17. Counter Current Immounoelectrophcoresis (CIEP)


A.Principle :


In CIEP, two parallel wells in agar gel (pH 8.0) are filled with antigen and antibody move towards each other at a faster rate. The antigen migrates towards the anode and the antibody moves in opposite direction as a result of electroendosmosis and a precipitin line is formed where they meet. 


B.Materials :


a.Noble agar


b.0.01 M Veronol buffer, pH 8.6 (Appendix I).


c.Normal saline


d.Amido black (1%)


e.2% acetic acid


f. Glass slides


g.Humid chamber


h.Electrophoretic tank with power supply


i.Whatman-3 filter papers


j.Template and gel punch.




C. Method:


1.1%  Noble  agar  in veronol sodium acetate buffer (0.01M pH 8.6) is layered over glass  slides and wells are made as described under double diffusion technique.


2.Fill  the  buffer  tank with  veronol  buffer and  see  that the  level of buffer in the two  compartments are same.


3.Arrange  the agar  slides  in electrophoretic  chamber  and connect  to  the buffer with Whatman-3 filter paper wicks.


4.Make pre-run for 15 minutes at a constant current of 7.5 mA per slide.


5.Disconnect the power supply and apply 5 µl of antibody in the anodic well and 5 µl of antigen in the cathodic well.


6.Connect  the electrodes  to  the power  supply  and run  electrophoresis  for 1 hr at a constant current of 7.5 mA per slide.


7.After the run, disconnect the power supply and incubate the slide in humid chamber at room temperature for 12 hr.


8.Observe  the slides  for  precipitin bands  and  the slides may be stained for improved sensitivity in 1% amido black as described under double diffusion.   


Reference :


Desowitz, R. A. & Una, S. R., J. Helminthol, 50 (1976), 53.