mrc7 at hermes.cam.ac.uk
Wed Jul 11 05:59:42 EST 2001
On Mon 09 Jul, Dr. Attila Tarnok wrote:
> We want to remove antibodies from from the serum of patients for further
> testing of the serum. I remember that this can be done by Protein A
> Can anyone from the list provide a method how to perform this depletion?
> Thank you.
> Attila Tarnok
Please be aware that Protein A will only bind a fraction of the antibodies
in the patients serum.
Protein A binds to human IgG1, IgG2 and IgG4 very well through the Fc
binding site. It will also bind to IgG3 of the G3m(s) and G3m(t) allotypes
(common asian alleles), but not G3m(b) or G3m(g) alleles.
Protein A will also bind to some other classes and subclasses of human
immunoglobulin through a Fab binding site which is partly determined by the
Variable region subgroups of the antibodies.
If you intend to remove all IgG from the serum then Protein G is a better
alternative as it does not show the subclass and allotype dependency of
Media and columns coupled to Protein A or Protein G (eg sepharose or
agarose acrylamide) are available from major suppliers. Methodoloy is very
simple and written up in many immunological methods texts.
1] Equilibrate your column in PBS.
2] Pass the serum through (capacity is usually around 2-10 mg of IgG per ml
of gel so use 5-10ml gel per ml of serum if you wish to deplete)
3] Collect the eluate which is depleted of the Ig
4] You can regenerate the column and elute bound Ig using a low pH wash
with for example 0.2M Glycine HCl at pH 2.5
For small volumes the process can be scaled down to micro-columns or even
batch methods in eppendorf tubes. Also commercial 'filters' are available
with Protein A or G attached to the membranes.
Mike Antibody website <URL:http://www.path.cam.ac.uk/~mrc7/>
M.R. Clark, PhD. Division of Immunology
Cambridge University, Dept. Pathology
Tennis Court Rd., Cambridge CB2 1QP
Tel.+44 1223 333705 Fax.+44 1223 333875
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