Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

Figures

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  Figure 1. Binding activities of selected bovine scFvs toward (A) FMDV type O (O1 Manisa) and (B) FMDV type A (A22 Iraq) antigens. scFv sequences selected by phage library screening of FMDV-immunized cattle were cloned into a pDR-OriP-Fc1 vector, which allowed scFv-Fc expression in mammalian cells. Serial dilutions of purified scFv-Fc were applied onto FMDV antigen-coated 96-well Maxisorp plate. Bound scFv-Fc proteins were detected using HRP-conjugated anti-human IgG (Fc-specific). Data are shown as mean ± SD of triplicate samples.

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  Figure 2. Cross-reactivities of bovine scFvs to seven serotypes of FMDV. Inactivated FMDV antigens for seven serotypes purchased from Pirbright Institute and BSA (negative control) were coated onto a 96-well Maxisorp plate. A total of 0.1 µg/mL of purified scFv-Fc was applied onto the wells for most clones except BvA3, of which 2 µg/mL was applied. Bound scFv-Fc proteins were detected using HRP-conjugated anti-human IgG (Fc-specific). Data are shown as mean ± SD of triplicate samples.

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  Figure 3. Epitope binning of bovine scFvs by cross-competition ELISA. Inactivated FMDV type O (O1 Manisa) (A) or type A (A22 Iraq) (B) antigens were coated onto a 96-well Maxisorp plate. A total of 10 µg/mL of purified scFv-Fc was added as a competitor, followed by incubation with scFv phage. Bound scFv phages were detected using HRP-conjugated anti-M13 antibody. The data represents normalized values obtained by dividing the absorbance values by that of scFv phage binding in the absence of competitor scFv-Fc. Strong binding inhibition is also represented by the darker background in the cell.

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  Figure 4. Detection of FMDV antibodies in the serum samples from vaccinated and virus-challenged animals using bovine scFv antibody-based SPCE. (A) For type O antibody detection in the serum samples from vaccinated cattle (n = 9, ○) and FMDV (O/Anseong/SKR/2002)-challenged pigs (n = 5, ●), BvO17 and BvOA7-based SPCEs were carried out as described in Figure S3, and their performance was compared with a parallel experiment using PrioCHECK type O antibody ELISA Kit. The results were expressed as a percentage of inhibition as described in Materials and Methods. (B) For type A antibody detection in the serum samples from vaccinated cattle (n = 9, □) and FMDV (A/Yeoncheon/SKR/2017)-challenged pigs (n = 4, ■), BvA3 and BvOA7-based SPCEs were carried out as described in Figure S3, and their performance was compared with a parallel experiment using PrioCHECK type A antibody ELISA Kit.