| Japanese encephalitis(JE) is a serious mosquito borne zoonotic disease and endemic in most Asian regions and the Western Pacific Region. In recent years, the virus has spread as far as northern Australia and Pakistan which showed the epidemic range is expanding. JEV can not only threaten human health seriously, but also contribute to great economic losses to the pig industry. Establishment of monoclonal antibodies(Mc Abs)-based assays for detecting JEV antigens or antibodies is an important means of preventing and controlling the virus disease.In previous study, we have generated a mammalian cell line which can stably produce a secreted form of pr M-E protein of JEV. The pr M-E protein can assemble into virus-like particles(VLP) with similar conformational structure as JEV virion. In this study, the JEV-VLP was purified by sucrose density gradient centrifugation and used to immunize 6~8 weeks old female BALB/c mice. Eight hybridoma cell lines designated as 1B5, 3A7, 4C6, 5B10, 5E7, 11C2, 12D10, 18A7 respectively were screened after the lymphocyte hybridoma technique, 3~5 sub-clone and indirect ELISA. They can stably secret Mc Abs against pr M-E structural protein of JEV. The results of indirect ELISA showed that the cell culture supernatant titers of Mc Abs were between 1:64~1:2 048 and the ascites titers were between 1:51 200~1:204 800. Identification of Mc Abs subtypes with HBT kit showed that the subtype of 12D10 was Ig G2 b, while others were Ig G1 and the light chains of all Mc Abs were κ type. Western blot and indirect immunofluorescence assay(IFA) indicated that eight Mc Abs can identify the conformational epitopes of JEV, and 5E7 is also able to identify the linear epitopes. At last, 4C6 and 12D10 were JEV specific Mc Abs and there was no cross-reaction with pr M-E of West Nile virus.There is no specific treatment for JE. Vaccination is the most effective measures for the prevention and control of JEV infection. JEV pr M-E antigen has strong immunogenicity, and the protein has the potential to be a subunit vaccine. In the process of subunit vaccine development, the detection of vaccine antigens is an important quality control method. An antigen capture ELISA(AC-ELISA) which can apply to quantitative estimation of subunit vaccine antigens was established with Mc Ab 5B10 as capture antibody and horseradish peroxidase(HRP) conjugated Mc Ab 5E7 as detection antibody. The optimum reaction conditions are as follows. Coating buffer was 50 mmol/L p H9.6 carbonate buffer, the dilution of capture Mc Ab 5B10 was 0.6 μg/well, the blocking solution was 0.5% polyving akohol(PVA)and the antigen diluent was 0.5% bovine serum albumin(BSA), the reaction time for blocking solution and antigen were all 2 h at 37℃, the HRP conjugated 5E7 dilution ratio was 1:500 and reacted 1 h at 37℃, TMB substrate reacted 8 min at room temperature. The detection limit of AC-ELISA was 48.44 ng/m L, and the linear quantitative range was 0.05~1.00 μg/m L, and the linear correlation coefficient of the standard curve can reach more than 99%, using JEV pr M-E protein purified by gel-filtration chromatography(GFC)as standard antigen. The coefficients of variation intra-assay and inter-assay were less than 10%. The AC-ELISA was specificity which had no cross-reactivity with other related pathogens of swine diseases. The ELISA plate coated antibody can bear at least a month at 37 ℃ with the antigen binding capacity had no change. The AC-ELISA kit stored at 4℃ could be maintained for 12 months.The preparation of Mc Abs against pr M-E structural protein of JEV laid the foundation for the study of new methods for detecting JEV antigens or antibodies. And the development of AC-ELISA kit provided technical support to the development and production of JEV pr M-E subunit vaccine. Development of Mc Abs against pr M-E protein of JEV and establishment of AC-ELISA for pr M-E protein detection has important public health implications for the prevention and detection of JE. |
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