Antigen Antibody Precipitation Reactions 5v514c

ANTIGEN- ANTIBODY REACTIONSPRECIPITATION REACTIONS

by Vinu P .V

ANTIGEN ANTIBODY REACTIONS Characterized as • Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate)

Bonds: • • • •

Hydrogen Ionic Hydrophobic interactions Van der Waals forces

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PRECIPITATION REACTIONS -(Immunologic tests) •Soluble Ag + Ab

opt T & pH

insoluble ppt

Normal saline

• Antibodies------Precipitins • Antigens -----Precipitinogen • Occurs in liquid media, or in gels (agar, agarose)

• Ppt rxns ------highly sensitive for detection of antigens & relatively less sensitive for detection of antibodies • Either qualitative or quantitative

Precipitin curve ( Marrack hypothesis) • •

Keeping Ab conc. fixed pptn occurs only at the zone of equivalence

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Applications • Diagnosis of infections • Identify blood , semen stains etc in criminal investigations

• Detection of food adulterants

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Types of ppt rxn’s 1. 2. 3. 4.

Ring test Slide test Tube test Immunodiffusion/ gel precipitation • • • •

Single diffusion in one dimension Double diffusion in one dimension Single diffusion in two dimension Double diffusion in two dimension

5. Immunoelectrophoresis 6. Electoimmunodiffusion • • •

Counter current immuno electrophoresis (CIEP) Rocket electrophoresis Laurells electro immunodiffusion in two dimension 6

Ring test • Antigen solution taken over column of antiserum in a test tube ppt at the junction

• Eg: CRP test , ascoli thrombo pptn test 7

Slide test • A drop each of antigen & antibody on a slide Mixed by rotation Floccules

Eg: VDRL test for syphilis

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Tube test • Antigen soln & antiserum (patients serum) taken in a test tube mixed Incubated & observed for ppt

• Eg : Kahn test for syphilis 9

Immunodiffusion / Gel precipitation PPtn rxns are allowed to occur in gel

Types •

Single diffusion in one dimension

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Double diffusion in one dimension

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Single diffusion in two dimension

•

Double diffusion in two dimension

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Single diffusion in one dimension (Oudin method)

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Double diffusion in one dimension (Oakley - Fulthrope procedure)

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Single diffusion in two dimension (Mancini method/ Radial immunodiffusion)

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Double diffusion in two dimension (Ouchterlony method)

• Molten agar poured in glass slides / petry dishes • Test soln of Ag & Ab diffuse frm the wells to form a ppt at the equilence zone

• If two ppt lines fuse ( V- shaped)  identical Ags • Ppt lines fuse and project (Y- shaped) partial identity • Ppt lines cross each other  non identity

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Immunoelectrophoresis •

resolution

• Used for analysis of Serum Protein & immunoglobulin

Test -Agar in a slide 1.

Well cut is filled with Ag mixture, electric current is applied  separation  separate spots based on electric charge on the proteins

2. Rectangular trough is cut parallel to the separation and filled with Ab  incubated 3. Arc shaped ppt bands, r better visualized by staining 15

Electoimmunodiffusion (Electro immuno assay) • Counter current immuno electrophoresis (CIEP) • Rocket electrophoresis • Laurells electro immunodiffusion in two dimension Counter current immunoelectrophoresis • Detect Ag & Ab with opposite charge • Move towards each other , in electric field line of ppt

Eg: Detection of HBS Ag in serum,

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Rocket electrophoresis • Quantitative estimation of human plasma antigens & viral antigens • Ab is added to molten agar, poured on glass slide • Wells r cut & filled with antigen soln

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• Current applied , allowed to diffuse, forms rocket like ppt lines

• Area of each antigen, its height ∞ initial conc of antigen

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Laurells electro immunodiffusion in two dimension • Used to quantify each Ag in a mixture • Ag mixture is first electrophoritically separated into diff components immunodiffusion

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REFERENCES 1. Ananthanarayanan and Paniker’s text book of microbiology, seventh edition, orient longman,page no:8284. 2. Prescott, Harley and Kleins Microbiology, page no:771-773. 3. Pharmaceutical Biotechnology by S.P Vyas , V. K . Dixith, Page No 466-471 4. www.wikipedia.com

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