[F1ANNEX D U.K. AFRICAN HORSE SICKNESS DIAGNOSIS
Textual Amendments
3. BLOCKING ELISA FOR THE DETECTION OF ANTIBODIES TO AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST) U.K.
The blocking ELISA is designed to detect specific AHSV antibodies in sera from any susceptible species. VP7 is the major, antigenic, viral protein of AHSV, and is conserved within the nine serotypes. Because the monoclonal antibody (Mab) is also directed against the VP7, the assay will give a high level of sensitivity and specificity. Further, the recombinant VP7 antigen is completely innocuous and therefore guarantees a high degree of safety.
The principal of the test is the interruption of the reaction between the recombinant VP7, as the antigen bound to the ELISA plate and the conjugated Mab specific for the VP7. Antibody in the test sera will block the reaction between the antigen and the Mab resulting in a reduction in colour.
The test described hereinafter is carried out in the European Community Reference Laboratory for African horse sickness in Algete, Spain.
3.1. Test procedure U.K.
3.1.1. ELISA plates U.K.
3.1.1.1. Coat ELISA plates with recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate overnight at 4 °C. U.K.
3.1.1.2. Wash the plates five times with phosphate buffered saline (PBS) containing 0,05 % (v/v) Tween 20 (PBST). U.K.
3.1.1.3. Stabilise the plate by treatment with a stabilising solution (in order to allow long term storage at 4 °C without loss of activity) and blot dry onto adsorbent material. U.K.
3.1.2. Test samples and controls U.K.
3.1.2.1.
| For screening: | dilute test sera and controls 1 in 10 directly on the plate in PBST to give a final volume 100 μl/well. Incubate for 1 hour at 37 °C. |
3.1.2.2.
| For titration: | prepare a twofold dilution series of test sera and positive controls (100 μl/well) from 1 in 10 to 1 in 1 280 across eight wells. Negative control is tested at 1 in 10 dilution. |
3.1.3. Conjugate U.K.
Add 50 μl/well of pre-diluted horseradish-peroxidase (HRP) -conjugated Mab (monoclonal antibodies specific for VP7) to each well and mix gently to ensure homogeneity. Incubate for 30 minutes at 37 °C.
3.1.4. Wash the plates five times with PBST and blot dry as above. U.K.
3.1.5. Chromogen/Substrate U.K.
Add 100 μl/well of chromogen/substrate solution (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).
3.1.6. Reading U.K.
Read at 405 nm in an ELISA reader.
3.2. Interpretation of the results U.K.
3.2.1. Assay validation U.K.
The test is valid when the optical density (OD) of negative control (NC) is higher than 1,0 and the OD of positive control (PC) is lower than 0,2.
3.2.2. Cut-off calculation U.K.
Where, NC is the OD of the negative control and PC the OD of positive control.
3.2.3. Interpretation of results U.K.
Samples with OD lower than positive cut-off should be considered as positives to AHSV antibodies.
Samples with OD higher than negative cut-off should be considered negatives for AHSV antibodies.
Samples with OD between these two values should be considered doubtful and the animals re-sampled after two to three weeks.]