COMPETITIVE LUMINEX IMMUNOASSAYS FOR THE DETECTION OF ANTIBODIES TO FOOT-AND-MOUTH DISEASE AND VESICULAR STOMATITIS VIRUSES IN MULTIPLE SUSCEPTIBLE HOSTS Charles Nfon, Diana Lusansky, Tim Salo, Melissa Goolia, Ming Yang, Kate Hole, Tom Hunt National Centre For Foreign Animal Disease, Canadian Food Inspection Agency Winnipeg, Manitoba

Introduction FMD and vesicular stomatitis (VS) are clinically indistinguishable in cattle & swine. Laboratory tests are essential to distinguish between the two Competitive ELISAs for FMD and VS exist The cELISAs can easily be converted into competitive Luminex-based immunoassays (cLIAs)

Objective and Rationale Objective: Develop competitive Luminex-based immunoassays (cLIAs) for FMDV and VSV Luminex-based immunoassays compared to ELISAs: are fast (results within 1.5 to 2 h). require less sample volume are relatively more sensitive can be multiplexed (high throughput)

Materials and Methods For FMDV 3ABC: Recombinant 3ABC antigen MagPlex bead set 44 Mouse anti-FMDV 3B monoclonal antibody Negative sera from pigs, cattle and sheep in Canada (FMD-free country) Positive sera from experimentally infected pigs, cattle and sheep

Materials and Methods For VSV New Jersey (VSV-NJ) Recombinant VSV-NJ glycoprotein MagPlex bead set 46 Mouse polyclonal anti- VSV-NJ antibodies Negative sera from pigs and cattle in Canada (VSV-free country) Positive sera from experimentally infected pigs and cattle

Percent inhibition (PI) Competitive Luminex Assay Ag-coated bead MagPlex beads Antigen + Mse Ab PE-anti- MFI 12,000 + Y Percent inhibition (PI) 75 (1-3000/12000)X100 25 (1-9000/12000)x100 3,000 + Y Mse Ab + pos serum Mse Ab + Neg serum 9,000 + Y

Assay performance characteristics for FMDV 3ABC 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 20 40 60 80 100 120 Sensitivity / Specificity Percent inhibition Sensitivity Specificity Neg Pos 20 40 60 80 100 Percent inhibition

FMDV 3ABC cLIA and cELISA are comparable cLIA (cut off = 58) cELISA (cut off = 50) cLIA (cut off = 58) cELISA (cut off = 50) cLIA (cut off = 58) cELISA (cut off = 50)

Summary for cLIA for FMDV 3ABC Successful development of a cLIA for detection of antibodies to FMDV 3ABC in porcine, bovine and ovine serum Sensitivity 90% and specificity 98% Comparable or slightly more sensitive than a cELISA

Assay performance characteristics for VSV – NJ glycoprotein Sensitivity / Specificity Percent inhibition 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 10 20 30 40 50 60 70 80 90 100 Sensitivity Specificity 20 40 60 80 100 Negative Positive Percent inhibition

Antibody response pattern in VSV-NJ infected animals 20 40 60 80 100 1 2 3 4 5 6 7 14 21 28 DPI Percent inhibition Pigs 20 40 60 80 100 1 2 3 4 5 7 9 11 14 21 28 DPI Percent inhibition Cattle

cLIA more sensitive than cELISA for VSV-NJ cLIA (cut off = 21) cELISA (cut off = 40)

Summary for VSV – NJ glycoprotein cLIA Successful development of a cLIA for detection of antibodies to VSV in porcine and bovine serum Sensitivity 93% and specificity 95.4% cFMIA more sensitive than a cELISA

Conclusion Highly sensitive competitive Luminex-based immunoassays for detection of antibodies to FMDV and VSV are feasible These assays can be multiplexed to simultaneously to obtain rapid results for multiple analytes in a single test

Next steps Further validation of the cLIAs Development of a similar assay for VSV-Indiana serotype Combining the 3 cLIAs into a multiplex assay

Acknowledgements NCFAD Animal Care Staff Amber Papineau for technical support Funding provided by CFIA In-kind support provided by BioVet Inc, Canada

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