| ObjectiveEnvelope domain ?(ED?)of dengue virus(DENV)is responsible for viral attachment to host cells,and is also the target of specific neutralizing antibodies.However,Antibodies elicited by one primary DENV serotype infection are not strongly protective against the other three serotypes.Conversely they may lead to the development of dengue hemorrhagic fever(DHF).It is known as Antibody dependent enhancement(ADE).Serotype-specific IgG antibodies can be detected by the plaque reduction neutralization test(PRNT),but it is time-consuming,labor-intensive and low throughput.For fast and convenient measurement of neutralizing antibodies,a new method is needed to replace PRNT.The main goal of this study was to establish a double antigen sandwich ELISA method for measuring type-specific neutralizing antibodies against dengue virus based on ED? protein.Methods1.ED? fragments of dengue virus 1 and 2 were amplified by PCR and cloned into vectors pET-28b(+).The recombinant vectors were transformed into BL21 to express protein under IPTG induction.The recombinant proteins were purified by affinity chromatography and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).2.The ED? proteins of dengue virus 1 and 2 were labeled with horseradish peroxidase(HRP)by sodium periodate method and purified by gel filtration chromatography.3.The antibody typing ELISA methods of dengue virus 1 and 2 based on HRP labled ED? were developed.The coating concentration of ED?,concentration of ED?-HRP,serum dilution and coloration time were optimized,respectively.4.Convalescent phase sera of 48 individuals with DENV1 infection and convalescent phase sera of 11 individuals with DENV2 infection were detected by the typing ELISAs.The results were compared with the Dengue IgG ELISA kit and PRNT test.The performance in serological diagnosis of dengue virus type-specific antibody was evaluated.Results1.The recombinant vectors pET28B-ED? of dengue virus 1 and 2 were constructed and induced by IPTG.High quantity and good quality recombinant ED? proteins of dengue virus 1 and 2 were successfully obtained.The proteins were identified by MALDI-TOF-MS and purified by Ni-NTA metal-affinity chromatography.2.ED?-HRP conjugate of dengue virus 1 and 2 were successfully obtained by sodium periodate labeling method and purified by gel filtration chromatography.3.The antibody typing ELISA of dengue virus 1 and 2 were developed successfully.The optimized coating concentration was 1 ? g/mL,concentration of ED?-HRP was 1 ? g/mL,serum dilution was 1:40,coloration time were 20min.4.The results of convalescent phase sera of DENV1 infected patients showed that the positive rate of DENV1 antibody typing ELISA was 83.3%(40/48)and it was the same as dengue IgG antibody ELISA kit.PRNT results showed the positive rate was 95.8%(46/48).It was more sensitive than the other two methods.There was a close correlation between the DENV1 antibody typing ELISA and PRNT test(correlation coefficient R2?0.4590,P<0.01);The positive rate of DENV2 antibody typing ELISA was 81.8%(9/11),it was more sensitive than dengue IgG antibody ELISA kit.The cross reaction rate of DENV2 antibody typing ELISA to sera with DENV1 infected patients was 22.9%(11/48),the cross reaction rate of DENV1 antibody typing ELISA to sera with DENV2 infected patients was 45.5%(5/11),the total cross reaction rate of the typing ELISA was 27.2%(16/59).ConclusionThe typing ELISA developed in this study showed it was valuable for the serologic typing of dengue virus antibody.The typing ELISA would provide essential materials and experimental basis for the development of next-generation diagnostic kit for dengue virus antibody.This method might be a promising alternative for serologic typing and seroepidemiologic survey of dengue infection. |
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