- Y Diweddaraf sydd Ar Gael (Diwygiedig)
- Pwynt Penodol mewn Amser (30/11/2009)
- Gwreiddiol (Fel y’i mabwysiadwyd gan yr UE)
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)
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Reagents for the enzyme-linked immunosorbent assays (ELISA) described below may be obtained from the European Community Reference Laboratory or the OIE Reference Laboratories for African horse sickness.
Competitive ELISA is used to detect specific AHSV antibodies in sera from any species of equidae. The broad spectrum, polyclonal, immune anti-AHSV guinea-pig serum (hereinafter referred to as ‘guinea-pig antiserum’) is serogroup specific and is able to detect all known serotypes of AHS virus.
The principle of the test is the interruption of the reaction between AHSV antigen and a guinea-pig antiserum by a test serum sample. AHSV antibodies in the test serum sample will compete with those in the guinea-pig antiserum resulting in a reduction in the expected colour (following the addition of enzyme labelled anti-guinea-pig antibody and substrate). Sera can be tested at a single dilution of 1 in 5 (spot test method) or may be titrated (serum titration method) to give dilution end-points. Inhibition values higher than 50 % may be regarded as positive.
The test protocol described hereinafter is used in the Regional Reference Laboratory for African horse sickness in Pirbright, United Kingdom.
or
then
Prepare the chromogen OPD (OPD = ortho-phenyldiamine) solution according to the manufacturers’ instructions (0,4 mg/ml in sterile distilled water) just before use. Add substrate (hydrogen peroxide = H2O2) to give a final concentration of 0,05 % (v/v) (1 in 2000 of a 30 % solution of H2O2). Add 50 μl of the OPD solution to each well and leave plates on the bench for 10 minutes at ambient temperature. Stop the reaction by the addition of 50 μl/well of 1M sulphuric acid (H2SO4).
Read spectrophotometrically at 492 nm.
The duplicate negative control serum wells and the duplicate blank wells should record PI values between + 25 % and – 25 %, and between + 95 % and + 105 %, respectively. Failure to be within these limits does not invalidate the plate but does suggest that background colour is developing.
Samples that record PI values above and below the threshold for the duplicate wells are considered doubtful. Such samples may be re-tested in the spot test and by titration. Positive samples may also be titrated to provide an indication of the degree of positivity.
–ve cont = negative control. + ve cont = positive control. GP cont = guinea pig control. | ||||||||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
+ ve cont. | Test sera | |||||||||||
A | 1:5 | –ve cont. | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
B | 1:10 | –ve cont. | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
C | 1:20 | Blank | ||||||||||
D | 1:40 | Blank | ||||||||||
E | 1:80 | GP cont. | ||||||||||
F | 1:160 | GP cont. | ||||||||||
G | 1:320 | GP cont. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
H | 1:640 | GP cont. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
–ve cont = negative control. + ve cont = positive control. GP cont = guinea pig control. | ||||||||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
+ ve cont. | Test sera | |||||||||||
A | 1:5 | –ve cont. | 1:5 | 1:5 | ||||||||
B | 1:10 | –ve cont. | 1:10 | 1:10 | ||||||||
C | 1:20 | Blank | 1:20 | 1:20 | ||||||||
D | 1:40 | Blank | 1:40 | 1:40 | ||||||||
E | 1:80 | GP cont. | 1:80 | 1:80 | ||||||||
F | 1:160 | GP cont. | 1:160 | 1:160 | ||||||||
G | 1:320 | GP cont. | 1:320 | 1:320 | ||||||||
H | 1:640 | GP cont. | 1:640 | 1:640 |
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.
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.
Colour development is stopped by adding 50 μl of 3N H2SO4 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.
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.
:
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.
:
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.
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.
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 % H2O2] 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).
Read at 405 nm in an ELISA reader.
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.
=
NC – ((NC – PC) × 0,3)
=
NC – ((NC – PC) × 0,2)
Where, NC is the OD of the negative control and PC the OD of positive control.
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.
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