An investigator wanted to produce rabbit antiserum specific for mouse IgG, so she injected a rabbit with purified mouse IgG. However, to her dismay, the antiserum reacted with all isotypes of mouse antibodies. Why? How could she modify the procedure to obtain IgG specific serum?
During the maturation of B-cells, different constant domains are spliced to the variable domain - therefore, any antibodies directed to epitopes within the framework part of the variable domains would recognize any isotype. To get an IgG specific serum, one might immunize with just the FC fragment of the IgG
During the maturation of B-cells, different constant domains are spliced to the variable domain - therefore, any antibodies directed to epitopes within the framework part of the variable domains would recognize any isotype. To get an IgG specific serum, one might immunize with just the FC fragment of the IgG
What happened to your colleague is normal. If she immunized with whole mouse IgG not only the variable domains with conserved frameworks (see above), but also the constant domains of the light chains were there. They can be recognized independently of the isotype. Even immunizing with the Fc there could be shared regions between some isotypes. One solution is to deplete the antisera in a column with immobilized antibodies of the undesired isotypes. Theoretically only IgG specific antibodies could pass through the column without being retained.
But what I would really suggest is to buy anti-IgG antibodies. Many companies produce them and some guarantee minimal residual recognition of other isotypes and other species IgG.
There is still an option to use the produced antiserum.
Negative affinity chromatography. Insolubilized murine IgM. This contains light chains and so the antibodies against L-chains are removed. Also the µ-chain is important to remove cross-reactive antibodies from the antiserum. Both specificities, the one against L-chains and the cross reactivities cause the observed phenomenom. Threrefor the results getting better, when also a negative affinity chromatography against murine IgA is included. The differences between the major types of heavy chains are not that large...
There is one company using this preparation scheme: http://www.seramun.com/home.html
Hi Patil
I agree with above answers. In this case, the antiserum is already obtained, therefore the negative adsorption against immobilized mouse IgM and IgA is the best option.
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