Council Directive 93/85/EECShow full title

[F1 4.3.

IF procedure: U.K.

(i)

According to test slide preparation in 4.1(i):

Prepare a set of twofold dilutions of the antibody in IF buffer. The first well should have 1/2 of the titre (T/2), the others 1/4 of the titre (T/4), 1/2 of the titre (T/2), the titre (T) and twice the titre (2T).

(ii)

According to test slide preparation in 4.1(ii):

Prepare the working dilution (WD) of the antibody in IF buffer. The working dilution affects the specificity.

Figure 1.

Preparation of the test slide according to 4.1(i) and 4.3(i)

	Dilutions of resuspended pellet
	1/100	1/1	1/1	1/1	1/1		Dilution of resuspended pellet
(T = titre)	T/2	T/4	T/2	T	2T		Twofold dilutions of antiserum/antibody
Sample 1
	1	2	3	4	5
Duplicate of sample 1 or sample 2
	6	7	8	9	10

Figure 2.

Preparation of the test slide according to 4.1(ii) and 4.3(ii)

	Working dilution of antiserum/antibody
	1/1	1/10	1/100	empty	empty		Decimal dilution of resuspended pellet
Sample 1
	1	2	3	4	5
Duplicate of sample 1 or sample 2
	6	7	8	9	10

4.3.1. Arrange the slides on moist paper. Cover each test window completely with the antibody dilution(s). The volume of antibody applied on each window must be at least the volume of extract applied. U.K.

The following procedure should be carried out in the absence of specific instructions from the suppliers of the antibodies:

4.3.2. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). U.K.

4.3.3. Shake the droplets off each slide and rinse carefully with IF buffer. Wash by submerging for 5 minutes in IF buffer-Tween (Appendix 3) and subsequently for 5 minutes in IF buffer. Avoid causing aerosols or droplet transfer that could result in cross-contamination. Carefully remove excess moisture by blotting gently. U.K.

4.3.4. Arrange the slides on moist paper. Cover the test windows with the dilution of FITC conjugate used to determine the titre. The volume of conjugate applied on the windows must be identical to the volume of antibody applied. U.K.

4.3.5. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). U.K.

4.3.6. Shake the droplets of conjugate off the slide. Rinse and wash as before (4.3.3). U.K.

Carefully remove excess moisture.

4.3.7. Pipette 5 to 10 µl of 0,1M phosphate-buffered glycerol (Appendix 3) or a commercially antifading mountant on each window and apply a coverslip.] U.K.

Textual Amendments

F1 Substituted by Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot.