Classical Swine Fever (CSF) is one of the most important infectious diseases of swine which can spread in an epizootic form as well as establishing enzootic infections in domestic and wild pig populations and cause significant economic losses to the pig industry all over the world (5). Classical swine fever virus (CSFV) belongs to the genus Pestivirus of the Flaviviridae family along with bovine viral diarrhoea virus (BVDV) (15).
Detection of virus-specific antibodies is a prere-quisite for aiding epidemiological surveys in tracing the spread of the virus and for monitoring in an eradication program. The neutralization test (NT) has been described for detection of specific antibodies to CSFV (18) but while it is reliable and sensitive, it is not readily applicable for large-scale screening in animal husbandry production because it is time-consuming. Several recombinant antigen ELISA have been developed (3, 11), but a more sensitive as well as specific and rapid indirect ELISA for screening of a large number of serum samples during an outbreak is still needed. Therefore, a CSFV indirect ELISA method using a recombinant E2 protein as antigen was developed in this study.
The recombinant expressed protein was 46.65 kDa in size including GST protein (26.0 kDa in size) and target protein (20.65kDa in size) as estimated, and could be observed by SDS-PAGE analysis both in supernatant and deposition. The ratio was approxi-mately 1:4 as measured by lamina-scan ananlysis (Fig. 1). After the recombinant protein in supernatant was purified with Glutathione Sepharose TM4B, the value of OD280/OD260, concentration and purity were 1.63, 4.752 mg/mL and 80%, respectively, and for the GST protein, were 1.80, 3.502 mg/mL and 90%, respectively (Fig. 2).
Figure 1. SDS-PAGE analysis of expression products of BL21/pGEX-E2. M, Low molecular weight protein Marker; 1-2, Expression products in deposition of BL21/pGEX-E2; 3, Expression products in supernatant of BL21/pGEX-E2; 4, Expression products of BL21/pGEX-4T-1.
Figure 2. SDS-PAGE analysis of the protein purified. M, Low molecular weight protein Marker; 1, GST protein purified; 2, Target protein purified.
Western blot analysis, revealed the band of the expressed protein was ~46.65 kDa in size but no band corresponding to GST protein was observed (Fig. 3), indicating that the target protein could react with anti-CSFV serum.
By checkerboard titration tests, the concentration of coating antigen was 0.6 μg/well, and the dilutions of the sera and HRP-IgG were 1:80 and 1:2000 respectively. A mean P/N of 1.461 with a standard deviation (SD) of 0.196 was obtained from 300 CSFV-negative sera, so the cut-off value was adjusted to 2.1 (mean+3 SD). Namely, 2.1 times the OD490 value obtained from reference negative serum was set as the criteria. A sample was considered positive if its OD490 value was over or equal to the criterion.
To determine the specificity and sensitivity of the iE2-ELISA, 100 serum samples were tested and com-pared with the results obtained with CSFV IHAT (Table 1). Of the 62 positive serum samples in the CSFV IHAT, 6 were negative in iE2-ELISA. For the corresponding negative serum samples, 2 out of 38 tested positive in iE2-ELISA. The comparison of the tests gave a calculated sensitivity of 90.3% and a specificity of 94.7%.
Table 1. Comparison of the iE2-ELISA with the whole Ag IHA
In this test, no cross-reaction with known positive sera to bovine viral diarrhoea virus (BVDV) was seen in the iE2-ELISA using E2 recombinant antigen, giving values well below the defined cut-off (Table 2).
Table 2. The result of cross-reaction test