Radial Immunidiffusion - For antigen quantification (Sep/23/2008 )
I want to ask you all that what can be the reason for failure of immunodiffusion technique.
1)overlay slide with agar which is mixed with desired antibody such that the concentration of agar remains 1.5% and that of antibody is equal to that of antigen under test.
3)Add variable concentration of std antigen along with the unknown
4)Keep in moist conditions at room temperature overnight.
But the Precipitin ring is not visible in any well..............
by mixing the antibody with the agar you are not going to allow the antigen to diffuse. it will precipitate at the surface of the punched hole.
when we performed radial immunodiffusion we had a central hole with either antibody or antigen (depending on which we were using as the unchanged parameter) and holes surrounding the central hole (equidistant from central hole) which contained the other. a precipitin band would form between the central and satellite holes.
generally, we had antigen central and different antibodies in satellite holes to see which would react with the antigen.
Radial immunodiffusion is an old method.
I think most people switch to ELISA, which is more sensitive and requires only a small amount of antigen and antibody.
Early tests for human IgG, M, A, D and light chain K and L were done by this method...Yes, Ab in the gel and ag in the well. the ring diameter proportional to the concentration of ag placed in the well. Unknowns were read from the dose response curve constructed by known standards.
So...does you ab recognize the ag? Is the ab polyclonal? We have to ppt the ag not only bind to it.
You can also try making an elisa or try another method.
Did you cool down the agar to 50/55 oC before the antibody was added?
Otherwise the possibility is there that the antibody was degraded en no longer functional.
Stupid question did you add the antibody?
In addition to what has been said above: What is the size of your antigen? Haptens and small polypeptides won't work as you can't bind multiple Abs, which won't give you a ring of precipitate. Of course your antigen could be a huge protein as well, unable to diffuse through the pores in the agar...