Development of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

Japanese encephalitis virus (JEV) transmitted by mosquitoes is caused reproductive failure in pregnant sow and the encephalitis in human. Therefore, it has been known as zoonosis. There are several methods to detect antibodies against JEV. These methods include hemagglutination inhibition (HI), viral neutralization test (VN), plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay (ELISA). HI test is considered the standard method to detect antibody against JEV. However, the complicated test procedure may limit the use of HI test. The VN and PRNT test are more reliable method. However, both of the tests take one week to get the results. ELISA test is the most rapid and easiest method. Many ELISA products related to JEV have been developed in human field but ELISA products for animal are rare. In this study, we developed indirect ELISA (I-ELISA) for swine and estimated the efficacy by compared with HI, VN and PRNT.

A total of 175 swine serum samples were collected from slaughterhouse and Yeonggwang province. KV1899 strain of the JEV which was isolated from swine blood in 1999 was used for HI test, PRNT₉₀ and I-ELISA. K87 strain which was isolated from mosquito was used for VN test in this study. HI test was carried out in 96 well microplates, using a little modified method of Clarke and Casals. The HI titer of 1: 20 or higher was considered positive. PRNT₉₀ titers ≥1:10 were considered positive. The antigen and swine serum was diluted from 4 to 1 µg/mL and 1:2 to 1: 256 to detect optimal concentration of antigen and serum dilution for I-ELISA, respectively. VN test was conducted by micro neutralization test technique. An antibody titer ≥ 1:2 was considered positive.

The optimal antigen concentration and serum dilution were found to be 2µg/mL and 1:100, respectively. The absorbance > 0.25 was considered positive. The absorbance results obtained from 175 swine serum samples were compared with the results of VN, HI and PRNT₉₀. The significant correlation was shown between VN (r=0.95), HI (r=0.93) and PRNT₉₀ (r=0.95). The sensitivity of I-ELISA was 95.0% with VN test, 91.8% with HI test and 94.7% with PRNT90, respectively. The specificity was 94.7%, 92.2% and 94.7% with the VN, HI and PRNT90, respectively.

ELISA test is the easiest method and is suitable for large number of samples compared with VN, HI and PRNT. In addition, it is easy to test many samples without special equipments at one time. According to the results, I- ELISA showed a high correlation with other methods, which suggests that I-ELISA can be used for the detection of antibodies against the JEV in swine.

Disclosure of Interest: None Declared