3.3.3. Running a JH RIA
1. To set up a standard curve (in duplicates), prepare 10 disposable borosilicate glass culture tubes (6 x 50 mm).
Eight of each series will receive aliquots of 0.5, 1, 2, 5, 10, 20, 50 and 100 µl (these corresponding to 25, 50, 100, 250, 500, 1000, 2500 and 5000 pg of JH- III) of the stock solution of unlabelled JH-III (see section 184.108.40.206).
Note that pipetting of the standard curve samples must be done with Hamilton-type syringes that are exclusively dedicated to use in this JH RIA.
2. The additional four tubes will not receive any unlabelled JH.
One of each series will receive antibody-free background solution (to assess non-specific binding) and the other receives radiolabelled JH only (maximum binding values).
3. Evaporate the solvent from all RIA vials, both the standard curve and the sample tubes.
4. Add 100 µl of the RIA tracer solution (220.127.116.11) to each vial.
5. Vortex vigorously for 10 s.
6. Add 100 µl of the antibody solution (18.104.22.168) to all tubes, except the two for background counting, which will receive an equal amount of the background protein solution (see section 22.214.171.124. step 2).
7. Gently mix the tracer with the serum components.
Do not vortex as this will cause bubbles to form.
8. Quickly (30 s) spin the tubes in a centrifuge (2000 x g) to remove any solution adhering to the walls of the vials.
9. Cover RIA test tubes with an adhesive sheet to avoid loss by evaporation.
10. Incubate samples overnight at 4 oC to attain a binding equilibrium.
Do not incubate for more than 24 h.
11. Add 200 µl of saturated ammonium sulphate solution (see section 126.96.36.199) to the test tubes to precipitate proteins, including the antibody-bound JH.
13. Store samples at 4 oC for 30 min.
14. Centrifuge at 7,500 x g for 15 min at 4oC.
15. Remove supernatant, taking care to leave the pellet undisturbed.
16. Add 400 µL of a 50 % ammonium sulphate solution [1:1 saturated ammonium sulphate/water (v:v)] to the test tubes to dissolve the pellet.
18. Store samples at 4 oC for 30 min.
19. Centrifuge at 7,500 x g for 15 min at 4 oC.
20. Remove supernatant, taking care to leave the pellet undisturbed.
21. Add 50 µl of water to the pellet.
22. Vortex to dissolve completely.
23. Transfer the dissolved precipitate to a 20 ml vial appropriate for liquid scintillation counting (LSC) vial.
24. Wash the RIA test tube with another 50 µl of water and add to the same respective LSC vial.
25. Add 5 ml of a biodegradable LSC cocktail (e.g. Optiphase Hisafe3, Perkin-Elmer).
26. Leave the counting vials for 1 h at room temperature before starting the counting process in a β-counter.
27. Counting times should be set to allow a 5 sigma level of confidence.
This period will vary depending on the number of counts in the sample, counting efficiency and instrument variables. Most LSC instrumentation includes variable counting to provide 5 sigma margins of error in their programs. As a rule of thumb for counting the RIA samples, counting times of 2 min should be sufficient, and the entire series should be counted at least twice