1. 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

2. 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)

3. 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)

4. 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

5. 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

6. 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

7. 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

8. 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)

9. 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

10. 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

11. Antibody response pattern in VSV-NJ infected animals20 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

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

13. 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

14. 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

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

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

17. THANK YOU

19. From Luminexcorp.com