3.4.3. Running an ecdysteroid RIA
1. To set up a standard curve (in duplicates), prepare two series of 10 disposable borosilicate glass culture tubes (6 x 50 mm); to eight tubes each, add serial aliquots of the stock solution of unlabelled 20E, this being 0.5, 1, 2, 7.5, 10, 15, 20, 40 µl (corresponding to 25, 50, 100, 375, 500, 750, 1000 and 2000 pg of 20E).
Note that pipetting of the standard curve samples must be done with Hamilton-type syringes that are exclusively dedicated to use in this ecdysteroid RIA.
2. The remaining two tubes in each series do not receive any unlabelled JH; one will receive antibody-free background solution (to assess non-specific binding) and one will receive radiolabelled JH only (maximum binding values).
All further steps (3-27) are the same as those described for the JH RIA in section 3.3.3.