F1ANNEX IVAFRICAN HORSE SICKNESS

DIAGNOSIS

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PART ASerological tests

1.Indirect ELISA for the detection of antibodies to African horse sickness virus (AHSV)

1.1.Test procedure

1.1.1.Solid phase

1.1.1.1.

Coat ELISA plates with recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate plates overnight at 4 °C.

1.1.1.2.

Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution). Gently tap the plates onto absorbent material to remove any residual wash.

1.1.1.3.

Block the plates with phosphate buffered saline (PBS) pH 7,2 + 5 % (w/v) skimmed milk (Nestlé Dry Skim MilkTM), 200 μl/well, for 1 hour at 37 °C.

1.1.1.4.

Remove the blocking solution and gently tap the plates onto absorbent material.