To describe the preparation of diluent buffer for Horseradish Peroxidase enzyme conjugates to be used in ELISA assays.




  • RMS-109 Bovine Serum Albumin (BSA) Protease free
  • RMS-110 Tris
  • RMS-107 Tween 20
  • RMS-108 Thimerosal
  • RMS-106 Sodium Chloride
  • RMS-111 Hydrochoric Acid
  • SOP-106 2N NaOH


  • Stir bar and Stir plate
  • Clean 500 ml Bottle with cap
  • 500 ml Graduated cylinder
  • Balance and weigh boats
  • 5 ml pipette with bulb
  • pH Meter with pH 7 and pH 10 standards



  1. Weigh 6.06 g Tris, 4.38 g Sodium Chloride and 50 milligrams Thimerosal. Add to a clean 500 ml bottle.
  2. Add 500 ml DI water and a stir bar. Stir on a stir plate for 30 minutes or until solids are dissolved.
  3. Calibrate pH meter with the pH 7 and 10 standards. While the pH electrode is immersed in the stirring buffer, slowly add 37% HCl until the pH stabilizes at pH 8. If you overshoot pH 8, add 2N NaOH to raise the pH back to pH 8.
  4. Remove the pH electrode and slow the stirring to a rate that a vortex is barely noticeable. Add 250 ul Tween-20 and the 5 g BSA. Stir for one hour or until the solids are dissolved.
  5. Filter the solution through a 0.45 um filter.
  6. Label the container with the contents and a batch number.
  7. Store the closed container at 2 - 8 C for up to one year.