Competitive ELISA

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DAY 1

1. Coat Nunc immuno-module plates overnight, in usual manner.

2. Set up competition assay between antibody and competing substance :-

Prepare a 1:2 serial dilution of the competitive substance from 480 to 0.4 ng/ml. Add 50ul of each of the above dilutions to 25ul of 3x strength titre of antibody (i.e. a solution 3 x the titre giving 50% inhibition on direct ELISA), in sealed tubes.

3. Incubate overnight at room temperature.

4. Set up competition between unknown concentration of competitive substance and antibody as above.

DAY 2

1. Wash plate with 4x 200ul/well PBS /Tween.

2. Add 50ul/well of antibody and incubate for 2hrs.

3. Wash 4x with 200ul/well PBS/Tween.

4. Add 50ul/well secondary antibody and incubate for 2hrs.

5. Wash 4x with 200ul PBS/Tween.

6. Add 50 ul peroxidase substrate (TMB) as for direct ELISA.

7. Read OD @ 450nm.

8. Plot standards against absorbance and determine unknown concentrations.