{
  "url": "https://openlegislation.uk/eu-regulation-2010-206/annex-I/part-6/division-5/division-A/division-8",
  "json_url": "https://openlegislation.uk/eu-regulation-2010-206/annex-I/part-6/division-5/division-A/division-8.json",
  "legislation": "eur/2010/206",
  "legislation_title": "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)",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2010-206",
  "path": "annex/I/part/6/division/5/division/A/division/8",
  "kind": "division",
  "number": null,
  "heading": "Titration of BTV ELISA antigen:",
  "text": "Bluetongue ELISA antigen is titrated by the indirect ELISA. Twofold dilutions of antigen are titrated against a constant dilution (1/100) monoclonal antibody 3-17-A3. The protocol is as follows:\n1.. Titrate a 1:20 dilution of BTV antigen in PBS across the microtitre plate in a twofold dilution series (50 μl/well) using a multichannel pipette.\n2.. Incubate for one hour at 37 °C on an orbital shaker.\n3.. Wash plates three times with PBS.\n4.. Add 50 μl of monoclonal antibody 3-17-A3 (diluted 1/100) to each well of the microtitre plate.\n5.. Incubate for one hour at 37 °C on an orbital shaker.\n6.. Wash plates three times with PBS.\n7.. Add 50 μl of rabbit anti-mouse globulin conjugated to horseradish peroxidase, diluted to a pre-titrated optimal concentration, to each well of the microtitre plate.\n8.. Incubate for one hour at 37 °C on an orbital shaker.\n9.. Add substrate and chromogen as described previously. Stop the reaction after 10 minutes by the addition of 1 Molar sulphuric acid (50 μl/well).\nIn the competitive assay, the monoclonal antibody must be in excess, therefore a dilution of antigen is chosen which falls on the titration curve (not on the plateau region) which gives approximately 0,8 OD after 10 minutes.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "X",
      "label": "X1",
      "text": "Substituted by Corrigendum to 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 (Official Journal of the European Union L 73 of 20 March 2010)."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2010/206/annex/I/part/6/division/5/division/A/division/8",
    "retrieved_at": "2026-11-01T14:50:53.580499+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}