Division Titration of BTV ELISA antigen:X1

Bluetongue ELISA antigen is titrated by the indirect ELISA. Twofold dilutions of antigen are titrated against a constant dilution (1/100) monoclonal antibody 3-17-A3. The protocol is as follows:

1. 1..

   Titrate a 1:20 dilution of BTV antigen in PBS across the microtitre plate in a twofold dilution series (50 μl/well) using a multichannel pipette.

2. 2..

   Incubate for one hour at 37 °C on an orbital shaker.

3. 3..

   Wash plates three times with PBS.

4. 4..

   Add 50 μl of monoclonal antibody 3-17-A3 (diluted 1/100) to each well of the microtitre plate.

5. 5..

   Incubate for one hour at 37 °C on an orbital shaker.

6. 6..

   Wash plates three times with PBS.

7. 7..

   Add 50 μl of rabbit anti-mouse globulin conjugated to horseradish peroxidase, diluted to a pre-titrated optimal concentration, to each well of the microtitre plate.

8. 8..

   Incubate for one hour at 37 °C on an orbital shaker.

9. 9..

   Add substrate and chromogen as described previously. Stop the reaction after 10 minutes by the addition of 1 Molar sulphuric acid (50 μl/well).

In the competitive assay, the monoclonal antibody must be in excess, therefore a dilution of antigen is chosen which falls on the titration curve (not on the plateau region) which gives approximately 0,8 OD after 10 minutes.