Sean Lauber:ELISA Development: Difference between revisions
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Revision as of 08:49, 11 July 2012
First you need to determine what reagents you will use for your ELISA. The protocol I use is based off of a generic R&D ELISA. The primary antibody binds to the plate (this should be monoclonal), the sample binds to the primary, then the secondary binds to the sample (this should be be biotinylated), then strep-HRP binds to biotin and the HRP is used for color development (see any R&D ELISA for the complete protocol).
Next you need to determine what concentration of primary and secondary will work best for giving good color development for a range of sample concentrations. I choose the range 0.25 - 8 μg/mL for the primary, 50 - 400 ng/ml for the secondary, and 10-1000 pg/ml for sample detection. Set up the plate as follows:
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