3.4.2.5. Antibody solution (RIA serum)

Prepare an adequate volume of RIA serum for the number of samples to be run. Typically, a minimum of 10 ml solution should be prepared, which would be sufficient for 100 samples (100 µl of this solution are required per sample). The solution should be stored at 4oC and should also be used within 4-6 weeks. For making 10 ml of antibody solution:

1. Dissolve 10 mg of bovine serum albumin (BSA, Fraction V) and 10 mg of IgG from rabbit serum (Sigma I5006) in 10 ml of 0.1 M phosphate buffer (3.3.2.2.).

2. Remove a 0.5-1 ml aliquot of this solution to later be used with the non-specific binding (background) tubes in the assay.

3. Add an appropriate volume of the JH antibody to the remaining RIA serum solution.

Note: The appropriate concentration of ecdysone antibody will have to be established in prior tests evaluating sensitivity through a dilution series of the respective antibody, remembering that an RIA is an isotope dilution assay whereby the specific antibody is the limiting factor. Ideal antibody concentrations should give maximal-binding cpm values corresponding to 35-40 % of the total radioactivity added to each sample (around 1800-2200 cpm under the above described conditions). Such tests also need to be run periodically to ascertain binding and, if necessary, adjust the antibody concentration.

 

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