| Japanese encephalitis (JE) is a serious zoonoses caused by the Japanese encephalitis virus (JEV) which is a mosquito-borne pathogen of the family Flavivirus. In general, JEV is maintained in a transmission cycle between amplifier swine and vector mosquitoes. As an important pathogen in swine, it induces terrible consequences in sows reproduction and death in piglets. At the same time, it also threatens public health. So accurate clinical diagnosis paves the important way in preventing and controlling JE infection effectively. However, the application of several developed laboratory methods for the detection of JEV antigens, such as virus isolation, RT-PCR, are limited by their requirements of laboratory operations, skilled technicians and special facilities. Therefore, it is important and urgent to establish a method for detecting JEV antigen simply, exactly, effectively.A double-antibody sandwich ELISA method and an ELISA kit for detecting JEV antigen in swine, human, mosquito and other clinical specimens were developed, in order to provide a rapid, specific and easily performed technique for JEV detection.The content of this thesis is as follows:1. The establishment of the JEV double-antibody sandwich ELISAA monoclonal antibody (MAb) against the E protein of JEV and a polyclonal antibody (PcAb) against JEV were produced and characterized for their specificity and reactivity. To develop the double-antibody sandwich ELISA assay, the polystyrene microtiter plates were coated with primary antibody E MAb to capture the JEV antigen, and the JEV PcAb was used as anti-antibody. A horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody was added for identifying detection antibody specifically. Finally, the tetramethylbenzidine was added. By checkerboard titration, optimal concentrations of the primary antibody (E MAb) was defined as5μg/mL, the optimal dilution of the detection antibody (PcAb) and the HRP-labeled antibody were1:10000and1:30000respectively. Sensitivity, specificity, repeatability and stability of the developed assay were evaluated. The sensitibity assay showed that the minimum virus amount for detection was1.0× 104PFU/mL. There was no cross-activity reaction observed among other viruses relating with popular viral diseases. The coefficients of variation of five samples tested in this assay were7.6%,6.7%,6.7%,7.6%and4.40%respectively, wihic were all less than10%. The OD630values of the standard positive control and the standard negative control were shown with no significant decrease for at least five days in the stability detection of the assay.The results demonstrated that the double-antibody sandwich ELISA method was capable in detecting JEV antigen with high sensitivity and specificity compared with conventional methods.2. The application of the JEV double-antibody sandwich ELISA60clinical samples were tested by this kit and by RT-PCR analysis, including20mosquito homogenate,24swine brain tissues and16cerebrospinal fluid of the human patients. Among which,14samples showed the positive result with coincidence rate of70%,46displayed negative result with coincidence rate of100%, and the total coincidence came up to100%compared to that of RT-PCR. It indicated that the developed double-antibody sandwich ELISA kit could be used as a convenient and specific method for the large-scale determination of JEV antigen in infected swine, human and mosquito samples with high sensitivity and specificity. |
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