Practical immunology
Immunological technologies
Dr. B.P. Nayak
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
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
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
Antibody specificity
Good: recognize and identify related antigens
Bad: Confuse one antigen with a related antigen
Affinity and Avidity
Property of the
antibody binding site
Property of the
valency
Antibody types
• Polyclonal 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
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
http://www.udel.edu/biology/Wags/histopage/illuspage/ilst/lymphspleenthymusppt.htm
Immunological assays
•Diagnostic:
• Assay for antigen
• Assay for antibody
•Research:
• Identify cells
• Identify cell products
• Isolate cells or macromolecules
• Assay cell function
Examples
Diagnostic assays Research techniques
• Hemaggluination • ELISA
• Hemagglutination inhibition • Immunohistochemistry
• Enzyme-linked Immunosorbant • Flow cytometry
Assay (ELISA) • Cell sorting
• Radioimmunoassay (RIA) • ELIspot
• Immunofluorescent assay (IFA) • Immunoblot
• Immunoprecipitation
• Mixed lymphocyte response
• Chromium release assay
Types of assays
• Precipitation tests: Detection based on precipitation
of complexes
• Colorimetric tests:
• Fluorescent
• Enzyme-linked
Precipitation reactions:
• Antibody-antigen complexes precipitate out at
equivalence
• Utility:
• Detection (visual precipitate)
• Quantification
• Dilution
• Diffusion
“Prozone”
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
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
Electrophoresis
• Differential charge of antibody and antigen
• Improves sensitivity of radial immunodiffusion
Electric current
• Antigen precipitates at equivalence
• Height of “rocket” is proportional to antigen concentration
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
Red blood cells: Antigen of interest
• Easy to see bound to red
• Bind to antigens blood cells
Agglutination
Sample Antibody Titer
a 1:32
b Undetectable No agglutination
c 1:16
d 1:128
e 1:256
f Undetectable
g 1:8
Prozone
h 1:32
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
Variations on ELISA
• Indirect ELISA
• Capture (Sandwich) ELISA
• ELIspot
• Competitive ELISA
• Radioimmunoassay
• Immunoblot (“Western” blot)
• Diagnostic kits
Labeled Unlabeled Secondary rabbit
anti-goat (labeled)
Primary goat anti-X
Competitive ELISA
“Western blot”
Immunochemistry and Immunofluorescence
• Solid-phase antigen:
• In tissue section
• Cytology
• Detect presence and distribution of:
• Surface marker
• Intracellular product
• Extracellular product
• Metabolic product
Immunofluorescence
Direct Indirect
Red: Bcl-6, germinal center B cells
Cell surface markers: Green: IRF-4: Plasma cells
B cells: CD45R
Plasma cells: IRF-4
Enzyme-linked and related assays
Assay Target antigen Detection Variants
ELISA Bound to well Chromogen Direct, Indirect,
Sandwich
ELIspot Product of Chromogen B cell or T cell
cultured cells products
Immuno- In tissue Chromogen, Direct or indirect
histochemistry Fluorochrome
Competitive ELISA Soluble Chromogen
Immunoblot Gel separated Chromogen,
proteins Fluorochrome,
Radioactive isotope
Antibodies as traps
• Fluorescence-activated cell sorting (FACS):
• Quantitation
• Isolation
• Plate-isolation of cell populations (“panning”)
• Magnetic bead isolation of cells and macromolecules
Cells coated with
fluorescent antibody
B cells: red
T cells: green
Macrophages: cyan
Lymphocyte assays
• Lymphocytes as tools:
• 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
(blastogenesis)
Magnetic bead isolation
Subculture and cloning
