Porcine reproductive and respiratory syndrome (PRRS) was first observed in America in 1987, and subsequently spread worldwide, becoming one of the main infectious diseases in the swine industry and the cause of substantial economical loss. PPRS is characterized by pregnant sow reproductive failure, dyspenea, high mortality in piglets and respiratory symptoms in growing pigs [5, 7]. The PRRS virus (PRRSV), is a small enveloped virus with a single-stranded RNA genome containing eight ORFs. These are the viral replicase encoded by ORF1, which contains ORF1a and ORF1b; the virus envelope glycoproteins GP2, GP3, GP4 and GP5 encoded by ORFs 2-5 respectively ; the viral unglycosylated membrane protein M encoded by ORF6; and the highly conserved nucleocapsid protein N encoded by ORF7[4, 3]. GP5 protein is a major, multifunctional glycoprotein of 25 kDa. It has six antigenic determinants that can induce the production of antibodies that neutralize viral infection in vitro[1, 8]. GP5 shows the greatest variability of the PRRSV proteins, and stimulates cellular and humoral immunity. Therefore, establishing ELISA detection methods using GP5 protein as the antigen is essential. In vivo, antibodies against GP5 protein appears in the late stage of PRRSV infection.
A rapid and accurate diagnostic method for PRRS is essential for avoiding both detection failure, and unnecessary and expensive implementation of control measures. The current diagnostic techniques for PRRS antibodies are the American IDEXX PRRSV-Ab ELISA kit and the French LSI PRRSV-Ab ELISA kit. Although effective and sensitive, these methods are expensive, which is an important limitation. To explore additional detection options, we cloned and expressed the genes for the PRRSV major glycoprotein gene GP5 from the recent PRRSV isolate LZh/07, in Escherichia coli. We used the recombinant proteins to develop a method to detect PRRS antibody in swine.
Western blotting for recombinant GP5 protein showed a specific protein band with a molecular weight of 42 kDa for the GP5 protein, after induction at 28℃ (Fig. 1). This was consistent with the expected sizes, since GST added 26 kDa to the molecular mass of 16 kDa for GP5. The expression level peaked at 12 h after induction. Chromatography scanning suggested that recombinant GP5 was 30% of the total protein content. The purity of the target protein after affinity chromatography reached 90%. Bands corresponding to the target proteins were observed by Western blot analysis (Fig. 1), indicating reaction with anti-PRRSV serum.
Figure 1. SDS-PAGE, Western blotting analysis of the expressed GP5 protein. A: SDS-PAGE detection the cell lysate of E. coli for expressing GP5 protein. Lane 1, The cell lysate of Escherichia coli expressed GP5 protein; 2, The uninduced bacteria harboring 6P-1-5/BL21; M, The protein marker. B: SDS–PAGE analysis. C: Western blot analysis of the puriﬁed recombinant protein. Lane 1, GP5 protein; M, Protein marker.
By checkerboard ELISA, the optimal concentration of coating antigen was determined to be 0.2 μg/well GP5 protein. The optimal antibody dilutions were 1:40 for serum and 1:40 000 for HRP-IgG. Optimal exposure time was 45 min for serum samples and 15 min for the conjugate at 37℃.
Receiver-operating characteristic curve analysis was used to set a cut-off value of 0.2, determined from a mean P/N of 1.516 with a SD of 0.056 for the 30 PRRSV negative sera. The speciﬁcity and sensitivity of the assay at three different cut-off values, 0.10, 0.2 and 0.3, are shown in Table 1. At the cut-off value of 0.2, the sensitivity and speciﬁcity were relatively high for three animal species. Thus, the cut-off values were established to be: positive≥ 0.3; suspicious, 0.2 -0.3; negative≤0.2. Serum samples classiﬁed as suspicious were re-tested, with the sample judged to be positive if the result was confirmed by the repeat test.
Table 1. Specificity and sensitivity according to three different cut-off values
The inter-assay CV ranged from 1.9% to 5.8%, and the intra-assay CV ranged from 1.4% to 5.0%, for 20 sera selected for validation testing (Table 2). When comparing the GP5-Ⅰ-ELISA with the LSI PRRSV Ab-ELISA, the same results were obtained in 266 of 300 samples for an the agreement rate of 88.7% (Table 3).
Table 2. Intra-and inter-assay coefficient of variation (CV) obtained from assessment of 20 sera
Table 3. Comparison of PRRSV GP5-Ⅰ-ELISA with LSIR PRRSV Ab-ELISA
No evidence of cross-reactivity with known positive sera to swine viruses was observed. The results show that four samples positive serologically for related swine viruses have no cross-reactivity. All tested sera gave values below the deﬁned cut-off point (Table 4).
Table 4. Results of cross-reactivity test