| 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.
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