TABLE OF CONTENTS
SECTION A: QUALITATIVE IMMUNOTECHNIQUES
A.1. Ouchterlony Double Diffusion 03
A.2. Counter Current Immunoelectrophoresis 06
A.3. Immunoelectrophoresis 09
A.4. Crossed Immunoelectrophoresis 12
A.5. Agarose Immunoslide Staining and Documentation 15
A.6. Blood Group Typing - The MN System 17
A.7. Fluorochrome (FITC) Conjugation to Antibodies 20
A.8. Enzyme Linked Immunosorbent Assay (ELISA) 24
A.9. Identification of Human B-Lymphocytes 27
A.10. Identification of Human T-Lymphocytes 30
A.11. Salt Fractionation of Antibodies 33
A.12. Affinity Purification of IgG 38
SECTION B: QUANTITATIVE IMMUNOTECHNIQUES
B.1. Single Radial Immunodiffusion 43
B.2. Rocket Immunoelectrophoresis 46
B.3. Titre Studies by Immunodiffusion 49
B.4. Titre Studies by Heamagglutination 52
B.5. Concentration (to increase the titre) of Antisera 56
- Solid Sucrose dialysis
- Ultra concentrators
B.6. Quantitative ELISA 61
B.7. Cell-ELISA / ELISPOT Assay 63
SECTION C: GENERATING POLYCLONAL ANTIBODIES
C.1. Introduction 69
C.2. Immunization, Schedule and Uses of Adjuvants 71
C.3. Sample Blood collection, Isolation and Preservation of Anti Serum 74
C.4. A Model Protocol to Generate PAbs in Rabbits 78
SECTION D: GENERATING MONOCLONAL ANTIBODIES
D.1. Introduction 83
D.2. Immunizations and Collection of Primed Lymphocytes 84
- “in vivo”
- “in vitro”
D.3. Somatic Cell Hybridization 89
D.4. Limiting Dilution and HAT Selection 94
D.5. Screening Techniques 98
D.6. Expansion of Positive Hybridoma Clones 101
D.7. Maintenance and Passaging of Hybridoma Cultures 105
D.8. Harvesting MAbs from Hybridoma Culture Supernatants 107
D.9. Preservation and Re-culture of Positive Hybridoma Clones 109
ANNEXURE I 113
ANNEXURE II 116
PREFACE
Immunoglobulins when taken the role of being antibodies are powerful molecules with exquisite specificities and a myriad of functions. Apart from conferring protection and defense from potentially harmful entities in vivo, antibody molecules have increasingly become indispensable for many applications ranging from Diagnostics, Therapeutics and Research. From many years of dealing with such molecules and constant interactions with students of Basic Research Sciences, we understand a bit too well the importance of techniques that utilize antibodies and the antigen-antibody interactions. Looking around, we see only textbooks, references and laboratory protocol material that are quite complex, of which only a select part might be of routine use to various persons such as a student, research scholar or a laboratory technician. From an understanding of this and inputs that we give and receive from students that we felt like bringing out a Practical Manual of Immunotechniques which deals with more basic and universal applications. A strong foundation in basics should sure be of immense help in honing the skills of any laboratory practitioner to perform more complicated and seemingly difficult techniques with ease.
Many people are instrumental in giving us the urge and confidence to bring out this manual. Our mentors and teachers, Dr. Seetharami Reddy, Dr. Krishnaswamy, Mrs. Uma R.N Kumar, Dr. Vikram R. Jayanth, Dr. P. Venkatachalam, Dr. A. Meenakshi, Dr. Alamelu Raja, Dr. Vinod Kumar Panikar, Shri. A. R. Sunderarajan and Shri. A. Natrajan are fundamental for our motivation. All our students and research scholars are a source of inspiration of which Mr. N. Venkateswaran, Mr. Govind Pai, Ms. Deepa Parvathi, Ms. Bairavi Swaminathan, Mr. U. Baraneedharan and Ms. Amritha Nair need special mention.
It is a pleasure to acknowledge Ms. Sukanya Shyama Sundar and Ms. Priya Sivaramakrishnan who continue to inspire us with their inherent ability to understand complex concepts, their dedication and positive approach to technical assignments, their intelligent and meticulous laboratory skills and a very progressive attitude. Their inputs in terms of Illustrations, Images and Annexures are invaluable and this manual would have been incomplete without them.
We thank the management of Sri Ramachandra University for their constant motivation and encouragement which always is our source of strength to provide with the best platform to our students towards their successful careers.
We thank Shri. K.R. Pillai and the entire team at Samanthi Publications, especially
Miss. M. Gurulakshmi and Mrs. K. Solagracia for their valuable inputs and excellent publishing standards which was of immense help for this manual to materialize. We also thank
Mrs. M.D. Sujatha Ravi for Proof Reading the Manuscript.
We sincerely hope this Manual on Basic Immunotechniques will be of use to various persons such as a student, research scholar and technician in the exciting areas of Immunology and Basic Research Sciences.
Dr. M. Ravi Ph.D.,
Dr. Solomon F.D Paul Ph.D.,
LIST OF IMAGES
1. Ouchterlony Double Diffusion
2. Counter Current Immunoelectrophoresis ..................08
3. Immunoelectrophoresis ..........................11
4. Crossed Immunoelectrophoresis ...........................................................14
5. Blood Group Typing MN System ...........................19
6. Blood Group Typing ABO System ..................19
7. Chromatogram of Antibody-FITC Conjugate ................23
8. Human B - Lymphocytes ..................29
9. Human T - Lymphocyte E-Rossets .............32
10. Chromatogram of 'De-Salting' ...............37
11. Chromatogram of IgG Affinity Purification .............40
12. Single radial Immunodiffusion ............45
13. Semi-Log Graph for Single Radial Immunodiffusion ........45
14. Rocket Immunoelectrophoresis ..........................48
15. Semi-Log Graph for Rocket Immunoelectrophoresis .................... 48
16. Titre Studies by Agarose Gel Immunodiffusion ...................... 51
17. Heamagglutination ...............................55
18. Antibody Concentration by Sucrose Dialysis ................................58
19. Antibody Concentrations by Ultra-Concentrators ...............................59
20. Standard Graph for Quantitative ELISA .....................................62
21. Culture Plate for Cell-ELISA .............................65
22. Immunodiffusion Studies for Polyclonal Antibody Responses ............80
23. in vitro Immunizations ............................88
24. Sp2/0 Cells .............................90
25. Initiation of Sp2/0 Cell Culture ....................91
26. Somatic Cell Hybridization Hybridoma Technology ...............92
27. Preparation of Feeder Cells Hybridoma Technology ...........96
28. Feeder Cells Hybridoma Technology ......................97 29. Limiting Dilution Culture Plate for Screening Hybridoma echnology 99
30. Hybridoma Clone 100
31. Expanding Hybridoma Clones 102
32. Expansion of Positive Clones Hybridoma Technology 103
33. Freezing of Hybridoma Cell Lines 111
34. Thawing and Reculture of Hybridoma cell Lines 112
ABOUT THE AUTHORS
Dr.M. Ravi obtained his Ph.D., In Human Genetics from Sri Ramachandra Medical
College and Research Institute. He is presently an Assistant Professor in the Department of Human
Genetics of Sri Ramachandra University, Chennai and fields of interest and specializations are in
Immunology, Immunotechniques and Research Methodology. He is also the Assistant Editor of Advanced Biotech, a
monthly magazine.
E mail : maddalyravi@hotmail.com
Dr. Solomon F.D. Paul obtained his Ph.D., From Madras University for research carried out in Biodosimetry
at IGCAR, Kalpakkam. He is the current Professor and Head of the Department, Departments of Human Genetics and
Biomedical Sciences. His research interests and expertise are in Biodosimetry. Human Genetic Diagnosis and
Genotoxicity studies.
E mail : wise_soly@yahoo.com