F1ANNEX DAFRICAN HORSE SICKNESS
DIAGNOSIS
F11.COMPETITIVE ELISA FOR THE DETECTION OF ANTIBODIES TO AFRICAN HORSE SICKNESS VIRUS (AHSV) (PRESCRIBED TEST)
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F11.1.Test procedure
F11.1.1.Preparation of plates
F11.1.1.1.
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F11.1.1.2.
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F11.1.2.Control wells
F11.1.2.1.
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F11.1.2.2.
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F11.1.2.3.
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F11.1.2.4.
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F11.1.3.Spot test method
F11.1.3.1.
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F11.1.4.Serum titration method
F11.1.4.1.
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F11.1.5.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).
F11.1.5.1.
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F11.1.5.2.
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F11.1.5.3.
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F11.1.5.4.
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F11.1.5.5.
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F11.1.6.Chromogen
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F11.1.7.Reading
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F11.2.Expression of results
F11.2.1.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.
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F11.2.2.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.
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Repealed by 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).