Antibody purification

Wofo novalidaddress at nurfuerspam.de
Wed Feb 9 18:08:05 EST 2005


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

http://www.biochem.unizh.ch/antibody/Introduction/IGGinteractions/Specificity.html


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)

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14725509

Best regards and good luck!

Wolfgang

At 19:46 08.02.2005, you wrote:
>Hi=20
>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 =3D 1.6/0.5 micro gm
>2. Flow through: ELISA O.D =3D 0.3/0.5 micro gm
>3. Eluted fraction: ELISA O.D =3D 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
>
>--=20
>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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