Antibody Purification

Biplab Bose biplabbose at gmail.com
Tue Feb 8 13:50:52 EST 2005


Hi 
I am using MAbTrap Kit with HiTrap Protein G HP 1 ml column(Amersham),
to purify a recombinant human antibody (IgG1). This antibody is
expressed in adherent CHO cells. I am using DMEM with 5% SYNSER. I am
not using any FCS in culture. SYNSER is a Hybridoma growth supplement
from Biological Industries, Israel. I am facing the following problem
in purification:

I performed an ELISA to check antigen binding with different fraction.
I used equal amount of each fraction for the ELISA ( 0.5 micro gm
/well). I measured the protein content of each fraction both by OD280
and by Bradford reagent BioRad.

1. Crude culture supernatant: ELISA O.D = 1.6/0.5 micro gm
2. Flow through: ELISA O.D = 0.3/0.5 micro gm
3. Eluted fraction: ELISA O.D = 0.6/0.5 micro gm

As far as I understand the purified eluted fraction should give more
ELISA reading than the original crude sample.

It seems, I am loosing lots of the antibody. But OD280 of Wash
fractions were near zero.
How can I increase the yield of purification?
Can the neutralization buffer, used during purification, creat problem
in ELISA?
Expecting your suggestions.
With regards
Biplab
biplabbose at gmail.com

-- 
Biplab Bose
Department of Biochemistry
All India Institute of Medical Sciences
Ansari Nagar
New Delhi, INDIA
PIN-110029
Ph.No: 2659-3314
http://www.biplabbose.wb.st



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