A. Principle :
This technique is based on the
ability of antibodies to form precipitin lines specifically with the antigen.
Free diffusion of both the antigen and antibody take place in agar gel and the
resulting precipitates are normally visible to the naked eye.
B. Materials :
a.Noble agar
b.0.05 M Phosphate buffer saline,
pH 7.2 (Appendix I)
c.Normal saline
d.1% Amido black
e.2% Acetic acid
f.Glass slides
g.Humid chamber
h.Whatman-3 filter paper
i.Template and gel punch
C. Method :
1.Take glass slides ( 25 x 75 mm ), clean with alcohol
and place on a horizontal level
surface.
2.Dissolve noble agar (1%) in PBS
(0.05M, pH 7.2) by indirect boiling.
3.To ensure good contact between
the gel and glass plate, the glass plate may be precoated with a thin layer of
agar solution (0.5%).
4.Pour 3 ml of 1% agar solution on
each glass slide avoiding trapping of air in the gel.
5.After polymerization (5 minutes
time), transfer the gel slides to humid chamber.
6.Punch wells of 3 mm diameter
with an interspace of 5 mm in agar gel using a template and a gel punch. The
punched gel is aspirated out.
7.Load 10 µl of
antigen and antibody
in separate wells
and keep the slide in humid chamber for 24 hr at room
temperature and at 40C for another 48 hr.
8.Note the development of
immunoprecipitate bands from time to time ( After optimal development of
precipitin lines as
visualized by naked
eye, the slide may be stained with 1% amido black for improved
sensitivity).
9.After incubation for an
appropriate period, keep
the agar slide in normal saline with
several hourly changes to remove unprecipitated proteins.
10.After washing the slide, dry
the gel at room temperature by keeping a wet Whatman-3 filter paper over it
without trapping air between the filter paper and the gel.
11.After drying, remove the filter
paper by slight wetting and immerse in 1% amido black for 10 minutes.
12.Transfer the
slide to destaining
solution ( 2 % acetic
acid ). Change the destaining solution 2 or 3 times till dark, sharp
protein bands are clear.
Reference:
Ouchterlony, O. Diffusion in gel
methods for immunological analysis. Prog. Allergy, 6, (1962), 30.