OUCHTERLONY DOUBLE DIFFUSION (FOR ANTIBODY TITRATION)Objectives
To learn the technique of ouchterlony double diffusion
Interaction between antigen (Ag) and antibody (Ab) at themolecular level forms the basis for several techniques that are useful inmodern day scientific studies and in routine clinical diagnosis.Ouchterlony double diffusion (ODD) or immunodiffusion technique is oneof the simplest techniques extensively used to check antisera for thepresence of antibodies for a particular Ag and to determine its titer.
100mg of agarose was boiled to dissolve in 10ml of 1X assaybuffer. Cool to 55°C.
5ml of the gel solution was poured onto a clean glass plate placedon a horizontal surface. The gel was allowed to set it tookapproximately 20-30 minutes.
The gel plate was placed on the template provided. Wells in the gelwas punched with the help of a gel punch corresponding to themarkings on the template. Gentle suction was used to avoid formingrugged wells.4.The test antiserum was serially diluted up to 1:32 dilution asfollows:
Take 20ul of 1x assay buffer in each of the five vials.
Add 20ul of test antiserum into the first vial and mix well. Thedilution of antiserum in this vial is 1:2.
Transfer 20ul of 1:2 diluted antiserums from the first vial into thesecond vial. The dilution in this vial is 1:4.
The dilution was repeated up to fifth vial.
10ul of the antigen was added to the center well and 10pl each of neat (undiluted), 1:2, 1:4, 1:16, 1:32 dilutions of antiserum into thesurrounding wells as shown in figure 3.
The plate was placed in a moist chamber and incubated at roomtemperature, overnight.
After incubation, opaque precipitin line between the antigen andantisera wells was observed.
The observation was recorded.