Division I.6.1 ELISA

A double antibody sandwich ELISA shall be performed in order to identify the virus isolate. Microwell plates shall be coated with 50 μl/well (0,9 pg) of proven quality protein-A purified immunoglobulins(Ig) from rabbit antisera against IHNV or VSHV diluted in carbonate buffer (pH 9,6) containing 15 mM sodium azide and incubated from 18 hours to 2 weeks at 4 °C.

On a dilution plate, each sample containing 1 % Triton X-100 and the positive controls shall be diluted with buffer solution (namely, phosphate buffered saline (PBS)-T-BSA, 1 % BSA) in a 4-fold dilution: undiluted, 1:4, 1:16, 1:64. The ELISA plates shall be washed in PBS containing 0,05 % Tween-20 (PBS-T) and 50 μl of each dilution shall be transferred from the dilution plate to the washed and coated ELISA-plate.

ELISA plates shall then be incubated for 30 minutes at 37 °C. Subsequently plates shall be washed and incubated for 30 minutes at 37 °C with specific monoclonal antibodies (namely for VHSV identification MAb IP5B11 and for IHNV Hyb 136-3, respectively). 50 μl of horseradish-peroxidase (HRP) conjugated rabbit anti mouse antibodies diluted 1:1 000 in PBS-T-BSA shall be transferred to the ELISA plate.

Finally, after renewed washing the reactions shall be developed adding 50 μl/well of ortho-phenylenediamine (OPD). The ELISA plates shall be incubated for 20 minutes at room temperature in the dark and the reaction shall be stopped by adding 100 μl/well 0,5 M H₂SO₄.

The absorbance shall be monitored at a wavelength of 492 and 620 nm in an ELISA reader. Samples shall be designated positive or negative after comparing the test results to the absorbance values for the positive and negative controls. In general, samples with combined absorbance (A) < 0,5 for undiluted material shall be considered negative, samples with A values between 0,5 and 1,0 shall be considered suspicious and samples with A values > 1,0 shall be considered positive.

Other ELISA versions with a proven similar efficacy may be used instead of those referred to in this point.