Development of a blocking ELISA employing a VP1-specific monoclonal antibody for the detection of DHAV3 antibodies

Figures

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  Fig. 1. DHAV3 VLPs Production and Characterization. A: Schematic diagram illustrating the construction of DHAV3 VLPs. B-C: Electron microscopy analysis of DHAV3 VLPs (scale bar = 400 nm). (B) Purified DHAV3 VLPs produced in Sf9 cells, stained with 1 % sodium phosphotungstate, showing virus-like particles. (C) Cellular sections of DHAV3 VLPs-infected Sf9 cells. a: DHAV3 VLPs showing cytoplasmic localization. b: Baculovirus-infected Sf9 cells showing rod-shaped virus particles and intact nuclear membrane. (D) Western blot analysis using mouse polysera against DHAV3 structural proteins (VP0, VP1, VP3). a: VP0; b: VP1; c: VP3. M: marker; 1: DHAV3 VLPs; 2:Bacmid. (E) Immunofluorescence assay (IFA) demonstrating the expression of DHAV3 structural proteins (VP0, VP1, VP3) in VLPs using mouse polysera. (F) Determination of DHAV3 VLP concentration (975 μg/mL) using the BCA method.

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  Fig. 2. Analysis of DHAV3 VP1-MAbs. A-C: Analysis of rVP1 protein. A: SDS-PAGE analysis of His-tagged rVP1 protein expression. M:Marker; 1: pCold-VP1; 2: pCold-TF. B: Western blot analysis of rVP1 protein with anti-His tag antibody. M:Marker; 1:-3 pCold-VP1; 4-5: pCold-TF. C: Western blot analysis of rVP1 protein with DHAV3-positive serum. M:Marker; 1: pCold-VP1; 2: pCold-TF. D: IFA analysis of VP1 MAbs. a: VP1 MAb-2B5 with rDHAV3-Bacmid infected Sf9 cells; b: VP1 MAb-2B5 with baculovirus-Bacmid infected Sf9 cells; c: VP1 MAb-4B8 with rDHAV3-Bacmid infected Sf9 cells; d: VP1 MAb-4B8 with baculovirus-Bacmid infected Sf9 cells. E: Specificity of VP1-MAbs. a: VP1 MAb-2B5; b: VP1 MAb-4B8. M: Marker; 1: DHAV3 SD01 strain; 2: DHAV3 HBGT strain; 3: ARV; 4: DTMUV; 5: DPV; 6: RA; 7: APEC; 8: H9N2; 9: NDV; 10: FAdV; 11: DHAV1 SH strain. F: The titer of MAbs.

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  Fig. 3. Optimal conditions for the bELISA. A: Different concentrations of coating antigen DHAV3 VLPs (4, 2, 1, 0.8, 0.5, 0.2, 0.1μg/mL) and dilutions of serum samples (1:2, 1:4, 1:8, 1:16, 1:32, 1:64) were tested by checkerboard. The coating time and temperature (B) were evaluated, and the optimal time and temperature was 37°C for 2h and then 4°C overnight. The blocking buffer (C) and the optimal blocking time (D) were evaluated. 5 % BSA, 5 % skimmed milk, and 10 % bovin serum were used as the blocking buffer, and the blocking conditions were 37°C for 2h. (E) Influence of detection serum reaction time. Serum samples was diluted 1:32 in PBST, and incubated 37°C for 30, 60, 90 and 120 min, respectively. Determination of the optimal dilution ratio (F) and reaction time (G) of MAb. The MAb was diluted (1:1000–1:5000), and incubation time was 37°C for 30, 60, 90 and 120 min, respectively. (H) Optimum the developing time. TMB was incubated with 37°C for 5, 10, 15, and 20 min, respectively, and then stopped in 2 M H2SO4. The PI value was calculated, and the highest PI value was the optimal work conditions of the bELISA.

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  Fig. 4. Determination of the negative cut off. A total of 100 negative serum samples were subjected to the optimized bELISA. The percentage inhibition (PI) values for these samples were calculated, and based on statistical analysis, the cutoff value was determined to be 38.22 %. Samples with a PI value greater than 38.22 % were considered positive, while those with a PI value less than 29.18 % were considered negative. Samples with PI values between 29.18 % and 38.22 % were considered suspected and required further re-identification.

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  Fig. 5. Validation of the bELISA. (A) Specificity. The sera against ARV, DTMUV, DPV, RA, APEC, H9N2, NDV, MDPV, FAdV, DHAV1 were used to evaluate specificity of the bELISA. (B) Sensitivity. Serum samples were diluted 1:100 to 1:1000 and used the bELISA to evaluate the sensitivity.