Council Directive of 26 June 1964 on animal health problems affecting intra-Community trade in bovine animals and swine (64/432/EEC)

[F1 B. Method for antigen standardization U.K.

Solutions and materials required U.K.

1. 40 ml of 1,6 % agarose in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

2. 15 ml of a bovine leukosis serum, having antibody only to bovine leukosis virus glycoproteins, diluted 1:10 in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

3. 15 ml of a bovine leukosis serum, having antibody only to bovine leukosis virus glycoproteins, diluted 1:5 in 0,05 M Tris/HCl buffer, pH 7,2 with 8,5 % NaCl; U.K.

4. four plastic petri dishes with a diameter of 85 mm; U.K.

5. a punch with a diameter of 4 to 6 mm; U.K.

6. a reference antigen; U.K.

7. the antigen which is to be standardized; U.K.

8. a water bath (56 ^o C). U.K.

Procedure U.K.

Dissolve the agarose (1,6 %) in the Tris/HCl buffer by carefully heating to 100 ^o C. Place in 56 ^o C water bath for approximately one hour. Also, place the bovine leukosis serum dilutions in a 56 ^o C water bath.

Now mix 15 ml of the 56 ^o C agarose solution with the 15 ml bovine leukosis serum (1:10), quickly shake and pour 15 ml into each of two petri dishes. Repeat this procedure with the bovine leukosis serum diluted 1:5.

When the agarose has hardened, holes are made in it as follows:

Addition of antigen U.K.

(i)

Petri dishes 1 and 3:

• well A — undiluted reference antigen,

• well B — 1:2 diluted reference antigen,

• wells C and E — reference antigen,

• well D — undiluted test antigen.

(ii)

Petri dishes 2 and 4:

• well A — undiluted test antigen,

• well B — 1:2 diluted test antigen,

• well C — 1:4 diluted test antigen,

• well D — 1:8 diluted test antigen.

Additional instructions U.K.

1. The experiment shall be carried out with two serum dilutions (1:5 and 1:10) in order to achieve optimal precipitation U.K.

2. If the precipitation diameter is too small with both dilutions, then the serum must be further diluted. U.K.

3. If the precipitation diameter in both dilutions is too large and faint, then a lower serum must be chosen. U.K.

4. The final concentration of the agarose must be 0,8 %; that of the sera 5 and 10 % respectively. U.K.

5. Plot the measured diameters in the following coordinate system. The dilution of the antigen to be tested with the same diameter as the reference antigen is the working dilution.] U.K.

Textual Amendments

F1 Substituted by Council Directive 97/12/EC of 17 March 1997 amending and updating Directive 64/432/EEC on health problems affecting intra-Community trade in bovine animals and swine.