1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. © 2008, YCMOU. All Rights Reserved.2 OC-SBT052-CP4-02 Introduction Programmes and Courses  SEP–SBT052–Unit-04

3. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.3 Credits  Academic Inputs by Arun Punaji More. M.Sc. (Microbiology) ‏ Experience: 11 Years arunmesh@hotmail.com

4. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 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.

5. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Describe the various precipitation reaction of antigens and antibodies. Describe the principle of precipitation reaction. Describe the applications of precipitation reactions in diagnosis of infections.

6. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.6 Introduction  The manifestation of antigens and antibodies reactions in vitro depend upon the chemical and physical nature of antigens and antibodies.  If the antigens are particulate in nature the manifestation of antigen antibody reaction in vitro is agglutination.  Whereas if the antigens are soluble, the manifestation of antigen antibody reaction in vitro is formation of precipitation.  In this module, the students will learn use of precipitation reaction in the diagnosis.

7. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.7 Immunoprecipitation-1  The soluble antigens when react with antibodies at equivalent concentration, precipitation of Ag-Ab complexes is formed.  Such precipitation reaction of Ag-Ab can be carried out in solution or in agar gel medium in which the precipit formed can be mobilized and the concentration of antigens or antibodies can be estimated.

8. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.8 Immunoprecipitation-2  Immunoprecipitation in agar gel:- Immunoprecipitation reactions (immunodiffusion) can be carried out in two ways  Single radial immunodiffusion  Double diffusion agar assay (Oouchterlony technique) ‏  Immunoelectrophoresis  Countercurrent-immunodiffusion

9. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.9 Immunoprecipitation-3  Single radial immunodiffusion:- In this technique the monospecific antibody is mixed with the molten agar gel and the mixture is either poured onto the clean glass slide or into the petri dish and allowed to solidify. The small wells are then cut into the solidified agar gel Unknown antigen is then added into such wells and is allowed to diffuse into the gel for 24 hrs. The antigen diffuse into the gel from the well radially at equivalent concentration with antibody form precipitation line in form of circle.

10. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.10 Immunoprecipitation-4  Single radial immunodiffusion:- The area of this precipitation circle is proportional to the concentration of antigen. By single radial immunodiffusion either antigen of antibody can be detected and quantified This technique is widely used to detect and quantitate antibodies, antigens, serum immunoglobulins, and complement proteins.

11. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.11 Immunoprecipitation-5  Single radial immunodiffusion:- To quantitate the antigen, first different dilutions of antigen is placed into wells of agar containing the fixed concentration of antibody. After the completion of diffusion the diameter of precipitation rings are measured for the different concentration of antigen and graph is plotted for the diameter of these precipitation rings versus concentration of the antigen. This standard plot is then used to find out the concentration of the same antigen in the other sample.

12. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.12 Immunoprecipitation-6  Single radial immunodiffusion:- Standard graph of diameter of precipitation ring and concentration of antigen.

13. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.13 Immunoprecipitation-7  Single radial immunodiffusion:-

14. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.14 Immunoprecipitation-8  Single radial immunodiffusion:- In single immuno diffusion titre of Ab can be estimated.

15. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.15 Immunoprecipitation-9  Double diffusion agar assay:- In this technique both antigen and antibody are added into the separate wells in the same agar gel. The antigen and antibody then allow to diffuse into the gel The antigen and antibody form line of precipitation at their equivalent concentrations.

16. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.16 Immunoprecipitation-10  Double diffusion agar assay:- The advantages of double diffusion technique over the single diffusion technique is that, the different antigens can be compared for their identity, non-identity and partial identity. If the two antigens are identical then they form the precipitation line at the same distance from their wells forming the V shaped precipitation ring. If the two antigens are partial identical then they form Y-shaped precipitation ring. If the two antigens are not identical then they form X-shaped precipitation ring.

17. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.17 Immunoprecipitation-11  Double diffusion agar assay:- Testing of identities of antigens--

18. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.18 Immunoprecipitation-11  Immunoelectrophoresis:- If the given solution is containing number of antigens, then such mixture is subjected to immunoelectrophoresis since many antigens in the mixture can not be resolved by the single or double diffusion techniques. In this technique, first the number of antigens in the mixture is separated from each other on the basis of their electric charge on the agar gel. Positively charged antigens move to the negative electrode and negatively charged antigens move towards the positive electrode. A trough is then cut parallel to the wells of antigens.

19. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.19 Immunoprecipitation-12  Immunoelectrophoresis:- This trough is filled with the antibody. The plate is incubated at 37 ° C overnight. The antigens and antibodies then diffuse toward each other and form precipitation arcs which can be stained. This technique is used to detect major blood proteins in serum for diagnosis of some diseases.

20. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.20 Immunoprecipitation-13  Immunoelectrophoresis:- Ag - + Ab Antigens loaded into the wells Antibody loaded into the trough Precipitation arc

21. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.21 Immunoprecipitation-14  Counter-current-immunodiffusion:- This technique is used when the antigens and antibodies are having opposite charges. In this technique the antigen and antibody migrate or diffuse toward each other under the influence of electric current. This technique is used to detect only a single antigen or antibody to diagnose a particular infection which predominantly stimulate humoral immune response. Thus technique is only qualitative in nature. The technique is rapid help rapid diagnosis.

22. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.22 Immunoprecipitation-15  Counter-current-immunodiffusion:- Ag Ab - + Positive end Negative end Immunodiffusion of antigen and antibody toward each other under the influence of electric current.

23. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.23 What We Learn…..  The various methods of immunoprecipitation reactions and  Their uses in diagnosis of infectious disease.  Mechanism of immunoprecipitation reaction

24. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.24 Critical Thinking Question  Why the immunoprecipitation is widely used in diagnosis of infectious diseases?

25. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.25 Hints For Critical Thinking Question  The nature of antigen determine which test is employed as serological test.  Serotyping

26. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.26 Study Tips  Book Title: Immunology Title: Immunology Author: Author: Richard A. Goldsby, Thomas Kindt, Barbara A. Osborne. Publication: Publication: W.H. Freeman and Company, New York.  Book Title: Textbook of Microbiology Author: Author: R. Anathanarayan & C.K.J. Paniker Publication: Publication: Orient Longman publication, Chennai.

27. © 2008, YCMOU. All Rights Reserved.27 End of the Presentation Thank You !