Council Directive 2009/156/EC of 30 November 2009 on animal health conditions governing the movement and importation from third countries of equidae (codified version) (Text with EEA relevance) (c. 156)

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

Textual Amendments

F1 Substituted by Commission Implementing Decision (EU) 2016/1840 of 14 October 2016 amending Annex IV to Council Directive 2009/156/EC as regards methods for African horse sickness diagnosis (notified under document C(2016) 6509) (Text with EEA relevance).

PART A U.K. Serological tests

2. Blocking ELISA for the detection of antibodies to African horse sickness virus (AHSV) U.K.

2.1. Test procedure U.K.

2.1.1. Solid Phase U.K.

2.1.1.1. Coat ELISA plates with 50-100 ng of recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate overnight at 4 °C. U.K.

2.1.1.2. Wash the plates three times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash. U.K.

2.1.2. Test samples and controls U.K.

2.1.2.1. Serum samples to be tested, and positive and negative control sera, are diluted 1 in 5 in diluent containing 0,35 M NaCl, 0,05 % (v/v) Tween 20 and 0,1 % Kathon, 100 μl per well. Incubate for 1 hour at 37 °C. U.K.

For titration, make a twofold dilution series of the test sera from 1 in 10 to 1 in 280 across 8 wells (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 five times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash. U.K.

2.1.3. Conjugate U.K.

2.1.3.1. Dispense 100 μl/well of horseradish peroxidase-conjugated Mab anti-VP7. In advance, this Mab has been diluted 1/ 5 000 -1/ 15 000 in a 1/1 solution of StabiliZyme Select® Stabilizer (SurModics. Reference: SZ03) in distilled water. Incubate for 30 minutes at 37 °C. U.K.

2.1.3.2. Wash the plates five times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST). Gently tap the plates on to absorbent material to remove any residual wash. U.K.

2.1.4. Chromogen/Substrate U.K.

Add 100 μl/well chromogen/substrate solution, i.e. 1 ml of ABTS (2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) 5 mg/ml + 9 ml of substrate buffer (0,1 M Phosphate-Citrate buffer of pH 4 containing 0,03 % H ₂ O ₂ ), and incubate for 10 minutes at room temperature. Colour development is stopped by adding 100 μl/well of 2 % (w/v) SDS (sodium dodecyl sulphate).

2.1.5. Reading U.K.

Read at 405 nm in an ELISA reader.]