16. Double Diffusion


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.





Ouchterlony, O. Diffusion in gel methods for immunological analysis. Prog. Allergy, 6, (1962), 30.