# Commission Regulation (EU) No 206/2010 of 12 March 2010 laying down lists of third countries, territories or parts thereof authorised for the introduction into the European Union of certain animals and fresh meat and the veterinary certification requirements (Text with EEA relevance), Division B

Extent: E+W+S+N.I.

The virus neutralisation test shall be carried out according to the following protocol:
Reagents Stock FMDV antigen is prepared in cell cultures or on cattle tongues and stored at - 70 °C or less or at - 20 °C after the addition of 50 % glycerol. This is the stock antigen. FMDV is stable under these conditions and titres vary little over a period of months.
Procedure The test is carried out in flat-bottomed tissue culture grade microtitre plates using susceptible cells such as IB-RS-2, BHK-21 or calf kidney cells. Sera for the test are diluted 1/4 in serum-free cell culture medium with the addition of 100 IU/ml neomycin or other suitable antibiotics. Sera are inactivated at 56 °C for 30 minutes and 0.05 ml amounts are used to prepare a twofold series on microtitre plates using 0,05 ml diluting loops. Pre-titrated virus also diluted in serum-free culture medium and containing 100 TCID50/0.05 ml is then added to each well. Following incubation at 37 °C for one hour to allow neutralisation to take place, 0,05 ml of suspension cells containing 0,5 to 1.0 × 10 6 cells per 1 ml in cell culture medium containing serum free of FMD antibody is added to each well and the plates are sealed. Plates are incubated at 37 °C. Monolayers are normally confluent within 24 hours. CPE is usually sufficiently advanced at 48 hours for a microscopic reading of the test. At this time a final microscopic reading may be made or the plates may be fixed and stained for macroscopic reading, for instance using 10 % formol-saline and 0,05 % methylene blue.
Controls Controls in each test include homologous antiserum of known titre, a cell control, a serum toxicity control, a medium control, and a virus titration from which the actual amount of virus in the test is calculated.
Interpretation Wells with evidence of CPE are considered to be infected and neutralisation titres are expressed as the reciprocal of the final dilution of serum present in the serum/virus mixtures at the 50 % end point estimated according to the Spearman-Karber method. (Karber, G., 1931, Archiv fuer Experimentelle Pathologie und Pharmokologie, 162, 480.). Tests are considered to be valid when the actual amount of virus used per well in the test is between 101,5 and 102,5 TCID50 and when the titre of the reference serum is within twofold of its expected titre, estimated from the mode of previous titrations. When the controls are outside these limits the tests are repeated. An end point titre of 1/11 or less is taken as negative.

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Source: https://www.legislation.gov.uk/eur/2010/206/annex/I/part/6/division/8/division/B (the official version)
Page: https://openlegislation.uk/eu-regulation-2010-206/annex-I/part-6/division-8/division-B
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.