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June 1990 on animal health conditions governing the movement and import from third countries of equidae (90/426/EEC) (repealed)

[F1 1. COMPETITIVE ELISA FOR THE DETECTION OF ANTIBODIES TO AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST) U.K.

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 ‘ 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.

1.1. Test procedure U.K.

1.1.1. Preparation of plates U.K.

1.1.1.1. Coat ELISA plates with AHSV antigen extracted from infected cell cultures and diluted in carbonate/bicarbonate buffer, pH 9,6. Incubate the ELISA plates overnight at 4 °C. U.K.

1.1.1.2. Wash plates three times by flooding and emptying the wells with phosphate buffered saline (PBS), pH 7,2 to 7,4 pH, and blot dry on adsorbent paper. U.K.

1.1.2. Control wells U.K.

1.1.2.1. Titrate the positive control sera in a twofold dilution series, from 1 in 5 to 1 in 640, across column 1 in blocking buffer (PBS containing 0,05 % (v/v) Tween-20, 5,0 % (w/v) skimmed-milk powder (Cadbury's Marvel ^TM ) and 1 % (v/v) adult bovine serum) to give a final volume of 50 μl/well. U.K.

1.1.2.2. Add 50 μl of the negative control serum at a dilution of 1 in 5 (10 μl serum + 40 μl blocking buffer) to wells A and B of column 2. U.K.

1.1.2.3. Add 100 μl/well of blocking buffer to wells C and D of column 2 (blank). U.K.

1.1.2.4. Add 50 μl of blocking buffer to wells E, F, G and H of column 2 (guinea pig control). U.K.

1.1.3. Spot test method U.K.

1.1.3.1. Add a 1 in 5 dilution of each test serum in blocking buffer to duplicate wells of columns 3 to 12 (10 μl sera + 40 μl blocking buffer). U.K.

or

1.1.4. Serum titration method U.K.

1.1.4.1. Prepare a twofold dilution series of each test sample (1 in 5 to 1 in 640) in blocking buffer across eight wells of single columns (3 to 12). U.K.

then

1.1.5. Add 50 μl of guinea pig antisera, pre-diluted in blocking buffer, to all wells except the blank wells of the ELISA plate (all wells now contain a final volume of 100 μl). U.K.

1.1.5.1. Incubate for 1 hour at 37 °C on an orbital shaker. U.K.

1.1.5.2. Wash plates three times and blot dry as before. U.K.

1.1.5.3. Add 50 μl of rabbit anti-guinea-pig horseradish peroxidase (HRP) conjugate pre-diluted in blocking buffer to each well. U.K.

1.1.5.4. Incubate for 1 hour at 37 °C on an orbital shaker. U.K.

1.1.5.5. Wash plates three times and blot dry as before. U.K.

1.1.6. Chromogen U.K.

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 = H ₂ O ₂ ) to give a final concentration of 0,05 % (v/v) (1 in 2000 of a 30 % solution of H ₂ O ₂ ). 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 (H ₂ SO ₄ ).

1.1.7. Reading U.K.

Read spectrophotometrically at 492 nm.

1.2. Expression of results U.K.

1.2.1. Using a software package print out the optical density (OD) values, and the percentage inhibition (PI) for test and control sera based on the mean value recorded in the four guinea pig control wells. The data expressed as OD and PI values are used to determine whether the test has performed within acceptable limits. The upper control limits (UCL) and lower control limits (LCL) for the guinea pig control are between OD values 1,4 and 0,4 respectively. The end-point titre for the positive control based on 50 % PI should be 1 in 240 (within a range from 1 in 120 to 1 in 480). Any plate that fails to conform to the above criteria must be rejected. However, if the positive control serum titre is greater than 1 in 480 and the test samples are still negative then the negative test samples can be accepted. U.K.

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.

1.2.2. The diagnostic threshold (cut-off value) for test sera is 50 % (PI 50 %). Samples recording PI values greater than 50 % are recorded as positive. Samples recording PI values lower than 50 % are recorded as negative. U.K.

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.

Spot test layout
–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

Test sera
–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

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).