DOUBLE IMMUNODIFFUSION TECHNIQUE PRACHEE RAJPUT
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DOUBLE IMMUNODIFFUSION TECHNIQUE
PRACHEE RAJPUT
SYNOPSIS Introduction Historical Background Types of Immunodiffusion Principle Ouchterlony Double Diffusion Required Material Methodology Interpretation Zonal reaction Application Example the test Recent research References
INTRODUCTION.
Immunodiffusion is a technique for studying reaction between antigens and antibodies by observing precipitates formed by the combination of specific antigens and antibodies that have diffused in a gel in which they have been separately placed.
HISTORICAL BACKGROUND
It was firstly developed by Dr MORRIS GOODMAN.
In 1948, Ouchterlony double diffusion technique was firstly developed by Dr ORJAN OUCHTERLONY.
TYPES OF IMMUNODIFFUSION
Immunodiffusion
Single Radial Immunodiffusion
Double Immunodiffusion
OUCHTERLONY DOUBLE DIFFUSION TECNIQUE
It is used extensively to check anti-sera for the presence and specificity of antibodies for a particular antigen.
Antibodies and Ag form lattice structures that will develop into visible precipitate.
Formation of Ab-Ag lattice depends on,• Ab must be bivalent.• Ag must be bivalent or polyvalent.
PRINCIPLE
In ouchterlony double diffusion, both antigen and antibody allowed to diffuse in to the gel. This assay is frequently used for comparing different antigen preparations. the method is called double since the antigen and antibody are allowed to migrate towards each other in a gel and a line of precipitation is formed where the two reactants are meet. This precipitation is highly specific and is used by people working with diagnosis and protein detection technique.
Cont…
The pattern of lines that from can be interpreted to determine whether the antigen are same or difference as illustrated below.
I. Pattern of identity AII. Pattern of partial identity BIII. Pattern of non identity C
REQUIRED MATERIAL
Chemicals :- Agarose, Assay Buffer(PBS) ,anti-serum, test antigens, (ph 7.2),0.05% Sodium azide.
Equipments:- incubator (37°C), micro pipette, tips, weighing balance, Gel punch with syringe.
Glassware's:- Glass plate, Template, conical flask measuring cylinder.
Reagents :- alcohol, distill water.
METHODOLOGY
Prepare 25 ml of 1.2 % agarose (0.3g/25ml) in assay buffer by boiling to dissolve the agarose completely.
Cool the solution 55 to 60°C pour 4ml/plate on to 5 grase free glass plates placed on a horizontal surface, allow get to set for 30 minutes.
Punch wells by keeping the glass plate on the template.
Fill the lower well 10 micro liter of anti-serum and the upper Two wells with 10 ml of each antigen.
Keep the glass plate in a moist chamber over night at 37°C.
After incubation, observe for opaque precipitin lines between the Antigen and anti-sera wells.
INTERPRETATION
If a pattern A or pattern of identity is observed between the antigen and the anti-serum it indicates the antigens are immunologically identical.
If a pattern B, it indicates that the antigen are partially similar or cross reaction.
If a pattern C, it indicates that there is no cross reaction between the antigen i.e. the two antigen are immunological unrelated.
ZONAL REACTION
Post zone reaction Pre zone reacton Equivalence zone
reaction
DOUBLE DIFFUSION
Double Immunodiffusion result
EXAMPLES
Home Pregnancy Test
It is a modification of an agglutination experiment.
It is called agglutination inhibition.
It is good because, it is sensitive to small amounts of Ag.
APPLICATIONS
Identification of Fungal antigen. Example- coccidiomycosis, Histoplasma.
It is used for measuring hormones serum proteins, drugs.
This test is commonly used in the clinical laboratory for the determination of Immunoglobulin levels in patient sample.
RECENT RESEARCH
Gomes S.F., Silva-Da-Marques H.S.
19 March 2012.
Effect of Pretreating serum samples on the Performance of a Latex agglutination test for Serodiognosis of Paracoccidioidomycosis.
Institute of Science Biologic and Evandro Chagas, Federal University of Para, Belem, Brazil.
REFERENCES
Baveja .C.P.,2007,Antigen-Antibody Reaction :In Text Book of Microbiology,2nd Edition Arya Publishing Company, pp:44-46.
Chirikjan.G.J., 1995,Anttibody Interactions With Antigens: In Plant Biotechnology Animal Cell Culture Immunology, Vol.1 Jones and Baraet Publishers,pp: 80-95.
Hay,C.F, Westwood, M.R,2002,Precipitation Reactions: In Practical Immunology, Black Well Publishing Compony,pp:92-95.
,Wilson, K, Walker, M.J, 1994,Electrophoresis Technique: In Principle and
Technique of Biochemistry,7th Edition, Cambridge University Press, pp:390-395.
CONT....
http://www.nhc.ed.ac.uk.(12/10/2012)
http://www.btprotocolsmaulikblogspot.com.(10/10/2012)
http://www.amrita.vlab.com.in(10/10/2012)
http://www.sbs.utexas.edu/sanders/Bio347/Lectures/htm.(8/10/2012)
http://www.ncbi.nlm.nih.gov.(12/10/2012)
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