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Antibody purification

Antibody purification - Protocols and Methods Forum

Antibody purification - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 02-08-2005, 06:46 PM
Biplab Bose
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Default Antibody purification



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
[Only registered users see links. ]

--
Biplab Bose
Department of Biochemistry
All India Institute of Medical Sciences
Ansari Nagar
New Delhi, INDIA
PIN-110029
Ph.No: 2659-3314
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  #2  
Old 02-09-2005, 11:08 PM
Wofo
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Default Antibody purification

Could be that something inhibits binding to the column. IgG1 should bind
perfectly to protein G actually (just checked it), for a nice chart see

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You might try ammonium sulfate precipitation of your cell supernatant in
order to remove small interfering molecules prior to any chromatography.

If you like, give us more details about the construct you're expressing and
your ELISA protocol. Could it be that there is something wrong with the
sequence (incomplete, frameshifts, mutants, no secretion signal sequence...)

You might read this papers by Roffler-S on purification of engineered
singlechain-ABs (scFv's)

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Best regards and good luck!

Wolfgang

At 19:46 08.02.2005, you wrote:

*^_^*

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