|
WORKPACKAGES
4.4. Work package descriptions
Workpackage 2 : Development and validation of confirmatory and new NSP-tests
PARTICIPANTS
Partners 2, 4, 5, 6, 7 and 9
Person-months per participant: 4 18 8 60 26.5 37
Start date or starting event: Month 1
OBJECTIVES
Objectives of this WP include the development and validation of new NSP-assays as confirmatory tests or as alternative primary assays in order to improve diagnostic performance.
Description of work:
- 2.1 Development and validation of confirmatory and new NSP-tests
- 2.1.1 Precise mapping of immuno-dominant regions of strategic NSPs by sequential pepscanning of sera from different species and infectious status (partner 7) (0 - 18 months). Production of screening grade multiple peptides and large-scale synthesis of peptides (by sub-contractor of partner 7). Use of selected peptides, either alone or in combinations, as antigens for immunoassays based on different principles and presentation.
- 2.1.2 Developing an indirect NSP-ELISA displaying the selected peptides, individually or in mixtures, to the reaction with sera (partners 5 and 7) (12-24 months).
- 2.1.3 Developing multispecies competition ELISAs using immuno-dominant peptides and/or recombinant NSPs as antigens and monoclonal antibodies against them as competitors (partners 4 and 7) (12 - 36 months).
- 2.1.4 Developing and full validation of a new enzymatic assay using chimeric biosensors (to be constructed, purified and biochemically characterised) based on engineered E. Coli b-galactosidases displaying selected peptides of different length (15-45 aa) spanning immuno-dominant epitopes of FMDV (partner 6).
- 2.1.5 Developing and full validation of a 3D ELISA - The recombinant 3D polyprotein will be expressed in baculovirus and monoclonal antibodies produced in order to develop ELISA tests. The 3D-ELISA will be evaluated as serotype-independent immunoassay by using sera from European and African ruminants and compared to results obtained with 3ABC ELISA and serotype-specific ELISAs (partner 9).
- 2.1.6 Developing an assay method for evaluating the potential of discriminating the polyclonal antibody responses against chimera marker vaccines - Serum derived from the cattle work in WP6 will be used to develop an assay method for discriminating the chimera vaccine antibody response with that of infected or normally vaccinated animals. A number of routes will be explored including competitive and direct methods, which will primarily be ELISA based, involving peptides, monoclonal antibodies to the A12 G-H loop region, and the chimera viruses themselves. Such development will also include consideration on the practicalities of large-scale use (partner 2).
- 2.1.7 Beside feasibility studies, analytical sensitivity and specificity of the new candidate assays will be evaluated using standard reference sera produced under WP1. More promising assays will be further investigated for the analysis of diagnostic sensitivity and specificity, using field and experimental sera achieved under other WP4, 5 and 6. Results will be compared according to the OIE validation principles and using latent Class analysis. According to the validation results the tests will be further developed as confirmatory test or primary test (partners 2, 4, 5, 6 and 7). In parallel to the above new approaches, improvements of the available NSP-ELISAs, either indirect or blocking ELISAs, will be evaluated to design confirmatory NSP-profiling ELISA (partners 2, 4, 5, 6 and 7).
- Development and full validation of serotype-specific and serotype-independent immunoassaysThe VP1 protein of each FMDV serotype will be expressed in the recombinant baculovirus system and analysed for their ability to distinguish between the seven serotypes. In parallel, part of the FMDV polyprotein (the structural P1 domain together with the 3C-protease) will be expressed in insect cells to produce virus-like particles (VLPs). The respective value of VP1 and the corresponding VLP as ELISA antigens will be studied.
Deliverables:
- D.8 - Peptides representing immunogenic regions of NSPs.
- D.10 - New indirect NSP-peptide ELISA (prototype).
- D.11 - ELISAs (indirect or competitive) using serotype-specific VP1s.
- D.15 - Multispecies competition-based NSP-ELISAs (prototype).
- D.16 - 3D ELISAs for serotype-independent antibody detection.
- D.25 - An enzymatic NSP-assay based on engineered E. coli b-galactosidase enzymatic sensor displaying immuno-dominant NSP-epitopes of FMDV.
- D.26 - Positive and negative discrimination of polyclonal antibody response against chimeric and natural virus response.
- D.27 - Validated new and confirmatory NSP assays.
- D.28 - ELISAs using FMD-VLP and comparison with the former VP1 ELISAs.
- D.50 - Reports: Scientific publications.
Milestones and expected result:
- M.7 - Identification of immuno-dominant NSP regions and selection of peptides for further steps (month 18).
- M.8 - Monoclonal antibodies produced against 3A, 3B and 3D (month 24).
- M.9 - Evaluated NSP-profiling ELISA as new or for confirmatory test (month 36).
- M.9 - Evaluated NSP-profiling ELISA as new or for confirmatory test (month 36).
- M.11 - Developed and validated ELISA for the detection of antibodies to differentiate sera from animals immunised with chimeric vaccines and sera from FMDV infected or normally vaccinated animals (month 48).
- M.12 - Developed and validated 3D ELISAs for serotype-independent antibody detection (month 36).
- M.13 - Developed and validated ELISAs (indirect or competitive) using serotype-specific VP1s (month 24).
- M.14 - Developed and validated ELISAs using FMD-VLP and comparison with the former VP1 ELISAs (month 48).
- M.15 - Evaluated diagnostic performances of most of the promising assays (month 48).
|
|