Use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease

Abstract

Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed by using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, United Kingdom. This serum panel has been used to assess the performance of other singleplex enzyme-linked immunosorbent assay (ELISA)-based nonstructural protein antibody assays. The 3ABC signature in the multiplexed assay showed performance comparable to that of a commercially available nonstructural protein 3ABC ELISA (Cedi test), and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex was acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promote further assay development and optimization to generate an assay for routine use in foot-and-mouth disease serological surveillance.

FIG. 1.

Schematic of bead-based, multiplexed NSP antibody (Ab) assay. A peptide or NSP is covalently conjugated to a Luminex bead. The peptide or recombinant protein captures antibodies to NSPs in serum samples from cattle infected with FMDV. The captured antibodies are subsequently detected by a secondary biotinylated detector antibody, followed by a fluorescent reporter molecule. The complex is analyzed in a flow cytometer. The beads are tested one at a time. A classification laser (635 nm) excites the dye molecules inside the bead and classifies the bead to its unique bead set. A reporter laser (532 nm) excites bound fluorescent reporter and quantifies the assay at the bead surface—only those beads labeled with a reporter molecule will fluoresce in yellow, and the signal is proportional to the captured-antibody concentration.

FIG. 2.

Responses of each signature in a multiplexed assay to a challenge with sera from 104 FMD-naive cattle to illustrate the expected variation in a naive population. Responses of assay beads are reported as MFI of the assay bead normalized by using the MFI of the NC BSA-coated bead in each sample. All signatures are shown on the same scale for visual comparison. Positive bars indicate a normalized MFI value above the cutoff. Cutoffs were determined from this naive population to give 95 to 97% specificity. Negative bars indicate a normalized MFI value below the cutoff. (a) 3A peptide, 97% specificity cutoff. (b) 3B peptide, 95% specificity cutoff. (c) 3D peptide, 97% specificity cutoff. (d) 3ABC protein, 95% specificity cutoff.

FIG. 3.

Responses of each signature in a multiplexed assay to a challenge with sera from 94 vaccinated cattle to illustrate the expected response of a vaccinated population. Black bars are responses to sera from cattle at 14 dpv; gray bars are responses to sera from cattle at 21 dpv. For a list of the vaccine strains used, see the supplemental material. Responses of assay beads are reported as the MFI of the assay bead normalized by using the MFI of the NC BSA-coated bead in each sample. All signatures are shown on the same scale for visual comparison. Positive bars indicate a normalized MFI value above the cutoff. Cutoffs were determined from a naive population to give 95 to 97% specificity. Negative bars indicate a normalized MFI value below the cutoff. (a) 3A peptide, 97% specificity cutoff. (b) 3B peptide, 95% specificity cutoff. (c) 3D peptide, 97% specificity cutoff. (d) 3ABC protein, 95% specificity cutoff.