1. Practical immunology
Immunological tools

2. Immunological methods: Antibodies as tools
Specific recognition of macromolecules
Cell types: surface molecules (CD markers)
Pathogens
Antibodies
Antibodies as labels
Antibodies as traps
Antibodies as competitors
Assays:
Detection
Localization
Quantitation

3. A brief history of antibodies
1891: The term “Antikörper” coined by Paul Ehrlich
Substance in the blood that confers immunity
"if two substances give rise to two different antikörper, then they themselves must be different”
“Lock-and-Key” theory
1920’s: Heidelberger and Avery identified antibodies as proteins
1940’s: Linus Pauling confirms lock-and-key theory
1948:Immunoprecipitation: use of antibodies for detection
1956: Glick and Chang-Bursa of Fabricius: Antibodies come from B cells
1976: Hozumi and Tonegawa, antibody gene rearrangement

4. Useful characteristics of antibodies
Specificity: Ability to recognize individual epitopes
Cross-reactivity:
Ability to bind to more than one epitope
Shared epitopes between different antigens
Strength of binding:
Affinity: Strength of binding between Fab and epitope
Avidity: Overall strength of binding of serum and antigen

5. Antibody specificity Good: recognize and identify related antigens
Bad: Confuse one antigen with a related antigen

6. Affinity and Avidity Property of the antibody binding site
Property of the valency

7. Antibody types Polyclonal antibodies: Monoclonal antibodies:
Isolated from immune serum
Many different idiotypes
Recognize many different epitopes
Strong avidity
Highly sensitive
Cross reactive: Less specific
Monoclonal antibodies:
Single idiotype
Single epitope
Highly specific
Lower avidity

8. Generation of antibodies as reagents
Polyclonal:
Purify antigen
Inject into rabbit or goat (or other species)
Collect serum
Purify immunoglobulin
Specificity depends on purity of antigen
Monoclonal:
Immunize a mouse
Culture spleen cells fused with myeloma cells
Select clones based on antibody specificity
Specificity to a single epitope

10. Immunological assays Diagnostic: Research: Assay for antigen
Assay for antibody
Research:
Identify cells
Identify cell products
Isolate cells or macromolecules
Assay cell function

11. Examples Research techniques Diagnostic assays Hemaggluination ELISA
Hemagglutination inhibition
Enzyme-linked Immunosorbant Assay (ELISA)
Radioimmunoassay (RIA)
Immunofluorescent assay (IFA)
ELISA
Immunohistochemistry
Flow cytometry
Cell sorting
ELIspot
Immunoblot
Immunoprecipitation
Mixed lymphocyte response
Chromium release assay

12. Types of assays
Precipitation tests: Detection based on precipitation of complexes
Colorimetric tests:
Fluorescent
Enzyme-linked

13. Precipitation reactions:
Antibody-antigen complexes precipitate out at equivalence
Utility:
Detection (visual precipitate)
Quantification
Dilution
Diffusion
images from

14. “Prozone”

15. Precipitin tests: Immunodiffusion
Double immunodiffusion:
Well or trough punched in an agarose gel
Antibody and antigen placed in separate well
Precipitate forms if antibody recognizes antigen
Identification of antigen or antigen mix

16. Radial immunodiffusion
Antibody incorporated into gel
Antigen added to wells
Zone of precipitation is proportional to concentration
Identification and quantification
Precipitation at antigen/antibody equivalence

17. Electrophoresis Differential charge of antibody and antigen
Improves sensitivity of radial immunodiffusion
Antigen precipitates at equivalence
Height of “rocket” is proportional to antigen concentration
Electric current

18. Quantification by titration
Principle:
Quantitation of antibody or antigen by serial dilution
Single concentration of antigen (or antibody )
Dilutions of antibody (or antigen)
Titer = the dilution at which precipitation occurs
Reflects but does not equal concentration
Assays:
Hemagglutination
ELISA

19. Antigen of interest bound to red blood cells
Easy to see
Bind to antigens
Antigen of interest bound to red blood cells
Agglutination
Sample
Antibody Titer a
1:32 b
Undetectable c
1:16 d
1:128 e
1:256 f g
1:8 h
No agglutination
Prozone

20. Colorimetric tests: Enzyme-Linked Immunosorbant Assay
ELISA
Principles:
Specific interaction between antibody and antigen
Solid-phase
Sensitive detection
Description:
Antigen bound to a surface
Enzyme-bound antibody
Colorimetric reaction
Semi-quantitative
Generate titer

22. Variations on ELISA Indirect ELISA Capture (Sandwich) ELISA ELIspot
Competitive ELISA
Radioimmunoassay
Immunoblot (“Western” blot)
Diagnostic kits

23. Primary goat anti-X Labeled Secondary rabbit anti-goat (labeled)
Unlabeled
Labeled

24. Competitive ELISA

26. “Western blot”

28. Immunochemistry and Immunofluorescence
Solid-phase antigen:
In tissue section
Cytology
Detect presence and distribution of:
Surface marker
Intracellular product
Extracellular product
Metabolic product

29. Immunofluorescence
Direct
Indirect

30. Cell surface markers: Red: Bcl-6, germinal center B cells
Green: IRF-4: Plasma cells
Spleen red:Bcl-6 germinal center B cells green: IRF-4 plasma cells (dark) B cells (light green)

32. B cells: CD45R
Plasma cells: IRF-4

33. Immunochemistry of enzymatic reactions
Activated Caspase 3

34. Immunochemistry Kit: “Vectastain ABC”

35. Enzyme-linked and related assays
Target antigen
Detection
Variants
ELISA
Bound to well
Chromogen
Direct, Indirect, Sandwich
ELIspot
Product of cultured cells
B cell or T cell products
Immuno-histochemistry
In tissue
Chromogen, Fluorochrome
Direct or indirect
Competitive ELISA
Soluble
Immunoblot
Gel separated proteins
Chromogen, Fluorochrome, Radioactive isotope

36. Fluorescence-activated cell sorting (FACS):
Antibodies as traps
Fluorescence-activated cell sorting (FACS):
Quantitation
Isolation
Plate-isolation of cell populations (“panning”)
Magnetic bead isolation of cells and macromolecules

37. B cells: red T cells: green Macrophages: cyan
Cells coated with fluorescent antibody
B cells: red
T cells: green
Macrophages: cyan

40. Lymphocyte assays Lymphocytes as tools: Assays: Easy to isolate
Survive well in culture
Assays:
ELIspot: B and T helper cell function
Lymphocyte blastogenesis (stimulation) test: T helper cells
Chromium release assay: Cytotoxicity

42. (blastogenesis)
Magnetic bead isolation
Subculture and cloning

44. That’s all, folks!!
that’s all folks