Appendix 75
Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain 1
New ELISAs for FMD diagnosis Emiliana Brocchi
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna
Brescia, ITALY
with collaboration of WRL
IZSLER
Open Session Vienna 2010 - Lecture Wilna Vosloo The changing landscape of diagnostics
“Progress/developments of ‘old’ ideas”
“Standardisation, Quality, Validation, Harmonisation”
EuFMD Open Session 2012
IZSLER
Why do we need “new generation” ELISA ? Robust & simple diagnostic kits are essential complements of the measures
for disease prevention and control
Particularly crucial in endemic countries adopting PCP
Emergencies, Diagnostics Bank ?
Limited availability for FMD diagnosis
restrictions in handling of FMD viruses
not easy accessibility to samples suited for assay validation
Properties
User-friendly Limited assay steps
Stability Robustness No need of sophisticated equipment
EuFMD Open Session 2012
IZSLER
Objectives
Develop “new generation” ready-to-use ELISA kits For FMDV antigen detection / serotyping
MAb based - sandwich ELISA
Lateral flow devices (LFDs)
For antibody detection
Ab to FMDV serotypes O, A, Asia 1 (SP antibody)
MAb based - Solid Phase Competitive ELISAs
Ab to FMDV NSP = already available commercially
EuFMD Open Session 2012
IZSLER
FMDV Antigen detection & serotyping Test purposes Direct testing of lesion materials − Vesicular epithelium or fluid
Indirect testing of all samples after amplification in cell cultures
Traditional ELISA format Serotype specific reagents for double-sandwich ELISA − Rabbit polyclonal capture antibody
- Guinea Pig polyclonal indicator antibody (+anti-GP IgG conjugate) - Control antigens (inactivated) supplied separately - Four incubation steps
EuFMD Open Session 2012
IZSLER
READY-TO-USE ELISA KIT for FMDV ANTIGEN DETECTION AND SEROTYPING
Simple sandwich ELISA based on Monoclonal Antibodies
Serotype-specific MAbs coated to microplate wells as catching antibodies Captured antigens detected by peroxidase-conjugated, cross-reactive MAbs
Detector MAb 1h RT
Target Ag 1h RT
Catching MAb ready-to-use
HRP HRP TMB Substrate
Fit for purpose: Rapid, direct diagnosis in vesicular epithelium or fluid
Identification of viruses isolated in cell cultures
EuFMD Open Session 2012
IZSLER
Appendix 75
Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain 2
Selection of typing MAbs (catching Abs) criterion: widest intra-type reactivity
FMDV N. isolates Serotype tested
Type A 130
Period
covered
1943
Final selection of
catching MAbs MAbs 4D12 & 5F6
N. isolates
2010
122
112
reactive
Type O 108
1950 2010
MAbs N. isolates
reactive
3B11 105
Type
Asia 1
53
MAbs 1954 N. isolates 2009 reactive
3D8 53
Type C 33
'60s
2005
MAbs N. isolates
reactive
3E9 32
Type
SAT 1
32
MAbs 1948 N. isolates 2009 reactive
HD7 + FC12 29 28
Type
45 SAT 2
MAbs 2H6 + 1F5
1948 N. isolates
2009 45 45 reactive
EuFMD Open Session 2012
IZSLER
WESTEURASIA Serotyping O, A, Asia 1, C
The test is fast - 2.5 hours - and simple: Microplates pre-coated with type-specific MAbs
Positive control antigens already incorporated into
corresponding wells
The operator handles a unique or at most two
immunological reagents, corresponding to cross-
reactive detector conjugates
Kit shelf-life estimated ≥ 1 years at 5°C Up to six samples can be tested in each plate
Layout flexible
AFRICA Serotyping O, A, SAT 1 , SAT 2
EuFMD Open Session 2012
IZSLER
FMDV Serotyping kit for WestEurasia - types O, A, Asia 1 & C Summary of diagnostic performances (Vienna 2010)
Sensitivity
FMDV
types
Type-specific MAbs-based ELISAs
N. POS Sens.
Polyclonal ELISA
POS Sens.
Pan-FMD ELISA
based on MAb 1F10
POS Sens.
O
136
116
85%
129
95%
118
87% A
103
67
65%
41
40%
67
65% Asia
30
22
76%
26
90%
21
72% C
29
19
66%
20
69%
20
69% TOTAL
Epithelium
298
224
75%
216
72%
226
76% suspensions
Specificity Epithelium suspensions Positive for O, A, Asia 1 and C cross-checked in heterologous tests
Cross-reaction of some FMDV isolates of type O with one type A catching Mab N. 24 SATs and SVDV + Negative epithelium suspensions
ALL NEGATIVE
EuFMD Open Session 2012
IZSLER
FIELD EVALUATION FMDV Serotyping kit for WestEurasia
Types O, A, C, Asia 1
External evaluation
Demonstration/Trainings
Supply (FAO & EuFMD programs)
FMDV Serotyping kit for AFRICA Types O, A, SAT1, SAT2
Initial feedback from 4 African Countries
The kit identified in vesicular epithelium samples
N. 12 samples pos type O N. 1 sample pos type A N. 7 samples pos type SAT1 N. 29 samples pos type SAT2 N. 2 samples pos types SAT2 & O
EuFMD Open Session 2012
IZSLER
FMDV Antigen detection - Penside tests Lateral Flow Devices
Results in 10-20 minutes In use or evaluated in field & laboratory
Pan-FMDV LFD
SAT2-specific LFD
Asia 1-specific LFD
Based on same catching and detector
MAbs of ELISA kits
Under development Type specific LFDs for missing serotypes
Multiplex serotyping in one strip
EuFMD Open Session 2012
IZSLER
FMDV antigen detection by Multiplexing testing system
LUMINEX TECHNOLOGY (POSTER)
Proof of concept: 7 different microspheres coupled with 7 different MAbs mixed for
simultaneous analyses of FMDV serotypes O, A, Asia1, C and SAT1 in a single
reaction
Represents an additional, flexible, though sophisticated tool, for FMD diagnosis Potential to increase the number of simultaneous analyses against many more MAbs-
coupled microspheres (up to 100) makes the test appropriate for accurate serotyping
and antigenic profiling
EuFMD Open Session 2012
IZSLER
Appendix 75
Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain 3
ELISAs for FMDV serotype specific serology
SP-Ab Test purposes
Diagnosis of suspect cases Estimating prevalence and substantiating freedom
Trade certification
Estimating vaccine efficacy and population immunity Current ELISA formats Serotype specific reagents for LPBE or SPCE − Rabbit polyclonal capture antibody
- Guinea pig polyclonal indicator antibody (+anti-GP IgG conjugate) - FMDV antigens (inactivated) - Five incubation steps
EuFMD Open Session 2012
IZSLER
“Ready to use” ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1
Three prototypes kits developed and evaluated
Single protocol for the three SP-ELISAs
Test is fast - 2.5 h - and simple
− Plates provided pre-coated with FMDV inactivated antigens − Only two incubation steps (sera & conjugated MAb) at RT
EuFMD Open Session 2012
IZSLER
NEW ready to use ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1
derived from in-house MAbs based SPC-ELISA
preliminary work
Conditions for stabilization of FMDV antigens trapped by homologous MAbs
>1 year the shelf-life of plates sensitized with FMDV captured by MAbs
Conditions for stabilization of conjugated MAbs (competition phase) shelf-life ranging from 1,5 to 3 years
Drawback : FMDV antigens tend to degrade to 12S during the stabilization
process need of initial excess of reagents
EuFMD Open Session 2012
IZSLER
NEW ready to use ELISA kits for SP-Ab Diagnostic SPECIFICITY
SP-type O Ab ELISA kit
SP-type A Ab ELISA kit
SP-type Asia1 Ab ELISA kit
Percentage inhibition
99.8% for SP-O Ab kit (4 false-positive in 2000 sera)
100% for SP-A Ab kit (1621 sera tested)
99.8% for SP-Asia1 Ab kit (5 false-positive in 2481 sera)
EuFMD Open Session 2012
IZSLER
NEW ready to use ELISA kits for SP-Ab Diagnostic SENSITIVITY
full dose vaccinated cattle (vaccine potency tests)
SP-ELISAs vs homologous VNT
Kit ELISA
EXTERNAL VALIDATION - WRL
vaccinated and/or infected animals positive to homologous VNT
Type O Bovine Ovine Porcine Total
Type O
Pos/Db VNT
Neg
Type A
VNT
Pos/Db Neg
89 11
3 6
Kit ELISA
Pos Neg
SP-ELISA O 85% VNT 92%
Concordance 0.872
SP-ELISA A 90%
VNT 96%
n.pos/n.tested 155/165 56/71 25/25 236/261 Sensitivity 94% 79% 100% 90%
Type A Bovine Ovine Porcine Total
n.pos/n.tested 163/196 // // 163/196 Pos/Db
or LPBE Neg
Type Asia1
Pos
42 5 Concordance 0.857
2 0
Kit ELISA
SP-ELISA Asia1 100% Pos Neg VNT 100%
20 0
Sensitivity 83% // // 83%
Type Asia1 Bovine Ovine Porcine Total n.pos/n.tested 49/49 4/4 // 53/53 Sensitivity 100% // // 100%
LPBE Neg 0 0
Concordance 1
EuFMD Open Session 2012
IZSLER
Ready-to-use ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1
CURRENT PROTOTYPES: adequate diagnostic performances, irrespective of the
strains that elicited antibodies
Possible improvement: use of recombinant capsid particles
EuFMD Open Session 2012
IZSLER
Appendix 75
Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain 4
Trainings
EuFMD Open Session 2012
Other perspectives for FMD serology
Multiplex immunoassays (LUMINEX technology) - Could enable profiling of humoral immunity in a single reaction
(Garnier et al., POSTER)
Pen-side tests (LFD) − For NSP Ab (Gonzales et al., Dho et al., POSTERs)
− For SP Ab
Conclusions FMDV ANTIGEN DETECTION & SEROTYPING
ELISA kit suited for EURASIA validated and available ELISA kit tailored for AFRICA: prototype under evaluation LFD for pan-FMD detection (O, A, C, Asia1 + some SAT2) in use
LFDs SAT2-specific and Asia1-specific launched
TO FOLLOW SAT3 typing still missing
Typing 6-7 serotypes in one ELISA kit (new plate layout) ? Complete serotype-specific LFDs and develop multiplex LFDs FMDV ANTIBODY DETECTION & SEROTYPING ELISA kits for SP-Ab to FMDV serotypes O, A, Asia 1 validated
TO FOLLOW Improve scale production for larger availability
ELISA kits for SATs-specific Ab (under development)
Recombinant virus particles
EuFMD Open Session 2012
IZSLER
Acknowledgements
IZSLER - Brescia Development & Production
• Santina Grazioli
• Franco de Simone
• Giovanna Dh0
• Edoardo Spagnoli
• Massimiliano Bugatti
WRL - Pirbright Collaboration & Validation
• Nigel Ferris
• Yanmin Li
• David Paton
EUFMD Consultant Feedback from trainings and evaluation in AFRICA • Kees Van Maanen
Discussion points
Antigen detection & serotyping ELISA New ready-to-use kit:
• Sensitivity similar to polyclonal ELISA • Better type-specificity • User-friendly
Pros: easier than RT-PCR, enables serotyping
Limit: sensitivity (lower than RT-PCR)
Serotype specific-Ab detection
Limit: • Imperfect type-specificity
• A different assay per each desired serotype
Kits expiring date − Usually kits can be used longer after expiration (check controls?)
EuFMD Open Session 2012
IZSLER