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READY-TO-USE KITS FOR THE DETECTION OF ANTIBODY TO FMDV SEROTYPES SAT1 and SAT2
G. Dho1, S. Grazioli1, M. Bugnetti1, G. Pezzoni1, F.F Maree2, J. Esterhuysen2, M. Chitray2, K. Scott2 and E. Brocchi1
1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy 2 Onderstepoort Veterinary Institute, Transboundary Animal Diseases Programme, Private Bag X05, Onderstepoort 0110, South Africa
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Objective: Complete the portfolio of ELISA kits for FMDV antibody detection
AVAILABLE: Kits for Ab to FMDV serotypes O, A, Asia 1 (Open Session 2012)
NEW: Kits for Ab to FMDV serotypes SAT1 and SAT2 Product: ready-to-use ELISA kits
User-friendly Limited assay steps Stability Robustness No need of sophisticated equipment
Test Principle: MAb-based Solid Phase Competitive ELISAs
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Single protocol for the five SP-ELISAs Serotypes O, A, Asia1, SAT1 and SAT2
Test is fast - 2.5 h - and simple
− Plates provided pre-coated with FMDV inactivated antigens trapped by capture mAbs
− Only two incubation steps (sera & conjugated MAb) at RT
IZSLER “ready to use” ELISA kits for FMDV anti-SP Ab
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Feasibility study
Selection of most suited MAbs, both for resistance to the stabilization process and for test performance
Stabilization process causes degradation of capsids in 12S-like particles
Evaluation of diagnostic performances
ROC analyses Threshold
Diagnostic Specificity
Diagnostic Sensitivity
Development and Validation process for the SAT1 and SAT2 Ab kits
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ROC curve: Kit for Ab to FMDV SAT1
2301 negative sera (naïve) (cattle, buffalo and small ruminant)
93 sera from exp. infected cattle (sequential samples from two animals)
4 cattle - 21 dpv and 28 dpv
2 cattle - 28 dpv
162 positive field cattle (Zimbabwe, herds C, D, E - positive to VNT)
100-specificity
sen
siti
vity
AUC = 0,986 P <0,0001
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100-specificity
sen
siti
vity
AUC = 0,986 P <0,0001
Sample size : 2567 Positive group : 266 Negative group : 2301
Threshold Se Sp
≥ 50% 98.1 94.4
> 57% 97.4 97.7
≥ 60% 96.6 97.8
≥ 70% 92.1 99.1
Selected threshold → ≥ 60%
Sensitivity → 96.6 (95% CI 93.7-98.4)
Specificity → 97.8 (95% CI 97.1-98.3)
Note: results of the ELISA kit were compared with those of VNT or LPBE obtained against homologous strains
ROC curve: Kit for Ab to FMDV SAT1
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ROC curve: Kit for Ab to FMDV SAT2
30 exp. Vaccinated cattle (SA) (21, 42 dpv, 28 days post-second vacc.)
10 buffalo (SA) (outbreak in 2008)
30 cattle - 36 dpv (Libya)
29 reference sera (SA) (pi and pv cattle)
272 field positive cattle (SA) (positive to LPBE)
237 field positive cattle (Zimbabwe) (herds A,B,C - positive to VNT)
2790 negative sera (cattle, buffalo and small ruminant)
100-specificity
sen
siti
vity
AUC= 0,954 P<0,0001
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100-specificity
sen
siti
vity
AUC= 0,954 P<0,0001
Threshold Se Sp
≥ 60% 91.4 89.0
> 66 % 88.1 96.4
≥ 70% 85.9 98.0
Selected threshold → ≥ 70% Sensitivity → 85.9 (95% CI 82.9-88.5) Specificity → 98 (95% CI 97.4-98.5)
Sample size : 3458 Positive group : 636 Negative group : 2822
ROC curve: Kit for Ab to FMDV SAT2
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Comparative results of SAT2-Ab kit and LPBE post-outbreak sera
+ -
+ 202 43
- 70 101KIT
LPBESAT 2
+ -
+ 232 4
- 0 1KIT
SAT 2VNT
416 field samples (South Africa)
Seroprevalence rate:
SAT-2 Ab kit 59% LPBE 65%
Concordance 0.728
237 field samples (Zimbabwe)
Seroprevalence rate:
SAT-2 Ab kit 98% VNT 99.5%
Concordance 0.983
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N. c
attl
e
0 dpv 2 dpv 4 dpv 7 dpv 9 dpv 11 dpv 14 dpv 21 dpv 28 dpv 42 dpv 56 dpv 70 dpv
Comparative results of SAT2-Ab kit and LPBE sequential samples from 30 experim. vaccinated cattle
Booster vaccination
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The availability of new ready-to-use ELISAs kits for detection of Ab to FMDV type SAT 1 and SAT 2 fills a gap in the spectrum of FMD diagnostic tools
The ready-to-use kits contribute to standardization and harmonization of results obtained in different laboratories
The validation process proved evidence of adequate or at least acceptable diagnostic performances.
The SAT-2 Ab kit was applied and evaluated in Ondesterpoort Lab proving satisfactory robustness
Conclusion
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Analysis of serotype-specificity
Investigations on how the antigenic divergence between antigens in the kits and FMD viruses eliciting Ab in the field may affect sensitivity
Identification of a protective threshold
Investigation whether different thresholds may be needed for different vaccine strains
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Thanks for the attention
IZSLER site - painting by L. Bozzetti 1954 IZSLER site, aerial photograph, ’90s