Division II.2.6 Immunofluorescence (IF)

ASK (pass 65 or lower) shall be grown in a L-15 medium containing 10 % foetal bovine serum, 2 % (vol:vol) 200 mM L-glutamine, and 0,08 % (vol:vol) 50 mM 2-mercaptoethanol in multi-well plates and used at greater than 50 % confluence. Other cell lines or growth medium of proven efficacy may also be used. 225 μl of putative virus-infected culture supernatant shall be added to each of two wells, mixed and 225 μl transferred to two further wells, namely a 1:5 dilution. Two additional wells shall be left uninoculated to act as controls. Samples from each fish farm site shall be handled on separate plates, as shall the virus control. A virus control shall be established using a reference isolate of ISAV.

Plates shall be incubated at 14 ± 2 °C and examined microscopically for up to 7 days. When early CPE is observed, or if no CPE is observed within 7 days, the next step shall be fixation. Wells shall be washed with Phosphate buffered saline (PBS) and fixed by incubation with 80 % acetone for 20 minutes at room temperature. Plates shall be air-dried and stained immediately or stored at 0 to 6 °C for no more than 24 hours prior to staining.

Replicate wells shall be stained with a mix of monoclonal antibodies (MAb) 3H6F8 and 1OC9F5 against ISAV, or other MAb of proven efficacy and specificity, diluted in PBS and incubated at 37 ± 4 °C for 30 minutes. MAb shall be removed and plates washed three times with 0,05 % Tween 20 in PBS. Anti-mouse IgG Fluorescein isothiocyanate (FITC) conjugate diluted in PBS shall be added to each well and incubated at 37 ± 4 °C for 30 minutes. The dilutions of different batches of MAb and FITC conjugate shall be optimised in each laboratory. Antibody shall be removed and plates shall be washed three times with 0,05 % Tween 20 in PBS.

Wells shall be examined immediately using an inverted microscope set up for fluorescence microscopy with a suitable filter for excitation of FITC. A test shall be considered positive if fluorescent cells are observed. For a test to be valid, the positive controls shall score positive and the negative controls shall score negative.