Thursday, April 19, 2007

Counter current immunoelectrophoresis

AIM: To check antisera for the presence and specifity of antibodies for a particular antigen by Counter current immunoelectrophoresis.

Counter current immunoelectrophoresis (CCIEP) is a rapid version of Ouchterlony double diffusion (ODD) technique. The technique is used to check antisera for the presence and specificity of antibodies for a particular antigen.


Prepare10 ml of 1.5% agarose(0.15g/10ml) in 1X reservoir buffer by adding dry agarose to the buffer and heating slowly to dissolve the agarose completely.
Mark the end of the slide that will be towards positive electrode during the electrophoresis.
Place the slide on a leveled tabletop and quickly pipette 7ml agarose onto 50X75mm slide, spreading while releasing the agarose. Allow solidifying for 15min.
Place the gel plate on the template holder and fix it for CCIEP. Punch 3mm wells with the gel punch at position indicated for CCIEP.
Place the slide in electrophoresis tank and fill the tank with buffer.
Add 10 l of antigen in the four wells towards –ve electrode and 10 l of positive control antiserum and three test antisera in wells towards positive electrode.
Apply 50v and allow the electophoresis to continue for about 45 min.
Observe for precipitin line between the antigen and antisera wells.


Here, pattern shows precipitin line between antigen and antisera wells.

Precipitin line indicates the presence of antibody the antigen in the test sera.
The presence of more than one precipitin lines indicate the heterogenicity of the antibody for the antigen in the test sera.
The absence of the precipitin line indicates the absence of any antibody for the antigen in the test sera


gowri said...

u can put pictures for expreminets and can also add trouble shooting wen carryin out the experiments and interpretation of results and antigen quantity is 10 microlitres and not 10 l :)

sudha said...
This comment has been removed by the author.
sudha said...

Hey I tried ur protocol it is giving good result but i feel you should have given some pictures to make the analysis more proper.........dont you think the concentration of antigen and antibody should be specified instead of the amount......the titre is important

gertrude said...

thanks alot its helping me and other students in my class

accumaximum said...

Many scientific supply companies can sell you pipettes, of nearly every kind, whether brand name or generic, but what is it exactly that your laboratory needs?


raja said...

What buffer i have to use to isolate antigen from nematode parasite in fish my mail id is kindly inform me the procedure

cusabio1 said...

Possible mechanisms of genetic factors in the development of IL-6 in T2DM, smoking, mental stress, obesity can cause oxidative stress enhanced oxygen free radicals increased pro-inflammatory transcription factor nuclear factor κB activation. NF-κB activation induced IL-6 promoter of the gene expression of IL-6 protein. Excess of IL-6 by interfering with the insulin receptor signal transduction and induce insulin Recombinant human Transcription intermediary factor 1-beta protein