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-CP4-04 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 radio-immunoassay Describe the mechanism of radio- immunoassay (RIA) ‏ Describe the applications of RIA.

6. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.6 Introduction - 1  Many biological substances such as hormones and enzyme need to be detected in order to diagnose may disorder involving these substances.  Such substances which are present in very micrograms concentration in the body can not be detected by the routinely used serological techniques such as immuno- diffusion and agglutination.  Radio-immunoassay is used to detect such substances.  In this module the students will learn the principle of RIA and its applications.

7. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.7 Radio-immunoassay-1  RIA is one of the most sensitive techniques used for the detection of either antigen of antibody.  RIA can measure antigenic substance upto picogram (10 -12 g).  Many substances such as hormones, enzymes and drugs present in very minute concentration in the body.  Such substances act as antigen in other species.  In RIA, the radio labelled antigen and same unlabelled antigen are subjected to competitive binding with their antibodies.

8. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.8 Radio-immunoassay-2  Mechanism:- In RIA the standard graph of various increasing concentrations of unlabelled antigen versus bound radioactivity in Ag-Ab complexes is prepared by allowing fixed radio- labelled antigen at its fixed concentration to react with its corresponding antibody in the presence of same unlabelled antigen. The labelled antigen and unlabelled antigen compete to bind with the antibody. When the concentration of unlabelled antigen is less than the labelled antigen, more labeled antigen will bind to the antibody.

9. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.9 Radio-immunoassay-3  Mechanism:- Labeled and unlabeled antigens compete for binding to Ab.

10. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.10 Radio-immunoassay-3  Mechanism:- When the concentration of unlabeled antigen is more than the labeled antigen, more unlabeled antigen will bind to the antibody and radioactivity in the Ag-Ab complexes will be more. In short the member of antigens will bind to the antibody whose relative concentration is more in the reaction mixture. In RIA to find out the titre of the antigen radio-activity in the Ag-Ab complexes is measured and the titre of the antigen is found out from the standard graph by finding the titre value of the antigen at the that radioactivity value.

11. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.11 Radio-immunoassay-4  Application:- RIA is used to detect and quantities the  hormones,  tumor markers,  IgE,  viral antigens and  drugs.

12. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.12 Immuno-fluorescence-1  Immuno-fluorescence technique used fluorescent dyes such as fluorescein or rhodamine.  In this technique these fluorochrome are conjugated with monoclonal antibodies against a particular antigen of our interest.  If the interested antigen is present on the tissue surfaces or in tissue, then these fluorescence dye labeled antibodies bind to them and emmit fluorescence making them visible under the microscope.

13. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.13 Immuno-fluorescence-2  There are two methods of immuno- fluorescence: Direct immuno-fluorescence Indirect immuno-fluorescence  Direct immuno-fluorescence: In direct immuno-fluorescence method the fluorochrome is directly conjugated to the antibody which react with the desired antigen in the tissues.

14. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.14 Immuno-fluorescence-3  Direct immuno-fluorescence: Sandwitched direct immuno-fluorescence

15. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.15 Immuno-fluorescence-4  Indirect immuno-fluorescence:- Indirect immuno-fluorescence is More sensitive than direct immuno-fluorescence technique since fluorescence amplification is more in indirect immuno-fluorescence technique.

16. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.16 Applications of Immuno-fluorescence Assay  Immuno-fluorescence technique is used to identify specific molecule in a cell, tissue and tissue fluid.  Immuno-fluorescence technique is used to detect the distribution of molecules of our interest in a cell or in a tissue.

17. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.17 What We Learn…..  Radio-immuno assay  Immuno-fluorescence assay  Mechanisms of radio-immuno assay and immuno-fluorescence assay  Applications of radio-immuno assay and immuno-fluorescence assay

18. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.18 Critical Thinking Question  Why higher radioactivity is inversely proportional to low titre of Ag in test sample?

19. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.19 Hints For Critical Thinking Question  Use of radioactively labeled and unlabeld Ags

20. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.20 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.

21. © 2008, YCMOU. All Rights Reserved.21 End of the Presentation Thank You !