Preparation of Analyte-depleted Serum for Alpha Assays
The following protocol can be used for serum immunodepletion. You may also be able to purchase analyte-depleted serum from a supplier such as Bioreclamation.
Materials
- Streptavidin Sepharose: GE Healthcare (catalog number 17-5113-01)
- Human serum: Cambrex (catalog number 14-402E)
Day 1
Calculations
- Determine the amount of analyte to be removed from the serum or plasma (based on literature or other assays).
- Use a 100:1 molar ratio of biotinylated antibody to analyte.
- Use 20-fold more biotin-binding sites than biotin.
Streptavidin Sepharose preparation
- Gently shake the bottle of SA-Sepharose to resuspend the matrix. (Do not vortex!)
- Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
- Wash the SA-Sepharose by adding 10 mL of 1X PBS. Invert to mix.
- Sediment the matrix by centrifugation at 2000 rpm for 5 minutes.
- Repeat washing and centrifugation steps twice more, and resuspend in 1X PBS buffer.
- Add antibody and incubate for 2 hours at 4ºC (with shaking).
Serum depletion
- Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
- Wash the SA-Sepharose by adding 10 mL PBS. (Note: Resuspend the matrix by inverting the tube; do not vortex.)
- Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
- Repeat washing and centrifugation steps once more.
- Add serum or plasma sample and resuspend the matrix gently.
- Incubate 18-24 hours at 4ºC with agitation.
Day 2
Serum recovery
- Distribute the SA-Sepharose / serum mix into Eppendorf tubes.
- Sediment the matrix by centrifugation at 12,000 rpm for 10 minutes.
- Recover as much sample as possible and transfer into new tubes.
- Repeat the centrifugation step.
- Recover as much sample as possible without aspirating any of the remaining SA-Sepharose and transfer into new tubes.
- Sediment any remaining matrix by centrifugation at 13,000 rpm for 10 minutes.
- Collect the depleted serum or plasma and store at -20ºC.