Conjugation of Antibody to HRP

Reagents and Materials

 PBS PBS: 0.05M EDTA PBS: 0.01 thimerosal Conjugate Storage Buffer SATA  DMF Hydroxylamine hydrochloride  NaOH Pellets PD10 Column or Sephadex G25 Sulfo-SMCC  Preparation of Thiolated Antibody

Concentrate the antibody to 5-7 mg/ml Dissolve 10 mg of SATA in 1 ml DMF (SATA:DMF) Add 3.0 ml SATA:DMF per mg of antibody Gently stir solution for 2 hours at room temperature. Dissolve 0.5 g hydroxylamine hydrochloride in 10 ml PBS:EDTA Add 0.25 g NaOH pellets to this solution to neutralize to approximately pH 7.0 Add to antibody solution at 6.49 ml/ml SATA. Gently stir for 30 minutes at room temperature. Equilibrate column with PBS:EDTA. Desalt thiolated antibody on column.  Collect in the smallest volume possible without collecting any SATA.  Allow free SATA (2nd peak) to come off the column. Store the thiolated antibody at 2-8oC for no more than one hour.  Re-equilibrate the column with PBS.  Preparation of Maleimide:HRP

Weigh out 1.067 mg HRP for each mg of thiolated antibody. Add 1.1 mg Sulfo-SMCC per mg of HRP Add PBS to a final concentration of 8 mg/ml HRP Mix for 2 hours on a rotator.  Desalt the HRP on PBS equilibrated column.  Collect the first peak.  Preparation of Ab:HRP Conjugate

 Immediately mix the HRP with the thiolated antibody at 1 mg HRP per mg antibody. Gently stir for 16-20 hours at 2-8oC. Dialyze against 3 changed of PBS, changing every 2 hours, 100X the conjugate volume.  Dilute the conjugate 2-fold in Conjugate Storage Buffer.  Sterile Filter.