Presentación de PowerPoint TMB Substrate Fit for purpose: Rapid, direct diagnosis in vesicular...

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


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


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


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


IZSLER

Appendix 75


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


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


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


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


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


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


IZSLER

Appendix 75


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


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


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


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)


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


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


IZSLER

Appendix 75


Trainings


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


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?)


IZSLER

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