Commission Regulation (EU) No 206/2010Show full title

[X1 C. The detection and quantification of antibody by ELISA shall be carried out according to the following protocol: U.K.

Reagents

:

Rabbit antisera to 146S antigen of seven types of foot-and-mouth disease virus (FMDV) used at a predetermined optimum concentration in carbonate/bicarbonate buffer, pH 9,6. Antigens are prepared from selected strains of virus grown on monolayers of BHK-21 cells. The unpurified supernatants are used and pretitrated according to the protocol but without serum, to give a dilution which after the addition of an equal volume of PBST (phosphate buffered saline containing 0,05 % Tween-20 and phenol red indicator) would give an optical density reading of between 1,2 and 1,5. The viruses can be used inactivated. PBST is used as a diluent. Guinea-pig antisera are prepared by inoculating guinea pigs with 146S antigen of each serotype. A predetermined optimum concentration is prepared in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is used at a predetermined optimum concentration in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Test sera are diluted in PBST.

Procedure: U.K.

1.

ELISA plates are coated with 50 μl of rabbit antiviral sera overnight in a humidity chamber at room temperature.

2.

Fifty microlitres of a duplicate, twofold series of each test serum starting at 1/4 are prepared in U-bottomed multiwell plates (carrier plates). Fifty microlitres of a constant dose of antigen are added to each well and the mixtures are left overnight at 4 °C. The addition of the antigen reduces the starting serum dilution to 1/8.

3.

The ELISA plates are washed five times with PBST.

4.

Fifty microlitres of serum/antigen mixtures are then transferred from the carrier plates to the rabbit-serum-coated ELISA plates and incubated at 37 °C for one hour on a rotary shaker.

5.

After washing, 50 μl of guinea-pig antiserum to the antigen used in point 4 is added to each well. The plates are incubated at 37 °C for one hour a rotary shaker.

6.

The plates are washed and 50 μl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37 °C for one hour on a rotary shaker.

7.

The plates are washed and 50 μl of orthophenylene diamine containing 0,05 % H ₂ O ₂ (30 %) w/v is added to each well.

8.

The reaction is stopped after 15 minutes with 1,25M H ₂ SO ₄ .

The plates are read spectrophotometrically at 492 nm on an ELISA reader linked to a microcomputer.

Controls

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For each antigen used 40 wells contain no serum but contain antigen diluted in PBST. A duplicated twofold dilution series of homologous bovine reference antiserum. A duplicate twofold dilution series of negative bovine serum.

Interpretation

:

Antibody titres are expressed as the final dilution of tests serum giving 50 % of the mean OD value recorded in the virus control wells where test serum is absent. Titres in excess of 1/40 are considered positive.

References

:

Hamblin C, Barnett ITR and Hedger RS (1986) ‘ A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA. ’ Journal of Immunological Methods, 93, 115 to 121.11. ]

Editorial Information

X1 Substituted by Corrigendum to Commission Regulation (EU) No 206/2010 of 12 March 2010 laying down lists of third countries, territories or parts thereof authorised for the introduction into the European Union of certain animals and fresh meat and the veterinary certification requirements (Official Journal of the European Union L 73 of 20 March 2010).