Competitive ELISA protocols require optimization for meaningful results to be obtained.
The most important factor is optimizing the amount of enzyme-conjugated antigen to add to the samples and this is achieved by producing a standard curve.
1. Follow the basic protocol (in Subheading 3.1.) until step 6.
2. Prepare a set of standards of known amounts of this conjugated antigen.
3. Incubate on the ELISA plate and complete the assay (see steps 7-14).
4. The resulting OD data are plotted and a curve of standards relating to the OD obtained.
5. The optimum antigen/enzyme conjugate concentration to use is the one giving approx 90% of the maximum value obtained using this curve.
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