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.