# 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: Preparation of BTV ELISA antigen:

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

1.. Wash 40-60 roux of confluent BHK-21 cells three times with serum-free Eagle's medium and infect with bluetongue virus serotype 1 in serum-free Eagle's medium.
2.. Incubate at 37 °C and examine daily for cytopathic effect (CPE).
3.. When CPE are complete in 90 % to 100 % of the cell sheet of each roux, harvest the virus by shaking any still-attached cells from the glass.
4.. Centrifuge at 2 000 to 3 000 rpm to pellet the cells.
5.. Discard the supernatant and re-suspend the cells in approximately 30 ml of PBS containing 1 % ‘ Sarkosyl ’ and 2 ml phenylmethylsulphonyl fluoride (lysis buffer). This may cause the cells to form a gel and more lysis buffer may be added to reduce this effect. (NB: phenylmethylsulphonyl fluoride is harmful - handle with extreme caution.)
6.. Disrupt the cells for 60 seconds using an ultrasonic probe at an amplitude of 30 microns.
7.. Centrifuge at 10 000 rpm for 10 minutes.
8.. Store the supernatant at + 4 °C and re-suspend the remaining cell pellet in 10 to 20 ml of lysis buffer.
9.. Sonicate and clarify, storing the supernatant at each stage, a total of three times.
10.. Pool the supernatants and centrifuge at 24 000 rpm (100,000 g) for 120 minutes at + 4 °C over a 5 ml cushion of 40 % sucrose (w/v in PBS) using 30 ml Beckmann centrifuge tubes and an SW 28 rotor.
11.. Discard the supernatant, drain the tubes thoroughly and re-suspend the pellet in PBS by sonication. Store the antigen in aliquots at – 20 °C.

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Source: https://www.legislation.gov.uk/eur/2010/206/annex/I/part/6/division/5/division/A/division/7 (the official version)
Page: https://openlegislation.uk/eu-regulation-2010-206/annex-I/part-6/division-5/division-A/division-7
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.