problems with Mab purification

Robert Burns burns at sasa.gov.uk
Wed Jun 5 10:43:26 EST 1996

Hi Folks, I wonder if you can help us. We have a particular Mab 
class IgG2a (Murine) that we are trying to purify. Routinely we
concentrate X10, then purify using protein G. This particular mab
yields about 30 mg/L but the activity of the antibody when determined
by indirect ELISA is severely reduced when compared with neat cell
supernatant. THe strange thing is that at high concentrations i.e.
neat, 1/10 and 1/100 etc (of 1mg/ml) a prozone is clearly visible 
although the signal is severely depressed. It looks almost as the 
the epitopes on the Mab to which the secondary ab (anti-mouse AP)
have been damaged by purification although the specific binding site
has not. The situation is even worse if purification is attempted
using Ammonium Sulphate. Has anybody any clues as to what is going
wrong and how to get round it as we don't particularly want to 
provide the Mab as Litres of neat cell supernatant!   


Robert Burns

Monoclonal Antibody Unit
Scottish Agricultural Science Agency
East Craigs

burns at sasa.gov.uk

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