mAb Purification Difficulties

[Patricia Keely]

Dear Gods of the Internet please convey your great wisdom upon this=20
troubled soul!

I'm attempting to purify a mouse monoclonal IgG1 from tissue culture=20
supernatent with little luck. I'm using proten A sepharose (HiTRAP column=
=20
from Pharmacia) and my yields are quite low. The [mAb] in all my supes=20
was determined by radioimmunodiffusion so I know what I should get in=20
terms of yield. I been able to purify the mAb to high purity but I'm=20
getting 1/10 the yield. My protocol is as follows:

Add NaCl slowly to the supernatent to reach 3M (no precipitation occurs)
Adjust the pH to 8.0 by diluting supe 1:1 with 1M Tris-HCl, pH 8.0.
Pass supe over a 1 ml HiTRAP column equilibrated with 50 mM Tris-HCl, pH=20
8.0, 3M NaCl at a flow rate of 1 ml/min at room temperature.
Wash column until A280 returns to baseline
Elute mAb with 0.1 M citric acid-NaOH, pH 3.5 collect 1 ml fractions=20
supplemented with 250 =B5l 1 M Tris-HCl, pH 9.0

Note: first two steps are reversed in order, pH first then add salt.

I've also tried protein G sepharose with  similiar results, ie low yield!=
=20
The only thing that has worked is thiophilic chromatography eg. T-gel=20
from Pierce.

Any suggestions as to where I'm messing this up would really be=20
appreciated.

Thanx for the input

David D. Shock dsho at med.unc.edu