JE is a severe zoonosis which induces fever, aseptic meningitis, acute flaccid paralysis or classic meningomyleoencephalitis, and up to 50% of persons who survive it may have prolonged neurological or psychiatric sequelae [15–18]. Currently, there is an urgent need of a rapid diagnosis method for detection of JEV infection in both humans and animals. Since the inoculation of JEV vaccines contributes to the result of antibody-positive, the accurate diagnosis still depends on the antigen detection. However, the application of conventional methods, such as RT-PCR and viral isolation, are limited by their requirements of laboratory operations, skilled technicians and special equipments. Therefore, a more simple and rapid method for the detection of JEV antigen is needed to be developed.
Herein, we reported a convenient antigen capture ELISA for the diagnosis of JEV infection by using the MAb 4D1 against E protein of JEV and the PcAb of JEV derived from the rabbits. Because the JEV-specific primary antibody and anti-antibody are used, the proposed ELISA in this report is shown to hold the characteristic of detecting JEV antigen specifically, which effectively reduces and clears the interference from other flavivirus for JEV diagnosis. In addition, as the major immunogenic gene, E gene is highly conservative in different JEV genotypes. It has been shown that the E MAb produced by Yaoming Li according to the nucleotide sequence of E gene of JEV genotype III possessed sensitive reaction with prevalence-predominant JEV genotype I , which suggested that our ELISA assay could be applied to clinical detection of different JEV genotypes.
Specific binding between antibody and antigen is the most important factor for the successful development of ELISA assay. In previous studies, specificity of the test has been modified by use of monoclonal antibodies of single strains or multiple strains. However, polyclonal antibodies are typically chosen to enhance sensitivity [19, 20]. Therefore, in this study, the polyclonal antibody rather than the monoclonal antibody against JEV is applied for conjugating with the antigen as detection antibody, since our results have showed that polyclonal antibodies could recognize more JEV strains, which improves the sensitivity of test.
Taken together, the ELISA assay developed in this study has following potential advantages. First, unlike RT-PCR and other viral isolation methods, our ELISA assay does not require expensive reagents and special facilities, and it can be routinely performed in any ordinary laboratory because of its simplicity and rapidity. Second, the ELISA assay has been confirmed with relatively high sensitivity. The minimum detection level of our ELISA assay (1.0 × 104 PFU) is lower than that of RT-PCR (3.2 × 102 PFU), but higher compared with that of immunochromatographic strip (ICS) (2.5 × 105 PFU) . Since the ineluctability of false positive reaction by RT-PCR and the low sensitivity of ICS always prevent the applying of these methods for accurate diagnosis of JEV, our ELISA assay may be developed as an alternative method for fast diagnosis of JEV infection. Additionally, the higher sensitivity with our capture ELISA for detecting JEV antigen than with traditional ELISA has been confirmed in our assay, it could be due to introducing of a horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody for specifically identifying detection antibody, which has amplified the reaction signals in whole system and modified the sensitivity. Most importantly, this ELISA assay provides a convenient way for detecting JEV in a large number of clinical specimens including swine, human and mosquito, which has not been reported before. Therefore, this method will have a broad application prospect in large-scale clinical diagnosis of JEV.
However, there are still some problems cannot be ignored. Samples collected at an appropriate time is required in this ELISA test when the virus in the detected samples has not been degraded. Because the timing of sample collection impacts on the ability to confirm the diagnosis of JEV  and that is necessary for successful detection by any technique. Moreover slight infection with JEV may not be capable of being detected by our ELISA test, it is due to the fact that some samples lightly infected with JEV have negative results with this ELISA but positive results by RT-PCR which has ability to detect lower virus load. Thus the ELISA test in this study is more suitable for diagnosis of severe JEV infection during an exhaustive outbreak in farm. It is necessary that these suspected samples without classical symptom of JEV infection should be further confirmed by other techniques, such as histological, immunological, or molecular assays.