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
• Hemaggluination
• Hemagglutination inhibition
• Enzyme-linked Immunosorbant
Assay (ELISA)
• Radioimmunoassay (RIA)
• Immunofluorescent assay (IFA)
Research techniques
• ELISA
• Immunohistochemistry
• Flow cytometry
• Cell sorting
• ELIspot
• 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
• Antigen precipitates at equivalence
• Height of “rocket” is proportional to antigen concentration
Electric
current

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

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

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

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

Cell surface markers: Red: Bcl-6, germinal center B cells
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
cultured cells
Chromogen B cell or T cell
products
Immuno-
histochemistry
In tissue Chromogen,
Fluorochrome
Direct or indirect
Competitive ELISA Soluble Chromogen
Immunoblot Gel separated
proteins
Chromogen,
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

Magnetic bead isolation
Subculture and cloning
(blastogenesis)