Font Size: a A A

Construction Of Double Antibody Sandwich Elisa For Dengue Antigen Detection Based On Its Ns1 Protein

Posted on:2021-01-16Degree:MasterType:Thesis
Country:ChinaCandidate:S X SunFull Text:PDF
GTID:2480306605993539Subject:Master of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Dengue fever is a global viral disease,mainly transmitted by the aedes mosquito.Over the past half century,dengue virus has become increasingly virulent,killing an estimated 12,000 people around the world every year.Dengue fever has become one of the most serious public health problems in the world.Infection of dengue virus can cause fever,rash and other clinical symptoms.Furthermore,infection in children or cross-infection of multiple serotypes can lead to bleeding,shock and even death.According to the antigenicity of envelope protein(E),dengue virus can be divided into four serotypes:?,?,? and ?,which are clinically difficult to distinguish.So far,there is no specific therapeutic drug or effective vaccine for humans.The clinical symptoms of dengue fever are not specific,and can not be differentiated from other similar pathogens.It is proved that early diagnosis and timely clinical treatment can effectively reduce the fatality rate of dengue fever.Therefore,the development of rapid,sensitive,reliable and standardized early diagnostic reagents in the laboratory is vitally significant for the effective prevention and treatment of dengue fever.This project involves the preparation of monoclonal antibody of non-structure protein 1(NS1)protein of type ? dengue virus and the establishment of double antibody sandwich ELISA for detecting NS1 protein of type ?-? dengue virus,which can be used for early diagnosis.The mice were immunized with the recombinant type ? NS1 protein and their spleen cells were fused with the mouse myeloma cell line SP2/0.The hybridoma cells were injected into the abdominal cavity of mice to prepare ascites,and the monoclonal antibody was purified from the ascites by protein G affinity chromatography.After screening by an indirect ELISA,9 hybridoma cell lines which secrete serotype ? NS1 specific monoclonal antibodies were finally selected out,including 4F11-C12,5E10-E5,6E8-E3,6F7-G4,8B4-F6,9G1-E9,12B3-H2,13A5-A10 and 15C11-H12.For the construction of double antibody sandwich ELISA to detect NS1 proteins with a full coverage of ?-? serotypes,other three antibody strains with ? serotype specificity,called 5H11-5H11,6D1-B6,and 6F7(polyclonal antibody),were incorporated for pair-matching.10 of 12 these clones showed confirmed reactivity to ?-? type NS1.However,5 of these 10 clones were removed due to their cross-reactivity with Zika NS1,and 5 clones were finally obtained.Double antibody sandwich ELISA was used to match each other,and 5H11-5H11/12B3-H2 combination,and 6F7-G4/12B3-H2 combination,for capture and detection antibodies,respectively,were finally established.This double antibody sandwich ELISA system can cover NS1 proteins of type ?-? dengue virus.The specificity and sensitivity of the system were evaluated,and the results showed that the system did not bind to Zika virus NS1,and the detection limit is lng/ml NS1 of serotype I.In conclusion,the double-antibody sandwich ELISA constructed in this study laid a foundation for immunological diagnosis of dengue fever.
Keywords/Search Tags:Dengue virus, NS1 antigen, Monoclonal antibody, Double antibody sandwich ELISA
PDF Full Text Request
Related items