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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

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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)
Introduction

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

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

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

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Competitive Luminex Assay
Percent inhibition
(PI)
0
75
(1-3000/12000)X100
25
(1-9000/12000)x100
Mse Ab
PE-anti-
Mse Ab
MFI
12,000
+ +
Mse Ab +
Neg serum
9,000+ +
3,000+ +
Mse Ab +
pos serum
Ag-coated beadMagPlex beadsAntigen
+

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Assay performance characteristics for FMDV 3ABC
Test Sensitivity Specificity PPV NPV
56.587 0.898 0.978 0.927 0.969
56.936 0.898 0.980 0.931 0.969
57.303 0.898 0.981 0.935 0.969
57.891 0.898 0.982 0.940 0.969
57.905 0.894 0.982 0.940 0.968
58.712 0.885 0.982 0.939 0.965
59.121 0.876 0.982 0.938 0.963
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 20 40 60 80 100 120
Sensitivity/Specificity
Percent inhibition
Sensitivity
Specificity
Neg Pos
0
20
40
60
80
100
Percentinhibition
Statistic Neg Pos
Nbr. of observations 737 273
Minimum 0 13
Maximum 71 99
Mean 29 79

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FMDV 3ABC cLIA and cELISA are comparable
cFMIA (cut off = 58) cELISA (cut off = 50)
DPI 0 3 4 5 7 14 21 28 0 3 4 5 7 14 21 28
c236 24 14 23 15 82 98 94 97 c236 0 0 0 4 90 99 97 97
c237 23 15 3 8 61 97 91 c237 0 0 0 0 79 98 100 100
c141 24 27 25 41 76 99 99 97 c141 0 0 0 0 67 103 100 100
c138 49 36 45 63 95 99 98 95 c138 13 16 25 48 98 99 101 99
c146 17 16 23 30 75 99 99 99 c146 0 0 0 0 77 100 100 99
c134 36 18 41 44 64 99 99 97 c134 0 0 0 0 22 101 100 101
c136 49 47 37 44 61 95 94 80 c136 0 23 15 14 60 101 100 100
c242 25 50 16 73 73 97 99 97 c242 5 12 8 5 55 94 97 94
cLIA (cut off = 58) cELISA (cut off = 50)
cFMIA (cut off = 58) cELISA (cut off = 50)
DPI 0 1 2 3 4 7 8 9 13 19 0 1 2 3 4 7 8 9 13 19
s991 12 31 29 24 35 38 46 66 71 61 s991 0 22 30 25 24 38 84 103 104 104
s268 33 29 23 39 38 65 81 98 98 96 S268 3 3 2 0 3 93 100 100 100 100
s804 28 22 22 15 0 43 68 86 96 95 s804 11 8 0 0 0 89 100 103 104
s68 48 56 50 46 45 87 87 85 89 S68 0 0 0 0 0 91 92 90 91
s205 20 23 28 33 11 24 68 88 86 s205 2 3 2 2 0 6 94 100 98
cLIA (cut off = 58) cELISA (cut off = 50)
cLIA (cut off = 58) cELISA (cut off = 50)
DPI 0 2 3 4 5 7 9 10 18 20 0 2 3 4 5 7 9 10 18 20
p42 22 29 49 26 31 50 97 99 p42 0 0 0 0 0 5 79 98
p3 28 26 14 27 83 95 p3 0 0 0 0 0 64
p6 22 14 19 22 31 45 72 59 p6 0 0 0 0 0 0 15 17 17
p71 45 45 42 34 32 36 37 71 p71 0 1 3 1 0 1 18 9 45
p13 25 34 25 24 27 41 31 95 93 p13 1 0 0 0 0 3 0 3 95
p45 26 20 22 25 33 21 16 24 91 84 p45 0 0 0 0 0 0 0 0 6 87
p20 14 14 15 27 26 23 24 24 83 94 p20 0 0 0 0 0 0 0 0 0 0
p7 55 37 34 31 78 93 96 p7 0 0 0 0 0 42 87

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

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Assay performance characteristics for VSV – NJ glycoprotein
Test Sensitivity Specificity PPV NPV LR+ LR-
18.903 0.930 0.945 0.808 0.982 17.038 0.0
20.221 0.930 0.948 0.816 0.982 17.984 0.0
20.960 0.930 0.951 0.825 0.982 19.042 0.0
20.999 0.930 0.954 0.833 0.982 20.233 0.0
21.387 0.919 0.954 0.832 0.979 19.980 0.0
22.020 0.907 0.954 0.830 0.976 19.727 0.0
22.254 0.907 0.957 0.839 0.977 21.042 0.0
Sensitivity/Specificity
Percent inhibition
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 10 20 30 40 50 60 70 80 90 100
Sensitivity
Specificity
0
20
40
60
80
100
Negative Positive
Percentinhibition
Statistic Neg Pos
Nbr. of observations 348 86
Minimum 0.0 2.7
Maximum 48.0 87.3
Mean 3.3 55.2

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Antibody response pattern in VSV-NJ infected animals
0
20
40
60
80
100
0 1 2 3 4 5 7 9 11 14 21 28
DPI
Percentinhibition
Cattle
0
20
40
60
80
100
0 1 2 3 4 5 6 7 14 21 28
DPI
Percentinhibition
Pigs

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cFMIA (cut off = 21) cELISA (cut 0ff = 40)
DPI 0 1 2 3 4 5 6 7 14 21 28 DPI 0 1 2 3 4 5 6 7 14 21 28
p41 0 0 0 0 0 0 3 0 28 55 60 p41 5 10 0 0 19 20 29 37 33 38 41
p42 0 0 14 0 0 0 0 0 70 68 68 p42 10 19 25 0 8 13 21 30 57 67 68
p43 0 37 0 0 0 0 0 17 42 64 57 p43 0 6 4 5 7 22 41 48 42 47 41
p44 0 0 0 0 0 0 45 5 65 p44 5 0 5 0 0 0 0 3 70 0 0
p45 0 0 0 0 0 0 0 0 3 8 p45 0 1 0 0 0 8 21 27 41 3 0
p46 0 0 0 0 0 0 0 4 21 26 p46 0 0.0 0.0 0 6 6 22 33 27 24 37
p47 12 0 0 1 25 13 8 24 23 29 p47 7 10 0 3 1 3 0 14 28 26 32
p48 0 0 10 20 14 0 50 28 69 69 69 p48 0 0 0 0 1 15 28 37 49 60 68
p49 0 7 0 2 0 12 2 6 50 66 13 p49 0 0 0 0 0 18 15 29 9 26 52
p50 0 0 0 0 0 0 0 0 12 3 9 p50 1 0 0 0 0 0 15 26 22 12 27
p51 5 2 4 13 0 0 15 32 64 70 61 p51 15 8 0 0 0 14 37 58 61 68 62
p52 0 0 0 0 6 0 0 34 58 67 p52 2 0 11 0 0 0 22 29 37 39 52
p53 0 16 0 0 0 8 43 48 63 68 p53 0 0 6 3 0 24 20 44 59 56 58
p54 0 0 20 0 0 0 0 0 24 14 p54 5 7 18 0 10 6 17 6 16 16 35
p55 0 0 0 0 0 0 14 25 52 44 p55 24 36 16 21 0 18 37 50 54 55 48
p56 0 0 0 0 0 0 14 0 63 64 52 p56 0 0 0 0 0 0 27 17 55 62 69
p57 0 23 6 0 0 0 0 15 67 76 68 p57 5 8 0 0 0 0 20 40 54 59 63
p58 0 0 0 10 0 0 28 5 25 51 26 p58 7 0 0 0 0 0 23 14 20 5 26
p59 22 20 22 45 3 9 0 0 55 59 3 p59 0 0 0 13 0 9 10 20 15 21 7
p60 11 8 0 0 0 0 15 18 31 54 45 p60 0 0 0 0 0 0 0 0 0 2 6
cLIA more sensitive than cELISA for VSV-NJ
cLIA (cut off = 21) cELISA (cut off = 40)

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

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

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

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

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THANK YOU

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From Luminexcorp.com