Antibody purification

Biplab Bose biplabbose at gmail.com
Tue Feb 8 13:47:36 EST 2005

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
Expecting your suggestions.
With regards
biplabbose at gmail.com

Biplab Bose
Department of Biochemistry
All India Institute of Medical Sciences
Ansari Nagar
New Delhi, INDIA
Ph.No: 2659-3314

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