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-CP4-05 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 enzyme linked immunosorbent assay or ELISA. Describe the principle of ELISA. Describe the applications of ELISA.

6. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.6 Introduction  Many time the the antigen or cell markers are so minute in the quantity that it very difficult, tedious and costly to isolate them in pure form in sufficient quantity so as to make their estimation possible.  Therefore the alternate approach is to detect their presence insitu.  The ELISA was developed as an alternate technique to fluorescent staining insitu due to its safety, cost effectiveness,simplicity and reliability.  In this module students will learn about the ELISA.

7. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.7 ELISA-1  The ELISA (enzyme linked immunosorbent assay is also called as enzyme immunoassay (EIA).  Principle:- In this technique either antigen or antibody is labeled or conjugated with an enzyme. The enzyme carry out breakdown of its substrate to a colorful product; the intensity of color is proportional to the product concentration. The intensity of color is measured by colorimeter or by UV spectrometer.

8. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.8 ELISA-2  Principle :- The enzyme carry out breakdown of its substrate to a colorful product; the intensity of color is proportional to the product concentration. The technique is standardized in such a way that when the substrate concentration is kept fixed then intensity of color developed is proportional to the enzyme concentration and thus ultimately to the antigen or antibody titre to which the enzyme is tagged.

9. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.9 ELISA-3  There are two types of ELISA Homogeneous ELISA Heterogeneous ELISA  The homogeneous ELISA is one step technique since there is no need to separate the free or unreacted antigens or antibodies from the reaction mixture.  In homogeneous ELISA all the reagents are added simultaneously.  The homogeneous ELISA is used for the detection of hapten such drug and not for microbial antigens or antibodies against them.

10. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.10 ELISA-4  The heterogeneous ELISA is two steps technique since the the free or unbound fraction of either antigens or antibodies have to be separated either by centrifugation or by absorption of bound antigens or antibodies to solid surface and washing the free fraction of either antigen or antibodies.  For the absorption of antigen or antibody the solid surface of cellulose, agarose, polystyrene, polyvinyl or polycarbonate materials is used.

11. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.11 ELISA-5  Heterogeneous ELISA can be carried out in by two different methods: Competitive ELISA Non competitive ELISA + ↔ Test + Patient’s sample Labeled Ag + In competitive ELISA labeled and unlabeled Ag compete for limited binding sites on Ab.

12. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.12 ELISA-5  Color development in ELISA test:-

13. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.13 Non-competitive ELISA  Non-competitive ELISA:- Solid Phase Ag Immobilized Ab in Patient’s sample Labeled Anti-Ig In non-competitive ELISA Ag-Ab complexes are conjugated with enzyme labeled anti -human immunoglobulin.

14. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.14 ELISA-6  Detection of rotavirus antigen in feces:- To detect the rotavirus infection, antibodies against the rotavirus is bound to the wells of microtitre plate; plate is washed. The fecal sample to be tested for rotavirus is then added to these wells and incubated overnight at 4 ° C and subsequently at 37 ° C for two hours. Then the wells are washed to get rid of free, unbound antigens. The guinea pig anti-rotavirus antibodies labeled with alkaline phosphatase enzyme are then added to the wells and incubated at 37 ° C for one hour.

15. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.15 ELISA-7  Detection of rotavirus antigen in feces:- The wells are then washed to get rid of the unbound antigens or antibodies and mixed with enzyme substrate, paranitrophenyl phosphate and incubated until the positive control developed yellow color. If the rotaviruses are present in the given fecal sample, then they will react with enzyme labeled antibodies; enzyme thus fixed will carry out breakdown of substrate to colorful product indicating positive ELISA test. This technique is also called as non- competitive sandwich ELISA. Competitive sandwich ELISA employ mixture of unlabeled and labeled antibodies.

16. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.16 ELISA-8  Using monoclonal antibodies by ELISA following pathogenic agents can be detected: Rotaviruse Hepatitis B virus HIV Respiratory syncytial virus

17. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.17 ELISA-10  Detection of HIV:- To detect the antibodies against the HIV-1 and HIV-2 viruses this test is carried out. The viral antigens are fixed onto the solid surface and different dilutions of test serum are added and incubated at 37 ° C. After incubation the reaction well are washed to remove unreacted Ab and mixed with antihuman immunoglobulin Ab conjugated with horse serum peroxidase enzyme. The reaction mixture is again incubated at 37 ° C and washed. The enzyme substrate O-phenylene diamine dihydrochloride is added and observed for color development.

18. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.18 ELISA-11  Detection of HIV:- The ELISA is routinely used as screening test to find out the high probability of HIV infection among high risk persons. The ELISA is used as qualitative as well as quantitative test. The test is rapid and cost effective to determined the HIV positiveness among the high risk patients. The limitations of ELISA in HIV infections is high probability of positive reaction among the patients undergone multiple blood transfusion and suffered malarial infection two three times.

19. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.19 What We Learn…..  The principle of ELISA test.  Procedure of conducting ELISA for detection of antibodies against viruses such as rotaviruses and HIV.  Limitations of ELISA in HIV infections.  Various applications of ELISA.

20. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.20 Critical Thinking Question  Why sensitivity of ELISA is more than agglutination reaction?  Why ELISA is the first choice of test in diagnosis of most of viral infections?

21. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.21 Hints For Critical Thinking Question  Nature of enzymatic reactions  Safe, high sensitivity

22. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.22 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 Ananthanarayan, C.K.J. Paniker Publication: Publication: Orient Longman Limited, Chennai.

23. © 2008, YCMOU. All Rights Reserved. 23 End of the Presentation Thank You !