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.
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.
Department of Biochemistry,
University of Nottingham,
The Medical School ,
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