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Reference Reagents
Guidelines for the preparation, validation and distribution of WOAH-approved reference reagents for diagnosis of infectious animal diseases (updated December 2024)
The following general criteria should be met for WOAH recognition of a reference material distributed by a Reference Centre for a diagnostic assay for the detection of a pathogen or disease-specific antibodies:
1. Fit-for-purpose
Positive reference standards (PRS): the material should contain the sample or analyte targeted by the diagnostic method under focus. PRS should be, at least, at the concentration needed for detection using the standard protocols accepted by WOAH Reference Centres as described in the WOAH Terrestrial Manual.
Negative reference standards (NRS): the material should be selected from animals or any other relevant material that have never been exposed to or vaccinated against the target pathogen. NRS should be free from specific antibodies, antigens and genetic material from organisms that might cross-react with the target pathogen in the
standard assay.
To be considered useful as a diagnostic standard and to respond to requests from diagnostic labs, a minimum number of 50 aliquots should be available.
Each batch should be appropriately labelled, with proven safety (point 2), homogeneity and stability (point 3).
2. Safety
The reference standards should be prepared so that they are demonstrated to be free of infectious material using a validated procedure. A certificate of safety should be made available upon request.
Standards should be subjected to appropriate innocuity tests as described in Chapter 1.1.9 of the Terrestrial Manual to ensure that they are free from detectable live agents.
3. Homogeneity and stability
Homogeneity should be assessed for each batch of reference material by repeated testing of independent aliquots (point 5). Considering stability, this must be monitored over time. The provider must ensure correct packaging, storage and transport conditions.
4. Origin
The material should be prepared by a Reference Centre in accordance with ISO 17034 standard or at least by a laboratory accredited with ISO17025 for the target pathogen.
A technical document (datasheet) describing the origin, preparation, validation data and storage requirements of the material should be provided in order that any laboratory can evaluate if the reference material has the characteristics required and does not pose any regulatory or biosafety/biosecurity issues.
5. Use and expected results
The technical document should provide the necessary information on how to prepare the material for its final use. For example, it could include proper instructions for reconstitution, method of storage after reconstitution, storage shelf life, and dilution method to reach the expected target value (e.g. for strong positives to be used to
prepare weak positives). The document should indicate the expected value (Ct for real-time PCR, % competition for C-ELISA, etc.) with a standard deviation at the target dilution, based on tests using standard protocols accepted by WOAH Reference Centres. The average expected value and associated standard deviation should be
first calculated by having tested three intra-assay replicates of eight aliquots of the material on four different days by more than one person. The methodology to be used to determine the average expected value and associated standard deviation is left to the discretion of the Reference Centre, guided by Chapter 1.1.6. of the Terrestrial Manual but it is recommended that this information is described in the technical document.
6. Approval of Reference Standards by WOAH
An International Reference Standard may not be issued under the name of WOAH unless it has been endorsed by the Biological Standards Commission.
The full technical and statistical data on the evaluation of the candidate reference standards, together with the full information as specified above (point 1 to 5) should be submitted to WOAH. The Biological Standards Commission will review the information. If the Commission approves, the reference standard will be added to the list of International Reference Standards available. This list will be supplied to all WOAH Members on request, and will also be accessed on the WOAH website.
Currently available WOAH-approved International Standard Reagents
| Disease | Test | Available from |
|---|---|---|
| Aujeszky’s disease | Enzyme-linked immunosorbent assay; Virus neutralisation | Dr Céline Deblanc Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) Laboratoire de Ploufragan/Plouzané/Niort Unité de Virologie Immunologie Porcines B.P. 53, 22440 Ploufragan France Tel: +33 (0)2 96 01 62 05 celine.deblanc@anses.fr |
| Bovine brucellosis (WOAHISS) | Complement fixation; Agglutination; Buffered Brucella antigen tests. Bovine antiserum against Brucella abortus adjusted to 1000 IU per ampoule. International Standard antiserum for use in standardising the rose bengal (RBT), serum agglutination (SAT) and complement fixation tests (CFT). Davidson et al. (1969). The Second International Standard for anti-Brucella abortus Serum. Bull. WHO, 40, 129–140. | Dr Adrian Whatmore Animal and Plant Health Agency New Haw, Addlestone Surrey KT15 3NB, United Kingdom Tel: (+44-1932) 35.73.50 [email protected] |
| Bovine brucellosis (WOAHELISASPSS, WOAHELISAWPSS, WOAHELISANSS) | For indirect ELISA, competitive ELISA and FPA in cattle, three WOAH ELISA Standard Sera are available for use. These are of bovine origin and consist of a strong positive (WOAHELISASPSS), a weak positive (WOAHELISAWPSS) and a negative (WOAHELISANSS) standard. McGiven et al. (2006). Harmonisation of European tests for serological diagnosis of Brucella infection in bovines. Rev. Sci. Tech., 25, 1039–1053 | Dr Adrian Whatmore (as above) |
| Contagious bovine pleuropneumonia | Both irradiated and non-irradiated sera are available. Please contact the Reference Laboratory for advice on their suitability for different types of serological test (complement fixation test, indirect or competitive enzyme-linked immunosorbent assay) | Dr Massimo Scacchia Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ Via Campo Boario, 64100 Teramo, Italy Tel: (39-0861) 33.24.05 [email protected] |
| Contagious bovine pleuropneumonia | Complement fixation test | Dr Ana Rosa Pombo Botelho Instituto Nacional de Investigação Agrária e Veterinária (INIAV, IP) Av. da República, Quinta do Marquês s/n, 2780-157 Oeiras, Portugal Tel: +351-21 440 35 14 [email protected] |
| Enzootic bovine leukosis | Agar gel immunodiffusion; Enzyme-linked immunosorbent assay | Prof. Thomas Vahlenkamp Institute of Virology Centre for Infectious Diseases Faculty of Veterinary Medicine Leipzig University, An den Tierkliniken 29 04103 Leipzig, Germany Tel: (49-341) 97.38.201 [email protected] |
| Equine infectious anaemia | Agar gel immunodiffusion | Dr Jose-Carlos Valle-Casuso ANSES Goustranville, 14430 Dozulé, France Tel : 33(0) 2 31.79.79.07 [email protected] |
| Equine influenza | Haemagglutination inhibition; single radial haemolysis | Dr Marie-Emmanuelle Behr-Gross European Directorate for the Quality of Medicines, Council of Europe 7 allée Kastner, F-67081 Strasbourg Cédex, France Tel: 33 (0)3 90.21.41.08 [email protected] |
| Equine influenza | Antigens and antisera for in-vitro standardisation of vaccines | NIBSC (National Institute for Biological Standards and Control) P.O. Box 1193, Blanche Lane South Mimms, Potters Bar Hertfordshire EN6 3QH United Kingdom Tel: (+44-1707) 64.14.67 [email protected] |
| Equine viral arteritis | Virus neutralisation | Dr Feng Li Maxwell H. Gluck Equine Research Center Department of Veterinary Science University of Kentucky, Lexington Kentucky 40546-0099 United States of America Tel: (1-859) 47 57 ext. 81094 f[email protected] |
| Foot and mouth disease | Enzyme-linked immunosorbent assay (antigen and antibody detection); Virus neutralisation | Dr Donald King The Pirbright Institute Ash Road, Pirbright Woking, Surrey GU24 0NF, United Kingdom Tel: (44-1483) 23.24.41 [email protected] |
| Foot and mouth disease | Nonstructural protein tests | Dr Edviges Maristela Pituco Centro Panamericano de Fiebre Aftosa OPS/OMS Av. Governador Leonel de Moura Brizola 7778, Sao Bento Duque de Caxias, ZC 25040-004 Rio de Janeiro, Brazil Tel: (55-21) 36.61.90.64 [email protected] |
| Infectious bovine rhinotracheitis | Virus neutralisation; Enzyme-linked immunosorbent assay | Dr Stephen Valas Laboratoire de Ploufragan-Plouzané-Niort Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) 60 rue Pied de Fond, CS 28440 79024 Niort Cedex, France Tel: 33 (0)5 49.79.61.28 [email protected] |
| Ovine epididymitis Brucella ovis (ISaBoS, International Standard 1985) | Complement fixation Each ampoule contains 1000 IU | Dr Adrian Whatmore (as above) |
| Ovine and caprine brucellosis (anti-Brucella melitensis [ISaBmS]) | Indirect and competitive ELISAs; fluorescence polarisation assay (FPA) Primary International Standard antiserum for use in standardising I-ELISA and FPA for small ruminants. It may also be used to standardise the C-ELISA for use in small ruminants although the WOAH ELISA standard sera can also be used. This is not a primary reference serum for the complement fixation test (CFT), rose bengal test (RBT) or modified (m)RBT. To standardise these tests for use in small ruminants, the WOAHISS should be used. McGiven et al. (2011). The first International Standard anti-Brucella melitensis Serum. Rev. Sci. Tech.,30, 809–819. | Dr Adrian Whatmore (as above) |
| Peste des petits ruminants | Virus neutralisation | Dr Arnaud Bataille CIRAD-Bios, Campus international de Baillarguet TA A-15/G, B.P. 5035, 34398 Montpellier Cedex 15, France Tel: 33 (0)4 67 59 37 98 [email protected] |
| Rabies | Fluorescent antibody virus neutralisation | Dr Florence Cliquet Anses Nancy, Laboratorie de la rage et de la faune sauvage Technopôle Agricole et Vétérinaire Bâtiment H, CS 40009, 54220 Malzéville, France Tel: 33 (0)3 83.29.89.50 [email protected] |
| Swine vesicular disease | Enzyme-linked immunosorbent assay (antibody detection);Virus neutralisation | Dr Donald King The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom Tel: (44-1483) 23.24.41 [email protected] |
| Trichinellosis | Enzyme-linked immunosorbent assay | Dr Gianluca Marucci Istituto Superiore di Sanita, Dipartimento di Malattie Infettive Viale Regina Elena 299, 00161 Roma, Italy Tel: +390-6 49.90.23.10 [email protected] |
The term ‘International Standard Reagent’ is synonymous with primary standard. It represents the standard by which all others (secondary standards) are compared and calibrated. Secondary standards may represent national standards or working standards that are in routine use at the diagnostic laboratory level. The secondary standard and not the International Standard are to be used on a daily basis to standardise testing. The International Standard Reagents listed above may be obtained, in small quantities, at the corresponding address. Some laboratories may make a charge for this service.
Should you contact one of these Reference Laboratories to obtain reference materials and get no response, please inform the Scientific Department.