1. IMMUNOLOGICAL METHODS
Dr. Gülderen Yanıkkaya Demirel

2. OUTLINE of THIS LECTURE
Definition of Immunology
Innate and Adaptive Immunity
Antigen/Antibody
Immunological Methods

3. Innate Immunity Ancient system - present in invertebrates
naïve, immediate, everyday immunity
Molecules recognize common features of pathogens
Lectin
Phagocytes, large lymphocytes (NK cells)
Complement

4. Adaptive Immunity specialized, late, long term immunity
Newer system - present in fish, birds, human
specialized, late, long term immunity
Molecules recognize specific features of pathogens
Antibodies
B and T cells - small lymphocytes
Immunological memory

5. Innate and Adaptive Responses
Pathogen independent
Immediate (hours)
Neutrophils
Macrophages
Mast cells
Eosinophils
Basophils
NK cells
“Large Lymphocytes” = NK cells
Adaptive
Pathogen-dependent
Slower (days)
Dendritic cells
B cells
T cells (CD4 or CD8)
“Small Lymphocytes” = B & T cells
Both systems “talk” to each other to modulate response
Both systems use leukocytes = white blood cells

6. IMMUNOLOGICAL METHODS depend on Ag Ab interactions

7. WHAT IS AN ANTIGEN ?
An antigen is any substance that elicits an immune response and is then capable of binding to the subsequently produced antibodies.
Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids can also be antigens.

8. How, Why and Where are Antibodies Produced
How, Why and Where are Antibodies Produced? (Massively condensed version)
Produced in vertebrate cells in response to a ‘non-self’ substance termed an antigen.
The process begins with antigen presenting cells roaming the bloodstream. They engulf ‘non-self’ molecules and then display portions of the engulfed molecules on their outer plasma membranes.
After many recognition steps B-cells are stimulated to produce specific antibodies.
The antibodies then go off and bind to the foreign substance thereby marking the substance for destruction.

9. Recognition modules of Adaptive immunity
B-cell receptor (BCR)
T cell receptor (TCR)
B cells
T cells
TCR is membrane bound
Antibody is a secreted form of BCR

11. IMMUNOLOGICAL METHODS
Agglutination/Hemaglutination
Immunoprecipitation
Immunodiffusion
Immunoelectrophoresis
RIA/ELISA
Immunoflorescence
Immunohistochemistry
Flow Cytometry
Molecular Methods
PCR
Sequence Analysis
Immunomics
Gene transfection

12. AGGLUTINATION/HEMAGGLUTINATION
Definition - tests that have as their endpoint the agglutination of a particulate antigen
Agglutinin/hemagglutinin Y + 
Qualitative agglutination test
Ag or Ab

13. AGGLUTINATION/HEMAGGLUTINATION
Quantitative agglutination test
Titer
Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7

14. AGGLUTINATION/HEMAGGLUTINATION
Definition
Qualitative test
Quantitative test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
Applications
Blood typing
Bacterial infections
Fourfold rise in titer
Practical considerations
Easy
Semi-quantitative

16. Radial Immunodiffusion
Method
Ab in gel
Ag in a well Ag
Ab in gel
Ag Concentration
Diameter2
Interpretation
Diameter of ring is proportional to the concentration
Quantitative
Ig levels

17. p. 152

19. Immunoelectrophoresis
Method
Ags are separated by electrophoresis Ag - + Ag Ab Ag Ab
Interpretation
Precipitin arc represent individual antigens

20. IMMUNE ELECTROPHORESIS

21. ELISA – Enzyme Linked Immuno Sorbent Assay

22. RIA – Radio Immuno Assay

23. ELISPOT

24. Immunofluorescence Direct Y
Ab to tissue Ag is labeled with fluorochrome Ag Y
Fluorochrome
Labeled Ab
Tissue Section

25. Immunofluorescence Indirect Y Qualitative to Semi-Quantitative
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled Ab
Qualitative to Semi-Quantitative

26. ImmunoHistochemistry

27. ImmunoHistochemistry
Arrows indicate localized CD3+ lymphocytes in tissue
Antibodies against surface receptors react with tissue on slide.
Secondary antibody with chromgenic probe recognizes first antibody.
Addition of substrate allows deposition of material and histological detection/localization of cell populations within tissue.

28. Mixed population containing CD3+ cells Add FITC-antibody specific for
Laser Excitation
Light Scatter
Detector
FITC
Fluor
CD3+
CD3+
CD3+
CD3+
CD3+
CD3+

29. p. 163

30. Immunoelectron microscopy

31. AUTOANTIBODIES

33. dsDNA

34. LYMPHOCYTE PROLIFERATION
Reactive Lymphocytes
Mitogen (ConA) added
Antigen added

35. MAGNETIC BEAD SORTING
p. 160

36. CELL SORTING

37. MOLECULAR METHODS

38. Western
blotting

39. GENE TRANSFECTION

40. SUGGESTED READING Abbas Immunology 2012 edition Kuby’s Immunology
Janeway Immunology
Temel İmmunoloji, İmmun sistemin İşlev ve Bozuklukları, 2007.