Immunological Methods AppendixIII

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Laboratory techniques commonly used in immunology:
Principles and examples
Karl Albert Brokstad
Antibody & antigen reaction basis of immunological assays
Detection, purification and quantitation of antigens
Majority antigens many epitopes heterogenous mixture of
polyclonal antibodies
Monoclonal antibodies derived from single clone to 1 epitope
(contains 4 MAbs)
(MAb derived from 1 plasma cell)
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Production of MAbs
1975 Georges Kohler and Cesar Milstein
1984 Nobel Prize in Physiology or
Medicine
Hybridoma
Fusion of myeloma cell with normal Ab
producing B cell
Result immortal B cell producing MAb Hypoxanthine Aminopterin Thymidine medium)
hypoxanthine-guanine phosphoribosyltransfera Lag phase naïve B cells undergo clonal selection, clonal expansion & differentiation into memory & plasma cells
Higher magnitude response
Higher affinity (IgG)
Shorter lag
Naïve lymphocytes
Large population memory cells,
easily activated
Memory lymphocytes
Immunoprecipitation in Gels (Ag & Ab form precipiate when in equivalence)
• Antigen in well, antibody in gel.
• Ring formed in area of
equivalence
• area of precipitation proportional
to concentration of antigen
• Antigen & antibody diffuse
towards each other
• Line of precipitation at
equivalence
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Agglutination
Antibodies to sheep RBC agglutination
Haemagglutination Inhibition
Influenza virus agglutinates red blood cells
Influenza specific antibodies inhibit agglutination
Radioimmunoassay
• Introduced 1960 by SA Berson and Rosalyn Yalow to
measure insulin in plasma (Nobel prize 1977)
• I125 (γ emitting) also 3H (β emitting)
• Competitive binding of radiolabeled antigen and unlabeled
antigen
• Solution or solid phase assays
• Wells coated constant
amount of antigen
• Serum & labeled hepatitis
surface antigen (HBsAg)
added
• Infected people less
radiolabeled HBsAg bound
• Standard curve used to
calculate concentration of
HbSAg in sample
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Enzyme Linked Immunosorbent Assay
(ELISA)
• Qualitative or quantitative measurement of
antigen or antibody
• Enzyme linked to detector antibody • Soluble chromogenic substrate Absorbance of product measured in an ELISA(spectrophotometer)
• Chemiluminesence
Measurement of light instead of absorbance Luxogenic substrate
Increased sensitivity (10-200 fold with
enhancers)
Amplification of signal
Biotin-avidin system
•  Amplification of signal
•  High affinity and specificity (avidin binds 4
molecules biotin)
•  Secondary antibody
•  Eg product detected MAB followed by
enzyme labeled anti-mouse antibody
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Multiplex Assays
• 
• 
• 
• 
Bead reader powered with Luminex xMAP technology
Reads 96-wells plates fully automated
Recognises multiple fluorescent signals, internal lasers
possible to measure up to 100 different biomolecules/well
Multiplex
•  Simultaneous quantitative analysis of up to 100 different
biomolecules from a single drop of sample
•  Increase dynamic range in concentration for all analytes,
because of the fluorescence and the superiour curve fitting
modules
•  Increase of sensitivity of all analytes compared to ELISA
•  Decrease volume requirements for precious sample
Bead-Based Technology
•  Assay occurs on bead surface
•  5.5µm bead diameter
  BASED ON BEAD COLOUR
•  Each bead contains different
concentration of RED and Infrared dyes
•  Up to 100 uniquely colored beads
• Used for detection of cytokines
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Purification of protein: Affinity chromatography
Purification of antigen A from complex mixture
Purification of protein: Immunoprecipitates collected by magnetic beads
Production of antibodies by genetic engineering Phage display library
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Western Blot
• Protein mixture treated with detergent SDS
• Separated by SDS PAGE - lower molecular weight migrate faster
• Proteins blotted from gel to nitrocellulose membrane under electric current
• Flood membrane with antibodies linked to enzyme used to visualise
antigens
• Developed insoluble substrate product deposited at site of reaction
• Chemiluminsence use film (increased sensitivity)
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Metabolic labeling
• All actively synthesised cellular proteins labeled radioactive
• amino acids
• Cells lysed detergent
• Precipitation specific proteins with MAbs on beads
• Elute proteins in SDS
• Run SDS PAGE
Immunohistology & Immunofluoresence
• used to visualize subcellular distribution of biomolecules • Immuno-labeled tissue sections studied using a (fluorescence)
microscope or by confocal microscopy
• antibodies or antigens labeled with enzyme (immunohistology)
or with fluorescent dyes (immunofluorescence) 8
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Direct and Indirect Immunofluoresence
Cells fixed to microsope slide
Collidal Immunogold staining in electron microscopy
Measuring lymphocyte responses
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Isolation of lymphocytes
Contains Sodium Diatrizoate & Polysaccharide aggregates erythrocytes (increasing
sedimentation rate)"
lymphocyte suspensions contaminated (1-5% erythrocytes)"
Separation of cells using magnetic beads
Positive selection selects for cells containing surface marker
Negative selection selects for cells other surface markers
Enzyme linked Immunospot Assay (ELISPOT)
• Modification of ELISA
• Quantification of number of cells producing antibody or • antigen e.g. Cytokine
• Insoluble chromogenic or chemiluminescence substrate
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Antigen coating
(influenza proteins)
Enzyme conjugated
avidin
Blocking
(FCS)
Colorless Substrate
(enzymatic reaction)
e
S
Sample
(Lymphocytes)
P
Colored Product
(insoluble - spots)
e
S
Detector antibody
(biotin conjugated)
P
An ELISPOT well
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What is Flow Cytometry?
•  Flow Cytometry is a powerful technique for cell
counting and surface marker analysis
•  Flow Cytometry can provide quantitative
information about cell surface markers
•  Based on immunofluorescence technique and
computer-aided analysis
•  Allows simultaneous multiparametric analysis of
cells
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Scatter Pattern of Human leukocytes
A flow cytometry
scattergram
Forward scatter (size)
Neutrophils
Monocytes
Lymphocytes
Side scatter (granularity)
10 3
CD4
102
10 1
10 1
102
CD4 -->
CD4 -->
CD4
10 3
10 4
10 4
Three Color Lymphocyte Patterns
CD3CD3 -->
10
3
10
4
10
1
CD8
10
1
10
2
CD8 -->
10
3
10
4
10 3
10 4
2
CD8 -->
102
10
CD8
1
10 1
10
10
2
CD3 -->
10
3
10
4
CD3
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Fluorescence-activated cell sorter
(FACS)
•  FACS is one version of Flow Cytometry,
which can sort cells by their surface markers
•  Individual cell is positively or negatively
charged based on their fluorescence color
•  When charged cells pass through an electric
field, they are deflected and hence separated
Flow Cytometric uses of CFSE
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CFSE or CFDA-SE: carboxyfluorescein diacetate, succinimidyl ester.
A fluorescein derivative which is cell permenant.
CFSE is partitioned equally among daughter cells with each division.
These properties allows simultaneous analysis of cell number, position, as
well as division status.
•  Fluorochromes compatible with fluorescein can be used to probe other
cellular properties.
Dilution of CFSE with cell division
Divisions:
3
2
1
�
Quantification of T cell responses:
Traditional methods
•  CD4 proliferation after
stimulation with specific
antigen
•  3H-Thymidine incorporation
into DNA
•  CD8 cytotoxic activity after
in vitro stimulation
•  Release of 51Cr from labeled
target cell killed by effector
•  Measures response of bulk
population
Modern Methods
•  Production of cytokines
after in vitro stimulation
with antigen
•  Intracellular staining
•  ELISPOT
•  Staining with MHC/
peptide complexes
(tetramers)
•  Measures single cell
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CD4 T cell proliferation assay
Polyclonal Activation
•  Activation of lyphocytes regardless of
antigen specificity
•  Lectins (plant proteins) bind to TCR
receptor stimulate T cells
•  E.g. Concanvalin A (Con A)
Phytohaemagglutnin (PHA)
•  Useful positive control in assays
Proliferation assay: Setup
1. Harvest PBMC
over Ficoll
2. Add killed virus
or peptide antigen
3. Add PBMC
~1x105
cells/well
Triplicate control wells:
+ve control = PHA
-ve control = no antigen
37OC 5% CO2
6 days
5. Add 1.0 µCi
3H-thymidine/well
37OC 5% CO2
18 hr
7. Obtain counts
in automated rack
β-counter
6. Automated harvest of
cellular DNA onto glass
filters
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Cytotoxic T cell assay
CTL assay set-up: in vitro restimulation
1. Harvest PBMC
over Ficoll
Infect PBMC with
virus incubate
3-4 hr at 37OC
Harvested live cells =
Effector CTL for 51Cr
release assay
Uninfect
PBMC
IL-2 50U/ml
Mix in
optimal
ratio
Incubate 37OC
5% CO2 7 days
CTL assay set-up: 51Cr release assay
Targets
Harvest 100 µl supe
for detection of
γ-emission cpm
Effectors
3 days
Weeks
OR
PHA-stim’d
autologous
blast cells
Triplicate wells:
E:T ratio =
40:1, 20:1, 10:1, 5:1
EBV-transf’d
lymphoblastoid
cell line
Label with 51Cr;
4-5 hr at 37OC
5% CO2
Targets +
0.1% Triton =Total lysis
medium = Spont. lysis
infect with virus or
pulse with peptide
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CD8 tetramer staining
MHC Tetramer staining of lymphocytes
Intracellular cytokine staining
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Intracellular cytokine staining
CD8+
- Brefeldin A
IFN-γ secreted
APC
CD8+
+ Brefeldin A
for 2 hr
Stimulate CD8+ T cell
with APC + peptide or
APC + (polyclonal stimulator)
PMA/ionomycin
for 5 hr
CD8+
IFN-γ
retained
•  Stain surface markers
•  Fix cells
•  Permeabilize with saponin
•  Stain intracellular marker
Analyze by
flow cytometry
Comparison of traditional and modern methods
Method
Time
Function
Phenotype
Cell no.
Labor
Proliferation
5-7 d
No
Requires
High
presorting
High
ELISPOT
2-4 d
Yes
Requires
Low
presorting
Moderate
Intracellular
Cytokine
staining
1 d
Yes
Yes
Moderate
CTL assay
6-8 d
Yes
Requires
High
presorting
MHC
Tetramer**
1d
No
Yes
Mod/
Low
V. High
Moderate
Low
**Requires HLA typing
•  Traditional assays
•  Modern assays
•  Labor intensive
•  Less labor intensive, overall
•  Requires extensive periods
of culture
•  Shorter or no stimulation
periods; fewer cells
•  Qualitative results
•  Exact cell numbers (and
phenotypes) determined
•  Data analysis and
interpretation not uniform
•  Data analysis more
straightforward?
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