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

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

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

Detection of food adulterants

Types of ppt rxns

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

Eg: CRP test , ascoli thrombo pptn test

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

Eg: VDRL test for syphilis

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

Eg : Kahn test for syphilis

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Immunodiffusion / Gel precipitation

PPtn rxns are allowed to occur in gel

Types

Single diffusion in one dimension

Double diffusion in one dimension

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
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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 Panikers 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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