Double immunodiffusion technique

by prachee rajput
Zoologist
Bhopal, INDIA

1. DOUBLE IMMUNODIFFUSION TECHNIQUE PRACHEE RAJPUT

2. SYNOPSIS Introduction Historical Background Types of Immunodiffusion Principle Ouchterlony Double Diffusion Required Material Methodology Interpretation Zonal reaction Application Example the test Recent research References

3. 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.

4. HISTORICAL BACKGROUND It was firstly developed by Dr MORRIS GOODMAN. In 1948, Ouchterlony double diffusion technique was firstly developed by Dr ORJAN OUCHTERLONY.

5. TYPES OF IMMUNODIFFUSION

6. 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.

7. 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.

8. Cont… The pattern of lines that from can be interpreted to determine whether the antigen are same or difference as illustrated below. Pattern of identity A Pattern of partial identity B Pattern of non identity C

9. 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.

10. METHODOLOGY

11. 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.

12. ZONAL REACTION Post zone reaction Pre zone reacton Equivalence zone reaction

13. DOUBLE DIFFUSION Double Immunodiffusion result

14. 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.

15. 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.

16. 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.

17. REFERENCES Baveja .C.P.,2007,Antigen-Antibody Reaction :In Text Book of Microbiology,2 nd 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,7 th Edition, Cambridge University Press, pp:390-395.

18. 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)