Development and characterisation of a fully recombinant competition ELISA for the diagnosis of foot‐and‐mouth disease virus infection

Figures

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  Figure 1. Affinity‐purified recombinant 3B and α‐3B‐AP and SEC analysis of purified α‐3B‐AP. (A) SDS PAGE analysis of purified recombinant 3B (lanes 3 and 4) and α‐3B‐AP (lanes 6 and 7) under reducing conditions. Each lane was loaded with approximately 0.5 or 0.25 μg of each respective protein. Molecular masses (kDa) are indicated against Precision Plus Protein Standards (Lane 1). (B) Chromatogram of affinity‐purified α‐3B‐AP analysed by size exclusion chromatography. Major peaks include aggregated material that eluted between 7 and 9 mL, dimeric α‐3B‐AP that eluted between 12.5 and 13.5 mL, and monomeric α‐3B‐AP that eluted between 13.5 and 15 mL. Absorbance and conductivity of eluted material are indicated on the y‐axes. System pressure, pH, and fraction numbers are also indicated.

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  Figure 2. Surface plasmon resonance analysis of 3B with α‐3B‐AP. Binding kinetics of immobilised 3B with (A) dimeric α‐3B‐AP at concentrations ranging from 0 to 640 nM or (B) monomeric α‐3B‐AP at concentrations ranging from 0 to 1.28 μM were assessed using SPR. Dissociation rates are given as SPR response (RU) versus time (s) (left panels) and the dissociation constants (KD) in each case were determined using the SPR response (RU) versus concentration (M) curves (right panels). Fluid phase samples were run in triplicate.

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  Figure 3. Chequerboard titration and analytical sensitivity of r3B cELISA. (A) Sigmoidal dose–response curve (variable slope) analysis of 3B titrated against various concentrations of α‐3B‐AP by indirect ELISA. Values shown are mean ± standard deviation of absorbance at 450 nm for each concentration of protein tested (n = 3). (B) Percentage inhibition values for positive serum serially 2‐fold diluted with negative serum (solid circles) or blocking buffer (open circles), or negative‐control bovine serum serially 2‐fold diluted with blocking buffer (solid triangles).

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  Figure 4. Receiver operating characteristic (ROC) curve analysis of r3B cELISA. (A) ROC curve analysis of results derived from 65 naïve and 227 cattle sera. Dotted lines, 95% confidence intervals. Circle (upper left corner), Youden index of 0.8111. AUC, area under the curve. (B) Plot‐versus‐criterion analysis for the r3B cELISA. The data set from (A) was used to plot the sensitivity (solid line) and specificity (dashed line) at different cut‐off values. Respective 95% confidence intervals are shown.