Radioimmunoassay

       Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in blood) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens.

     A. Materials and preparation for sample extraction

         Materials

  • SEP-COLUMN containing 200 mg of C18.
  • Buffer A (BUFF-A): 1% trifluoroacetic acid (TFA, HPLC Grade). (Acidifies plasma sample to remove interfering proteins such as albumin)
  • Buffer B (BUFF-B): 60% acetonitrile (HPLC Grade), 1% TFA, and 39% distilled water. (Elutes peptide from column). 

         Withdrawal and Preparation of Plasma

  1. Collect blood samples (2 - 6 ml) into a chilled syringe and transfer into a polypropylene tube containing EDTA (1 mg/ml of blood) as an anticoagulant and Aprotinin (500 KIU/ml of blood) as a protease inhibitor at 4°C. Do not use heparinized tubes as they may interfere with the assay. Vacutainers with EDTA are acceptable.
  2. Centrifuge blood at 1,600xg for 15 minutes at 4 °C.
  3. Collect the top (plasma) layer.
  4. Proceed to extraction immediately or freeze at -70 °C for later use. 

     B. C18 Sep-Column sample extraction

  1. Add an equal amount of Buffer A to the plasma.
  2. Centrifuge at 6,000xg to 17,000xg for 20 minutes at 4 °C.
  3. Transfer supernatant to a new tube discarding any pellet that may be present.
  4. Equilibrate a SEP-COLUMN by washing with 1 ml Buffer B followed by 3 X 3 ml Buffer A.
  5. Load the plasma solution onto the equilibrated SEP-Column.
  6. Slowly wash the column with Buffer A (3 ml, twice) and discard the wash. A light vacuum (10 sec/drop) may be applied to the column.
  7. Elute the peptide slowly with Buffer B (3 ml, once) and collect elutant in a polypropylene tube. A light vacuum may be applied as in previous step.
  8. Freeze-dry elutant to dryness using a dry ice/methanol bath to freeze the sample and a centrifugal concentrator to evaporate it.
  9. Dissolve the residue in a suitable volume of RIA buffer such that the concentration of the substance of interest will fall close to the IC50 (within the measuring range).

     C. Reagents and equipment for radioimmunoassay

          Reagents

  • RIA buffer 4x concentrate (50 ml). Store at 4 °C before and after dilution.
  • Standard peptide lyophilized powder. Store at -20 °C.
  • Specific rabbit antiserum lyophilized powder. Store at -20 °C.
  • 125I-tracer: at least 1.5 µCi (on preparation day). Store at -20 °C.
  • Secondary polyclonal antiserum lyophilized powder, e.g. goat anti rabbit (GARGG) antiserum. Store at 4 °C before and after rehydration.
  • Normal rabbit serum lyophilized powder. Store at 4 °C before and after re-hydration.
  • Standard diluent (8 ml) of peptide-free stripped frozen serum (for extraction-free kits only). Store at -20 °C.

          Equipment

  • 12 x 75 mm polystyrene tubes
  • Gamma counter for the above tubes
  •  Centrifuge for the above tubes
  • Curve fitting software (optional)
  • Various other standard laboratory items

     D. Radioimmunoassay procedure

  1. Dilute the RIA buffer concentrate to 200 ml.
  2. Sample extraction is recommended especially for serum samples. It may not be as important for some tissue culture samples. The concentration of the target molecule must be within the measuring range of the kit (in a region around the IC50). If you cannot estimate the concentration range of your sample you can prepare it at different concentrations such that one of the samples may be within the measuring range.
  3. Reconstitute the standard in 1ml RIA buffer and make serial dilutions covering the kit's range as stated in the enclosed datasheet. The standards and the samples should be in the same diluent. If extraction was done the diluent should be RIA buffer. Otherwise, for extraction-free kits use your own diluent if possible or use the included standard diluent for standards to match the serum or plasma samples.
  4. Reconstitute the antiserum in 13 ml RIA buffer.
  5. Mix 100 µl standards or samples with 100 µl antiserum. Add no standard or sample to controls TC, NSB, and TB. Add only 100 µl diluent and 100 µl antiserum to TB. Add 100 µl diluent plus 100 µl RIA buffer to TC and NSB.
  6. Vortex and incubate overnight (16-24 hrs.) at 4 °C.
  7. Prepare (a) the tracer stock and (b) the diluted tracer solutions. a) Tracer stock. Add 1 ml of RIA buffer to the shipped 125I-tracer vial and mix thoroughly. b) Diluted tracer. From the tracer stock make a working diluted tracer solution in RIA buffer at 10,000 to 12,000 cpm / 100 µl (value for reference only).
  8. Add 100 µl diluted 125I tracer (e.g. ~10,000 cpm) to all tubes.
  9. Vortex and incubate overnight (16-24 hrs.) at 4 °C
  10. Prepare the secondary antibody and serum. Reconstitute the secondary antibody (GARGG) with 13 ml of RIA Buffer and reconstitute the normal serum (NRS) with 13 ml of RIA Buffer.
  11. Add 100 µl of secondary antibody and 100 µl of normal serum to each tube and vortex.
  12. Incubate at room temperature for 90 minutes
  13. Add 500 µl of RIA Buffer to each tube and vortex.
  14. Centrifuge for 20 minutes at 1700 x g.
  15. Set aside the TC tubes. DO NOT ASPIRATE THE TC TUBES. Aspirate all other tubes being careful not to disturb the pellet.
  16. Count, plot and analyze the cpm data. Use a calibrated gamma counter and collect the cpm data for all tubes.