Antibody Purification: better method than western technique?
dspinella at chugaibio.com
Fri Feb 27 11:47:31 EST 1998
> I have serum from an immunized bunny.
> Is there a more gentle or efficient method for the purification of
> antigen-specific antibodies from total serum than the technique
> in which you western blot your antigen and affinity purify the specific
> antibodies folloed by a nasty pH elution?
> -any suggestions appreciated!
I think a lot depends on two factors: 1. How pure is your intial
antigen preparation? and 2. What are you going to do with the purified
If your antigen is relatively pure, it can obviously be conjugated to
Sepharose beads and used to construct a traditional affinity column over
which you can run whole serum. Low pH elution (Glycine-HCl, pH 2.5
followed by neutralization of all fractions with 1M Tris) is actually
fairly gentle for most antibodies. High pH elution can also work in some
cases. When run on an affinity column it is also quite efficient. I
presume however that if you are trying to purify by Western blotting and
removing the bound antibodies from a certain region of the blot (is that
truly what you mean?), there are manny contaminating antigens in your
initial immunogen. I think purifying antibodies this way has to be very
tedious -- and your yield must be awfully low.
You have several options. One is to purify the antigen before
immunization. This can be done by simply cutting out a band from a
stained PAGE gel, (no need to elute the band as acrylamide turns out to
be a pretty fair adjuvant), grinding it up with some Freund's and
injecting. Alternatively, you can try adsorption techniques to remove
contaminating antibodies. This requires that you have some sort of
complex antigen preparation lacking the antigen of interest but having
most or all of the contaminating antigens. Since I don't know what
system you are working in, its hard to know whether this will be
possible. Finally, if you truly need pure antibody, the best bet is
still to make a monoclonal in mice. In this way you don't have to worry
about the purity of the antigen prep -- as long as you selcet out a good
clone of hybridoma, you're set.
Sorry this anwer is a bit vague. If you provide more details about your
system perhaps I can be of more help.
More information about the Methods