Ouchterlony double diffusion

Ouchterlony double diffusion

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  OUCHTERLONY DOUBLE DIFFUSION METHOD PresentedTo: Dr. R. Saranya Assistant professor Department of Microbiology Sacred Heart Scared (Autonomous) Tirupattur Presented By: A. R. Deborah (BP211501) I M.Sc. Applied Microbiology Sacred Heart College (Autonomous) Tirupattur
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  PRINCIPLE  Ouchterlony doubleimmunodiffusion (also know as passive double immunodiffusion) is an immunological technique used in the detection, identification and quantification of antibodies and antigens. (Analyzing the antigen and antibody)  Immunodiffusion in gel are classified as single and double diffusion. In ODD, double diffusion of both antigen and antibody are allowed to diffuse into the gel.  This assay is frequently used for comparing different antigen preparation each containing single antigenic species are allowed to diffuse from specific separate well against the antiserum.  Depending on the similarity between the antigen different geomatical patterns are produced between antigen and antiserum.  The pattern of line that form can be determine whether the antigen are same or different.
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  MATERIALS REQUIRED  Equipment: Incubator (37 ͦ )  Glassware : Conical flask, Measuring cylinder, Glass plate.  Reagents : Alcohol, Distilled water, Agarose, 10X assay buffer, Antiserum, Test antigens.  Other Requirements : Micropipette, Moist Chamber tips, Gel puncher with syringe, Template.
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  PROCEDURE  Preparation ofAgarose: 1% of Agarose was boiled to dissolve in 1X assay buffer, cool to 55 ͦ to 60 ͦ C, Take clean glass slide and pour the agarose and allow it to set.  By using gel puncher cut the wells by keeping the glass plate on template.  Fill the wells with 10 µl each of the antiserum and antigens.  Incubate the glass plate in the moist chamber overnight at 37 ͦ C.
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  OBSERVATION AND INTERPRETATION The Presence of precipitant lines between the antigen and antiserum wells were observed like,  Pattern of identity  Pattern of partial identity  Pattern of Non – identity
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  Pattern of IdentityA The antibody in the antiserum react with both the antigens resulting in the continuous line of precipitate. The antibody can’t distinguish the two antigen (the two antigens are immunological identical)
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  Pattern of partialIdentity B The pattern of partial identity the antibodies in the antiserum react more with one of the antigen than the other.
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  Pattern of Non– Identity C The pattern of non – identity none of the antibodies in the antiserum react with antigenic determinant that may be present in either antigen.
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  THANK YOU !