Purification of Rat Anti-Mouse mAbs

  • The mAb MTS 35, was purified using a protein G sepharose 4 Fast Flow column which has the capacity of 7mg mAb/ml of gel bed matrix.
  • Briefly, the column was pre-eluted with 0.1M glycine-HCL (pH 2.7) until baseline absorbance at 280nm was obtained.
  • After re-equilibrating the gel bed with running buffer (20mM sodium phosphate, pH 7.2), hybridoma supernatant was added to the column at a flow rate of 1ml/min.
  • After the total sample had passed through the column, the gel bed was washed with running buffer to ensure a baseline absorbance reading of 280nm.
  • Once this was established, the mAb was eluted from the gel bed with 0.1M glycine-HCL (pH 2.7).
  • The eluate was collected until the 280nm absorbance reading returned to baseline level, and immediately neutralised to pH 7.0 by the addition of 1M Tris-HCL (pH 9.0).
  • The sample was then dialysed overnight against PBS at 4°C, with at least one change.
  • The concentration of purified mAb was determined using an Hitachi U-2000 spectrophotometer (Hitachi, Japan) at 280nm, using a value of 1.4 for the absorbance of 1mg/ml solution,
  • If the mAb needed to be further concentrated this was achieved using a minicon concentrator (Amicon Div. Beverley, M.A. U.S.A.).
  • Generally, purified mAbs were stored at a concentration of 2mg/ml.
  • The thymic reactivity of each purified mAb was determined by either indirect immunofluorescence and/or flow cytometry before use.