Development of a Competitive ELISA for Detecting Antibodies Against Pseudorabies Virus Glycoprotein D

Figure 1. Schematic representation of the competitive enzyme-linked immunosorbent assay (cELISA) method. The schematic illustrates the principle of cELISA: antigen coating, blocking, simultaneous addition of test serum and enzyme-labeled monoclonal antibody (mAb), competition for binding sites, and detection. Created in BioRender. Li, L. (2025) https://BioRender.com/ozel05q.

Figure 2. Purification and characterization of PRV gD protein and production of monoclonal antibodies. (a) SDS-PAGE analysis of purified gD protein. gD protein purified via Ni-NTA affinity chromatography from the supernatant of HEK 293F cells stably expressing pB513B-gD; NC: supernatant from untransfected HEK 293F cells. (b) Western blot analysis of purified gD protein. (B1) Probing with mouse anti-Flag primary antibody (1:5000 dilution). (B2) Probing with serum from a gD-immunized mouse (1:2000 dilution). (c) Serum antibody titers of mice following three immunizations with gD protein, determined by direct ELISA. Mouse 1–5: mice immunized with gD protein; control: negative control mouse. (d) Screening of hybridoma clones stably secreting gD-specific antibodies by direct ELISA (representative clone 1D11 shown). The S/N ratio was calculated as the OD450 value of the sample well (S) divided by the OD450 nm of the SP2/0 supernatant control well (N). Samples with S/N > 2.1 were considered positive.

Figure 3. Characterization of monoclonal antibody reactivity. HEK 293T cells were transfected with plasmid pB513B-gD encoding the PRV gD ectodomain (aa 1–355). Reactivity of 1D11 hybridoma supernatant with gD protein was assessed by immunofluorescence assay (IFA) (a) and western blotting (b). Mouse anti-Flag antibody served as a positive control. SP2/0 supernatant served as a negative control. Mouse anti-GAPDH antibody served as a loading control. Vero E6 cells were infected with PRV HeN1 strain at an MOI of 0.01. Reactivity of 1D11 hybridoma supernatant with virus-infected cells was assessed by IFA (c) and WB (d). SP2/0 supernatant served as a negative control. Mouse anti-GAPDH antibody served as a loading control.

Figure 4. Broad reactivity of the monoclonal antibody with different PRV strains. (a) Amino acid sequence alignment of the gD protein from 34 different PRV strains analyzed using MegAlign software, demonstrating high conservation within 25 variant strains. (b) Vero E6 cells were infected with PRV strains HeN1, TJ, SC, or Bartha K61 at an MOI of 0.01. Reactivity of 1D11 with the different PRV strains was assessed by IFA.

Figure 5. Purification of the monoclonal antibody and assessment of its competitive activity. (a) Purification of mAb 1D11 using Protein A/G magnetic beads. M, protein molecular weight marker; R1 and R2, purified mAb under denaturing and nondenaturing electrophoresis conditions, respectively. (b) Evaluation of HRP conjugation efficiency to mAb 1D11 by direct ELISA. PRV gB protein-coated wells served as a negative control. (c) Assessment of the competitive activity of mAb 1D11-HRP using cELISA. Four PRV-positive swine serum samples (#038, #059, #075, and #033) and three PRV-negative swine serum samples (#9232, #L004, and #306) were tested.

Figure 6. Receiver operating characteristic (ROC) analysis for determining the cut-off value using positive and negative serum samples. (a) Interactive dot diagram of cELISA results. The Y-axis represents the percentage inhibition (PI) values for 94 negative and 110 positive serum samples. The X-axis represents the detection results from the IDEXX commercial kit (1, positive; 0, negative). (b) ROC curve analysis for the cELISA. The area under the curve (AUC) was 0.995 (95% confidence interval [CI]: 0.973–1.000, p < 0.001). The diagnostic sensitivity and specificity of the cELISA were 96.4% and 97.9%, respectively, with an optimal cut-off value of 46.1564% PI.

Figure 7. Analysis of the specificity and sensitivity of the cELISA method. (a) Specificity analysis of the mAb 1D11-based cELISA. (b) Sensitivity analysis of the cELISA. The dashed line indicates the established cutoff value of 46.1564% PI.