# Commission Implementing Decision (EU) 2015/1554 of 11 September 2015 laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods (notified under document C(2015) 6188) (Text with EEA relevance), Division I.6.2: Immunofluorescence- IF

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

The identification of listed pathogens VHSV and IHNV shall be performed by infecting cells in ‘Black’ 96-well plates, conventional 24-well plates or cover slips into 24-well plates. When IHNV or VHSV or both are identified by infecting cells on cover slips, the following protocol shall apply:
(a)cover slips shall be seeded with cells at a density leading to between 60 % and 90 % confluence after 24 hours of cultivation. EPC cells shall be used where possible for this purpose because of their strong adherence to glass surfaces, but other cell lines such as BF-2, RTG-2 or FHM may be used as well. 150 μl cell culture supernatant in two different dilutions (1:10 and 1:1 000) shall be inoculated in duplicate onto 1-day-old monolayers and incubated at 15 °C for 24 hours;
(b)subsequently, cell culture medium shall be removed, and the infected cell monolayers fixed with 0,5 ml ice-cold, aqueous acetone solution (80 % vol:vol). Fixation shall take place in fume hood for 15 minutes at room temperature, then the acetone solution shall be removed and the cover glasses shall be air dried for at least 30 minutes. At this stage, the plates shall either be processed immediately or stored at – 20 °C for further use;
(c)specific monoclonal antibodies (namely for VHSV identification, MAb IP5B11 and for IHNV, Hyb 136-3 respectively) shall be diluted in 0,01 M PBST, pH 7,2 in the dilution recommended by the provider of the MAbs; 50 to 100 μl/well shall be added to the fixed monolayer and plates shall be incubated for one hour at 37 °C in a humid chamber;
(d)cover glasses shall be washed gently three times with PBS containing 0,05 % Tween-20 (PBS-T), and the buffer shall be removed completely after the last rinse. The cells shall subsequently be incubated for one hour at 37 °C with fluorescein isothiocyanate (FITC) — or tetramethylrhodamine-5-(and-6-) isothiocyanate (TRITC)-conjugated antibodies against mouse immunoglobulin used as the primary antibody, diluted according to the supplier instructions, washed again in PBS-T, and dried. Stained cultures shall be mounted onto glass slides using glycerol saline and examined under incident ultraviolet (UV) light. Use 10 × or 12 × eyepieces and a × 25 or × 40 objective lens with numerical apertures > 0,7 and > 1,3 respectively.
Other IF techniques, with regard to cell cultures, fixation and antibodies of reference quality, of proven similar efficacy may be used instead.

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Source: https://www.legislation.gov.uk/eudn/2015/1554/annex/II/part/1/division/I/division/I.6/division/I.6.2 (the official version)
Page: https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-1/division-I/division-I.6/division-I.6.2
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.