Ouchterlony Double Diffusion

33 EXPERIMENT NO: 15. DATE 6/12/22 DUCHTERLONY DOUBLE DIFFUSION AIM To study the reaction pattern of an ontigen with a set of artibodies by Duchter long rouble diffusion method PROCEDURE repace 10ml of 1% agreese (as given in important instructions) to 1. cool the solution to 55-60°C 4 pour 5ml / plate on : grease free glass plates placed on holigental surface Allow the gel to set for 30min. 3. Place the glass plate on the template provided 4. punch wells with the help of the gel punches coresponding to the markings on the template . lese gentle suction to avoid terming of lugged well. Add 10rl each of the antiserum 4 the corresponding antigens 5. the wells as shown to keep the glass plate in a moist chamber overnight lines at 37°c 6. After incubation. observe for opaque precipitin 7. between the antigen El antisierm wells. ABC. OBSERVATION It was observed that the antiseem A showed Arc (Identity pecipitin Pattern) / Antiserum B showed a spur (Partial odentity Peecipitin patterns) cl Anoserum C showed a interset (Non Identity - precipitin pattern) RESULT A full dencity (ie / a continuous line) - Line of precipi- tation at their junction forming an are represents serologic identity or the presence of a common epitope in antigens when Antiselum A was added

34 Partial identity (ie, a continuous line with a spue at one end ) - The 2 antigens shall a common epitope in antigers when antisirum B was added Non identity (ie, the 2 lines cross completely) -A pattern al crossed lines demonstrates 2 separate reactions unrelated of that the compared antigens antisierm are C was indicates shall no common epitopes when al added DISCUSSION Ouchterlong immunodiffusion assay developed by Swe dish physician Orjan Ouchterlony is used the for the detection of antigens El antibodies 4 determination of homologies between antigens Ouchteclong double diffusion is frequently used for comparing different antigen preparation It the andiger solution contains several diff antigens that an react with the antibo- dies of the antiserum then multiple lines of peccipitation will be produced. The method is called 'double' referring to the fact that in this procedure / antigen 4 antibody all allowed to migrate -towards each other in a gel al a line of percipitation is formed where the 2 reactants meet this precipitation reaction is highly specific. R