1. Online Counseling Resource YCMOU ELearning Drive…
Dec 18, 2007
Online Counseling Resource YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra Open University, Nashik – , India

2. OC-SBT052-CP4-01 Introduction
Dec 18, 2007
OC-SBT052-CP4-01 Introduction
Programmes and Courses
SEP–SBT052–Unit-04
© 2008, YCMOU. All Rights Reserved.

3. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Dec 18, 2007
Credits
Academic Inputs by
Arun Punaji More.
M.Sc. (Microbiology)‏
Experience: 11 Years
© 2008, YCMOU. All Rights Reserved. 3

4. How to Use This Resource
Dec 18, 2007
How to Use This Resource
Counselor at each study center should use this presentation to deliver lecture of minutes during Face-To-Face counseling.
Discussion about students difficulties or tutorial with assignments should follow the lecture for about minutes.
Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.
Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.
Appear several times, for all the Self-Tests, available for this course.
Student can use handouts for last minutes preparation just before end exam.
© 2008, YCMOU. All Rights Reserved.

5. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Learning Objectives
After studying this module, you should be able to:
Define affinity, avidity and specificity of antibody
Describe various methods of agglutination reaction
Describe applications of agglutination reaction in diagnosis of diseases.
© 2008, YCMOU. All Rights Reserved.

6. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Introduction
When immune system mount an immunological response to an invading pathogen or its toxic product, effector immune cells are produced to kill invading pathogen and antibodies are produced to neutralize the pathogen and its toxic product.
Since the antibodies have got specificity with its corresponding antigen, antigen antibody reaction in vitro is used to diagnose an infection.
In this module the students shall learn the underlining principle of diagnosis involving Ag-Ab reaction in vitro.
© 2008, YCMOU. All Rights Reserved.

7. Nature of Antibody-Antigen Reaction-1
Dec 18, 2007
Nature of Antibody-Antigen Reaction-1
The antibody react with its corresponding antigen at optimum pH and electrolyte concentration in reversible manner.
Antibody react with its corresponding antigen which has stimulated its production very specifically.
The antigen antibody combining involve following week forces:
Hydrogen bonds
Electrostatic bonds
Hydrophobic bonds
Van der Waal forces
© 2008, YCMOU. All Rights Reserved.

8. Nature of Antibody-Antigen Reaction-2
Dec 18, 2007
Nature of Antibody-Antigen Reaction-2
Each antigen-antibody reaction is unique in its following properties
Affinity of Ag-Ab reaction
Avidity of Ag-Ab reaction
Specificity of Ag-Ab reaction
© 2008, YCMOU. All Rights Reserved.

9. Nature of Antibody-Antigen Reaction-3
Dec 18, 2007
Nature of Antibody-Antigen Reaction-3
Affinity of Ag-Ab reaction:-
Strength between single antigen determinant and a single antibody combining site is expressed in term of affinity of Ag-Ab reaction.
Affinity of Ag-Ab is also product of attracting and repulsive forces between Antigen and antibody.
© 2008, YCMOU. All Rights Reserved.

10. Nature of Antibody-Antigen Reaction-4
Dec 18, 2007
Nature of Antibody-Antigen Reaction-4
Avidity of Ag-Ab reaction:-
The total overall strength between antigen and antibody combining is termed as avidity of antigen antibody reaction.
The avidity is dependent on the valency of antigen as well as valency of antibody.
© 2008, YCMOU. All Rights Reserved.

11. Nature of Antibody-Antigen Reaction-5
Dec 18, 2007
Nature of Antibody-Antigen Reaction-5
Affinity and avidity of Ag-Ab reaction:-
High affinity means interaction or association of antigen and antibody remain stable thus can be easily detected in vitro.
High avidity means interaction or association of antigen and antibody remain stable thus can be easily detected in vitro.
© 2008, YCMOU. All Rights Reserved.

12. Nature of Antibody-Antigen Reaction-6
Dec 18, 2007
Nature of Antibody-Antigen Reaction-6
Particulate antigens when react with antibodies at equivalent concentration, particulate antigens get coagulated forming visible clumps of particulate antigens; this clumping of particulate antigens is called as agglutination of antigens.
When pathogenic organisms enter into the body antibodies are produced against number of surface antigens of the pathogens.
When the serum containing the antibodies against such surface antigens is mixed with the culture suspension of the pathogen, the antigens get cross linked by the antibodies resulting in the agglutination of the pathogens .
© 2008, YCMOU. All Rights Reserved.

13. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-1
The clumping formation of the particulate
antigens is explained with lattice of
antigen formation when the antigen
molecules get cross linked at the
equivalent concentration of antigens
and antibodies.
© 2008, YCMOU. All Rights Reserved.

14. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-1
The agglutination is carried out in number of ways and accordingly there are different types of agglutination tests
Direct agglutination test
Indirect agglutination (or passive agglutination test)‏
Haemagglutination
Haemagglutination inhibition test
Coombs' test
© 2008, YCMOU. All Rights Reserved.

15. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-2
Direct agglutination test:-
The direct agglutination test is carried out to detect particulate antigens such as red blood cells, bacteria and fungi by serum antibodies.
In this test fixed amount of known particulate antigen solution is mixed with different solution of serum antibodies having serially diluted twofold times to determine the relative titre of the antibodies in the serum.
© 2008, YCMOU. All Rights Reserved.

16. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-3
Passive agglutination test:-
Very microscopic antigens such as originated from viruses though crossed linked they do not form visible agglutination;
Therefore such antigens are bound to some larger inert particles and are subjected to agglutination test.
Here the micro latex particles are coated with such antigens of infectious agents and are mixed with anti-sera which bring about visible agglutination of these latex particles making the test visible.
© 2008, YCMOU. All Rights Reserved.

17. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-4
Haemagglutination test:-
The antibodies against the surface antigens of red blood cells result into cross linking of the cells resulting in the agglutination which is called as haemagglutination.
Haemagglutination is used to determine blood types or blood groups.
© 2008, YCMOU. All Rights Reserved.

18. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-5
Haemagglutination inhibition test:-
Like antibodies against the surface antigens of red blood cells cause agglutination of these cells, certain viruses are also having capability of bringing about the agglutination of red blood cells.
To diagnose the infections caused by such viruses haemagglutination inhibition test is carried out.
The viruses which cause haemagglutination and are diagnosed by haemagglutination inhibition test are influenza, measles, mononucleosis, mumps viruses, etc.
© 2008, YCMOU. All Rights Reserved.

19. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-6
Haemagglutination inhibition test:-
In this test anti-sera is mixed with fresh red blood cells and then with suspected virion, if antibodies against these suspected viruses are present the antibodies against them will neutralize them and therefore they can not bring the haemolysis of red blood cells thus inhibition of haemagglutination is positive indication of particular viral infection.
© 2008, YCMOU. All Rights Reserved.

20. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-7
Coomb's test:-
This is modified agglutination test.
Some antibodies can not agglutinate the particulate antigen though they react with the antigens.
Such antibodies are called as non agglutinating antibodies.
Therefore the antibodies against such non agglutinating antibodies are produced in rabbit; these are called as anti- immunoglobulins.
These anti-immunoglobulins when added into the antigen and antibody mixture, bring about the agglutination of the particulate antigens.
© 2008, YCMOU. All Rights Reserved.

21. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-8
Coomb's test:-
The Coomb's test is used to detect the antibodies against the Rh antigen on red blood cells in mother's blood.
Coomb's test is routinely used to detect Rh incompatibility.
In the indirect Coomb's test Rh antibodies are detected in Rh negative mother's blood.
Whereas the direct Coomb's test detect the Rh positive red blood cells of Rh positive fetus.
© 2008, YCMOU. All Rights Reserved.

22. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-9
Direct Coomb's test:- + ↔ Y
Patient’s RBCs
Coombs Reagent
(Antiglobulin)‏
© 2008, YCMOU. All Rights Reserved.

23. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Agglutination-Test-10
Indirect Coomb's test:- Y
Patient’s
Serum
Target
RBCs + ↔
Step 1 + ↔ Y
Coombs Reagent
(Antiglobulin)‏
Step 2
© 2008, YCMOU. All Rights Reserved.

24. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
What We Learn…..
Various methods of agglutination reactions.
Mechanisms of agglutination reactions.
Applications of agglutination reactions.
Coomb's test and its applications.
Haemagglutination inhibition test.
© 2008, YCMOU. All Rights Reserved.

25. Critical Thinking Question
Dec 18, 2007
Critical Thinking Question
Why agglutination reactions are more widely used over the other serological reactions in bacterial infections?
© 2008, YCMOU. All Rights Reserved.

26. Hints For Critical Thinking Question
Dec 18, 2007
Hints For Critical Thinking Question
Particulate antigens
Specificity
Sensitivity
Reliability
Time of test
© 2008, YCMOU. All Rights Reserved.

27. © 2008, YCMOU. All Rights Reserved.
Dec 18, 2007
Study Tips
Book
Title: Immunology
Author: Richard A. Goldsby, Thomas Kindt, Barbara A. Osborne.
Publication: W.H. Freeman and Company, New York.
Title: Textbook of Microbiology
Author: R. Anathanarayan & C.K.J. Paniker
Publication: Orient Longman publication, Chennai.
© 2008, YCMOU. All Rights Reserved.

28. End of the Presentation
Dec 18, 2007
End of the Presentation
Thank You !
© 2008, YCMOU. All Rights Reserved.