Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies

Figure 1. Identification of pseudorabies virus (PRV) gE and gB proteins from the pMAL-c5x plasmid via SDS-PAGE. (A) M, molecular weight marker; lane 1, untreated E. coli pMAL-c5x; lane 2, E. coli pMAL-c5x after induction by IPTG; lane 3, untreated E. coli pMAL-c5x PRV gE; lane 4, precipitate of E. coli pMAL-c5x PRV gE after induction by IPTG and ultrasonic disruption; lane 5, supernatant of E. coli pMAL-c5x PRV gE after induction by IPTG and ultrasonic disruption. (B) M, molecular weight marker; lane 1, untreated E. coli pMAL-c5x; lane 2, E. coli pMAL-c5x after induction by IPTG; lane 3, untreated E. coli pMAL-c5x PRV gB; lane 4, precipitate of E. coli pMAL-c5x PRV gB after induction by IPTG; lane 5, supernatant of E. coli pMAL-c5x PRV gB after induction by IPTG and ultrasonic disruption; lane 6, precipitate of E. coli pMAL-c5x PRV gB after induction by IPTG and ultrasonic disruption.

Figure 2. Identification of PRV gE and gB proteins from the pMAL-c5x plasmid via immunoblotting. (A) Detection of PRV gE by His tagging. M, molecular weight marker; lane 1, supernatant of E. coli pMAL-c5x PRV gE after induction by IPTG and ultrasonic disruption. (B) Detection of PRV gE by MBP (Maltose binding protein) tagging. M, molecular weight marker; lane 1, supernatant of E. coli pMAL-c5x PRV gE after induction by IPTG and ultrasonic disruption. (C) Detection of PRV gB by His tagging. M, molecular weight marker; lane 1, supernatant of E. coli pMAL-c5x PRV gB after induction by IPTG and ultrasonic disruption. (D) Detection of PRV gB by MBP tagging. M, molecular weight marker; lane 1, supernatant of E. coli pMAL-c5x PRV gB after induction by IPTG and ultrasonic disruption.

Figure 3. Identification of PRV gE and gB recombinant proteins after purification by SDS-PAGE. (A) Purification of PRV gE recombinant protein washed with different concentrations of imidazole solution and analyzed by SDS-PAGE. M, molecular weight marker; lane 1, recombinant PRV gE eluted with 80 mM imidazole solution; lane 2, recombinant PRV gE eluted with 100 mM imidazole solution; lane 3, recombinant PRV gE eluted with 200 mM imidazole solution; lane 4, recombinant PRV gE eluted with 300 mM imidazole solution; lane 5, recombinant PRV gE eluted with 400 mM imidazole solution; lane 6, recombinant PRV gE eluted with 500 mM imidazole solution. (B) Purification of PRV gB recombinant protein washed with different concentrations of imidazole solution and analyzed by SDS-PAGE. M, molecular weight marker; lane 1, supernatant of recombinant PRV gB after induction by IPTG and ultrasonic disruption; lane 2, recombinant PRV gB eluted with 100 mM imidazole solution; lanes 3 and 4, recombinant PRV gB eluted with 20 mM imidazole solution; lanes 5 and 6, recombinant PRV gB eluted with 40 mM imidazole solution; lanes 7 and 8, recombinant PRV gB eluted with 60 mM imidazole solution; lane 9, recombinant PRV…

Figure 4. Antigenicity verification of PRV gE and gB recombinant proteins. PRV gE recombinant proteins were detected via immunoblotting using: (A) PRV gE-positive serum and (B) pig negative serum as the antibodies. PRV gB recombinant proteins were detected via immunoblotting using (C) PRV gB-positive serum and (D) pig negative serum as the antibodies.

Figure 5. Median fluorescent intensity (MFI) value for PRV gE/gB positive and negative sera: (A) the MFI value of the fluorescent-microsphere immunoassays (FMIAs) was determined to test the PRV gE, and gB positive/negative sera were detected for feasibility analysis. (B,C) Samples were diluted 10-, 20-, 40-, 80-, 160-, and 320-fold using PBS, and the MFI value of FMIA was determined. At the same time, the optimal serum dilution was determined.

Figure 6. Antigenic cross-reactivity detection by dual FMIA: (A) the MFI value using the PRV gE IgG method to detect gE-positive serum was >20,000, while other common pig pathogens and negative serum had low MFI values; and (B) the MFI value using the PRV gB IgG method to detect gB-positive serum was >18,000, while other common pig pathogens and negative serum had low MFI values. PCV (porcine circovirus), CSFV (classical swine fever virus), PRRSV (Porcine Reproductive and Respiratory syndrome virus).

Figure 7. The optimal concentration of the recombinant protein was determined: (A) the determination of the optimal concentration of gB recombinant protein coupled with microspheres; (B) the determination of the optimal concentration of gE recombinant protein coupled with microspheres.

Figure 8. Evaluation of the dual FMIA detection methods: the comparison of MFI values detected from single and multiplex assays for (A) the PRV gE IgG detection and (B) the PRV gB IgG detection.

Figure 9. Statistical analysis of the PRV gE and gB FMIA results. Reactivity of the PRV gE sera according to (A) the receiver operating characteristic (ROC) curve analysis with (B) the criterion value (MFI > 5991.5 is positive and MFI < 5991.5 is negative) of the assay (5991.5) denoted by *. Reactivity of the PRV gB sera according to (C) the ROC curve analysis with (D) the criterion value of the assay (2862) denoted by *. We used the SigmaPlot 10.0 for the analysis of the sensitivity and specificity of the assay. +LR is positive likelihood ratio; −LR is negative likelihood ratio.

Figure 10. Statistical analysis of PRV gB and gE ELISA results. Reactivity of the PRV gB sera according to (A) the ROC curve analysis with (B) the criterion value (OD > 0.399 is positive and OD < 0.399 is negative) of the assay (0.399) denoted by *. Reactivity of the PRV gE sera according to (C) the ROC curve analysis with (D) the criterion value of the assay (0.324) denoted by *. The results showed that the cut-off value of gB ELISA was 0.399, the sensitivity was 95.83%, and the specificity was 91.37% (Figure 10B); the critical value of gE ELISA was 0.3191, the sensitivity was 91.7%, and the specificity was 88.89% (Figure 10D). We used SigmaPlot 10.0 for the analysis of the sensitivity and specificity of the assay. +LR is positive likelihood ratio; −LR is negative likelihood ratio.