Detection of Bovine Antibodies against a Conserved Capsid Epitope as the Basis of a Novel Universal Serological Test for Foot-and-Mouth Disease

FIG 1. FMDV heterotypic-reactive MAbs recognize the N terminus of VP2. (a) Overlapping peptides representing 45 VP2 N-terminal amino acids. “(K)6” denotes the addition of 6 lysine residues at the C terminus of the peptide to increase peptide solubility. (b) Peptide ELISA showing cross-reactive MAbs recognize peptides equivalent to the 15 (N15), 30 (N30), and 45 (N45) N-terminal amino acids of FMDV VP2. The 45 N-terminal amino acids of human rhinovirus VP4 (HRV-VP4) was used as a negative control; the concentration of peptides was 2 μg/ml. (c) MAb 4A3 epitope mapping (using peptides shown in panel a) identifies the cross-reactive epitope at the N terminus of VP2. (d) Reactivity of MAb 4A3 with capsid protein VP2 of all 7 serotypes in Western blotting. The 4A3 MAb produced a clearly intense band for VP2 and a weaker reaction for VP0. (e) Immunofluorescence microscopy using MAb 4A3 to detect FMDV serotype O-infected IBRS-2 cells.

FIG 2. Sera from animals infected with any serotype of FMDV reacted with VP2 peptides. (a) Reactivity of serum from an animal experimentally infected with FMDV serotype O with peptides equivalent to the N-terminal 15 (N15), 30 (N30) or 45 (N45) amino acids of FMDV VP2, or the 45 N-terminal amino acids of human rhinovirus VP4 (HRV-VP4, negative control). (b) Reactivity of sera from animals vaccinated with vaccine strains of the seven serotypes with the FMDV VP2N-45 peptide.

FIG 3. Testing reference negative and positive serum samples to determine the specificity and sensitivity of the assay. (a) Reactivity in VP2 ELISA (OD at 490 nm) of negative (Neg; black squares, n = 100) and positive (circles represent infections [inf], triangles represent vaccinations [vac], serotypes are represented by colors as indicated; n = 72) reference sera. Peptide was used at 2 μg/ml, and sera were diluted 1 in 100. (b) Distribution plots comparing results of positive sera tested with VP2 ELISA (same as data in panel a; cutoff 0.4 OD) and with homologous (n = 37) and heterologous (n = 72) VNT (cutoff = log titer 1.65). Each sample was tested in triplicate, and at least two independent experiments were performed. (c) Distribution plots comparing results of positive sera tested with VP2 ELISA (same as data in panel a; cutoff 0.4 OD) with homologous (n = 30) and heterologous (n = 72) LPBE (cutoff = log titer 1.95). (d) Distribution plots comparing results of positive sera tested with VP2 ELISA (same as data in panel a; cutoff 0.4 OD), with SPCE (n = 30, cutoff = 40% of inhibition) and PrioCHECK ELISA kits (n = 29, cutoff = 50% of inhibition).