Council Directive of 26 June 1990 on animal health conditions governing the movement and import from third countries of equidae (90/426/EEC) (repealed) (c. 426)

[F1 ANNEX D U.K. AFRICAN HORSE SICKNESS DIAGNOSIS

Textual Amendments

F1 Substituted by Commission Decision of 21 February 2002 amending Annex D to Council Directive 90/426/EEC with regard to diagnostic tests for African horse sickness (notified under document number C(2002) 556) (Text with EEA relevance) (2002/160/EC).

2. INDIRECT ELISA FOR THE DETECTION OF ANTIBODIES TO AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST) U.K.

The test described hereinafter is in accordance with the test description in Chapter 2.1.11 of the OIE Manual of Standards for Diagnostic Tests and Vaccines , fourth edition, 2000.

The recombinant VP7 protein has been used as antigen for AHS virus antibody determination with a high index of sensitivity and specificity. Other advantages are that it is stable and not infective.

2.1. Test procedure U.K.

2.1.1. Solid phase U.K.

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

2.1.1.2. 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. U.K.

2.1.1.3. Block the plates with phosphate buffered saline (PBS) + 5 % (w/v) skimmed milk (Nestlé Dry Skim Milk ^TM ), 200 μl/well, for 1 hour at 37 °C. U.K.

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

2.1.2. Test samples U.K.

2.1.2.1. Serum samples to be tested, and positive and negative control sera, are diluted 1 in 25 in PBS + 5 % (w/v) skimmed milk + 0,05 % (v/v) Tween 20, 100 μl per well. Incubate for 1 hour at 37 °C. U.K.

For titration, make a twofold dilution series from 1 in 25 (100 μl/well), one serum per plate column, and do the same with positive and negative controls. Incubate for 1 hour at 37 °C.

2.1.2.2. Wash the plates as described in step 2.1.1.2. U.K.

2.1.3. Conjugate U.K.

2.1.3.1. Dispense 100 μl/well of horseradish-peroxidase (HRP) -conjugated anti-horse gamma-globulin diluted in PBS + 5 % milk + 0,05 % Tween 20, pH 7,2. Incubate for 1 hour at 37 °C. U.K.

2.1.3.2. Wash the plates as described in step 2.1.1.2. U.K.

2.1.4. Cromogen/Substrate U.K.

2.1.4.1. Add 200 μl/well of chromogen/substrate solution (10 ml of 80,6 mM DMAB (dimethyl aminobenzaldehyde) + 10 ml of 1,56 mM MBTH (3-methyl-2-benzo-thiazoline hydrazone hydrochlorid) + 5 μl H ₂ O ₂ ) U.K.

Colour development is stopped by adding 50 μl of 3N H ₂ SO ₄ after approximately 5 to 10 minutes (before the negative control begins to be coloured).

Other chromogens such as ABTS (2,2'-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), TMB (tetramethyl benzidine), or OPD (ortho-phenyldiamine) can also be used.

2.1.4.2. Read the plates at 600 nm (or 620 nm). U.K.

2.2. Interpretation of the results U.K.

2.2.1. Calculate the cut-off value by adding 0,6 to the value of the negative control (0,6 is the standard deviation derived with a group of 30 negative sera). U.K.

2.2.2. Test samples giving absorbance values lower than the cut-off are regarded as negative. U.K.

2.2.3. Test samples giving absorbance values greater than the cut-off + 0,15 are regarded as positive. U.K.

2.2.4. Test samples giving intermediate absorbance values are doubtful and a second technique must be employed to confirm the result.] U.K.