Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry

Figures

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  Figure 1. (a) Biotinylated peptides (blue) are loaded onto the streptavidin-coated Octet biosensor tip (lime) prior to dipping into antibody samples (green). (b) Amino acid sequences of GH-loop peptides relevant to this study. (c) Cartoon depicting the strategy of loading biotinylated O1 Manisa recombinant FMDV capsids (red) onto Octet biosensors.

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  Figure 2. The impact of biotinylation upon the antigenicity of recombinant capsids.

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  Figure 3. Biosensor loading with FMDV antigens. (a) BLI curves reflecting the loading of biotinylated peptide (ligand) onto the sensor surface. (b) Association and dissociation curves reflecting the interactions between Octet biosensors loaded with peptide and the D9 monoclonal antibody. Note: the 150 and 75 nM curves overlap. (c) Response levels observed when loading biosensors with different concentrations of recombinant capsid (2.5–20 µg/mL). (d) Reactivity of O1 Manisa polyclonal sera with biosensors functionalised with the different amounts of recombinant capsid.

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  Figure 4. Non-specific binding (NSB) to Octet biosensors. (a) Un-referenced BLI association/dissociation curves indicating the difference between the loaded and unloaded sensors (dark colours) highlighting NSB for three bovine sera (3204, 58 and 268). (b) Double referenced curves following the subtraction of the unloaded reference sensors. (c) Non-specific interactions in serum from sample 3204 (diluted 1:100) with unloaded sensors in kinetics buffer modified with various concentrations of Tween 20 giving a range of 0.00625–0.05%.

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  Figure 5. Measurement of specific interactions using a peptide surrogate of the GH loop. ELISA reactivity of the monoclonal antibodies D9 (a) and 2H6 (b) targeting antigenic sites 1 (GH loop) and 2, respectively, with complete recombinant capsids. ELISA reactivity of the monoclonal antibodies D9 (c) and 2H6 (d) targeting antigenic sites 1 (GH loop) and 2, respectively, with GH-loop peptides. BLI association and dissociation data for antibodies D9 and 2H6 when using the GH loop as a ligand (e,f).

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  Figure 6. Binding and avidity characteristics of FMDV seropositive bovine serum. (a) The response values (nm) observed in BLI for different sera plotted against virus neutralisation titre (VNT). (b) Neutralisation titres of sera derived from vaccinated and vaccinated/challenged animals. (c) BLI binding values for sera derived from vaccinated and vaccinated/challenged animals. (d) Dissociation rates for sera derived from vaccinated and vaccinated/challenged animals. (e) Correlation between neutralisation titre and BLI dissociation rates.