclonal antibody; duck tembus virus; cross reaction;
Abstract: Japanese encephalitis is a mosquito borne zoonosis caused by Japanese encephalitis virus( JEV),which threats both human and several kinds of livestock and has important impact in public health in China. In the present study,monoclonal antibodies( MAbs) against JEV envelope protein were prepared by cell fusion technology. The purified cap and ED3 fusion protein which form viral like particle was used to immunize BALB / c female mice. For the screening of envelope specific MAbs,a soluble recombinant ED3 protein were expressed in E. coli BL21 cells. Analyzed by immunogical methods,MAbs 1B10 and 3F5,4h1 and 5F9 were selected on the basis of their reactivity. All of the four MAbs could recognize naive JEV NJ2008 strain in the western-blotting and IFA tests. The titers of antibodies secreted into the culture supernatant and ascitic fluid were measured by indirect ELISA and the highest antibody titer of ascitic fluid was 1 ∶ 409 600 for 5F9. However,none of the four MAbs was identified to have neutralizing activity against JEV NJ2008 strain in the plaque reduction neutralization test.The result of additional ELISA indicated that 3F5 and 4H1 recognized the same antigen epitope while 1B10 and 5F9 reacted with other antigen sites. Furthermore these MAbs had no cross reactivity with duck tembus virus( DTMUV) except 5F9. The preparation of the four MAbs against JEV envelope protein provides a basis for development of ELISA kits for JEV antigen or antibody.
Pages: 1-9
Year: 2016 Issue: 7
Journal: Animal Husbandry & Veterinary Medicine
Keyword: Japanese encephalitis virus; envelope protein; monoclonal antibody; duck tembus virus; cross reaction;
Abstract: Japanese encephalitis is a mosquito borne zoonosis caused by Japanese encephalitis virus( JEV),which threats both human and several kinds of livestock and has important impact in public health in China. In the present study,monoclonal antibodies( MAbs) against JEV envelope protein were prepared by cell fusion technology. The purified cap and ED3 fusion protein which form viral like particle was used to immunize BALB / c female mice. For the screening of envelope specific MAbs,a soluble recombinant ED3 protein were expressed in E. coli BL21 cells. Analyzed by immunogical methods,MAbs 1B10 and 3F5,4h1 and 5F9 were selected on the basis of their reactivity. All of the four MAbs could recognize naive JEV NJ2008 strain in the western-blotting and IFA tests. The titers of antibodies secreted into the culture supernatant and ascitic fluid were measured by indirect ELISA and the highest antibody titer of ascitic fluid was 1 ∶ 409 600 for 5F9. However,none of the four MAbs was identified to have neutralizing activity against JEV NJ2008 strain in the plaque reduction neutralization test.The result of additional ELISA indicated that 3F5 and 4H1 recognized the same antigen epitope while 1B10 and 5F9 reacted with other antigen sites. Furthermore these MAbs had no cross reactivity with duck tembus virus( DTMUV) except 5F9. The preparation of the four MAbs against JEV envelope protein provides a basis for development of ELISA kits for JEV antigen or antibody.
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