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

[F1 [F2 B. Method for antigen standardisation U.K.

1. Solutions and materials required: U.K.

(a)

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

(b)

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;

(c)

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;

(d)

four plastic petri dishes with a diameter of 85 mm;

(e)

a punch with a diameter of 4 to 6 mm;

(f)

a reference antigen;

(g)

the antigen which is to be standardised;

(h)

a water bath (56 °C).

2. Procedure: U.K.

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

Now mix 15 ml of the 56 °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 shall be made in it as follows:

3. Addition of antigen: U.K.

(a)

petri dishes 1 and 3:

(i)

well A — undiluted reference antigen;

(ii)

well B — 1:2 diluted reference antigen;

(iii)

wells C and E — reference antigen;

(iv)

well D — undiluted test antigen;

(b)

petri dishes 2 and 4:

(i)

well A — undiluted test antigen;

(ii)

well B — 1:2 diluted test antigen;

(iii)

well C — 1:4 diluted test antigen;

(iv)

well D — 1:8 diluted test antigen.

4. Additional instructions: U.K.

(a)

the experiment shall be carried out with two serum dilutions (1:5 and 1:10) in order to achieve optimal precipitation;

(b)

if the precipitation diameter is too small with both dilutions, then the serum shall be further diluted;

(c)

if the precipitation diameter in both dilutions is too large and faint, then a lower serum shall be chosen;

(d)

the final concentration of the agarose shall be 0,8 %; that of the sera 5 and 10 % respectively;

(e)

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.] ]

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.

F2 Substituted by Commission Decision of 15 December 2009 amending Annex D to Council Directive 64/432/EEC as regards diagnostic tests for enzootic bovine leukosis (notified under document C(2009) 9951) (Text with EEA relevance) (2009/976/EU).