Download Immunochemical methods

Immunochemical methods
Generation and use of antibodies and antibody-based reagents for:
•Detection
•Quantification
•Localization
•Purification
Henrik Wernérus
[email protected]
Antibodies (immunoglobulins)
• Proteins capable of binding a molecule in a highly
specific manner
• Generated by the immunesystem in response to foreign
substance
• Important in vivo for inhibition or "labelling for destruction"
of foreign substances
ALSO:
• Can be generated to almost any molecule for diagnostic,
therapeutic or research interest
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The antibody molecule
Fab
Binding function
Fv (scFv)
Fc
H
C
1
V
L
V
C
DR
3
CD
R
C
2
D
R
1
Effector function
CH 2
CH 2
CH 3
CH 3
L
C
C
L
1
CH
H
Fv
Fab
IgG
Fc
Variablity located in the CDR regions
• The sequence variability of the VL and VH domains is concentrated in three
hypervariable regions that form the antigen-binding site.
•Also called complementarity-determining regions (CDRs)
•The remainder of the VL and VH domains form the framework regions (FRs)
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Structure of the immunoglobulin fold
• Diagram of an immunoglobulin light chain showing the structure of its constant (CL)
and variable (CV) domains.
•The CDR loop regions are shown in blue
•The β-pleated sheets are held together by hydrophobic interactions and conserved
disulfide bonds.
The nature of antibody-antigen interactions
•Cooperative effect from numerous
attractive forces
•Non-covalent interactions operates
over a very small distance (1Å)
•Shape complementarity is important!
Basis for the specificity of
antibody-antigen interactions.
3
Interaction between antibody and antigen
• Interaction between antibody and influenza virus antigen
(Coleman and Tulip, 1993)
Generation of antibody subfragments by enzymatic digestion
•For some applications the use of antibody
fragments rather than the intact molecule are
favoured.
•Prevent binding to Fc receptors on the cell
Surface (leukocytes)
•Pepsin, Papain are the most commonly used
proteolytic enzymes for generation of antibody
fragments
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Generation of polyclonal antibodies
Antigen
Immunization
Blood
Blood cells
Plasma
Coagulation factors
Antiserum
Enrich antibodies
(specific or unspecific)
Antigen
Protein A/G Class specific methods
(ex. Anti-IgE antibodies)
General characteristics of the antibody response
General characteristics of all specific immune responses
A. Self/non-self discrimination
B. Memory
C. Specificity
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Immunogen
+
Adjuvant
Immune response
Immunogen
Immune response
Adjuvants
An adjuvant enhances the immune response upon immunization through one
or several mechanisms including:
• providing a slow-release of immunogen (depot effect)
• stimulating T-cell help
• beneficial presentation of immunogen
Examples of commonly used adjuvants
Adjuvant
Composition and use
Freunds complete
adjuvant (FCA)
Mineral oil containing heat-killed mycobacteria. Used as emulsion with aqueous antigen
Used as emulsion with aqueous antigen
Freunds incomplete
adjuvant (FIA)
Mineral oil
Used as emulsion with aqueous antigen
Alum
Complex aluminium salts. There are various versions of the adjuvant: some can be
purchased ready for use (e.g. Alhydrogel); others can be prepared in the laboratory by
mixing various salts. Aqueous antigen is absorbed to gel.
Quil A
Saponin derived from Quillaja saponana Molina (South american tree). Mixed to form
a complex with aqueous antigen (ISCOMS).......
Bacillus pertussis
Killed organism mixed with aqueous antigen
FCA/FIA is probably the most potent adjuvant but may be inappropriate for
some purposes.
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Antibodies from chickens (IgY)
Chickens (birds) are evolutionary more distant from humans
than mice or rabbits, making it more easy to obtain an
immune response towards well conserved antigens.
A single chicken can produce high amounts of antibody, up
to 3 grams of IgY per month, which is 10-20 times the
amount of a rabbit
Antibodies are present in the yolk of the egg.
Compared to rabbits, chickens produce antibody much
quicker—high-titre antibody is available from eggs as early
as day 25.
Monoclonal vs Polyclonal antibodies
•The polyclonal antiserum produced in response to a complex antigen contains
a mixture of antibodies each specific for one of the epitopes present on the antigen.
•A monoclonal antibody, derived from a single plasma cell, is specific for one
epitope on a complex antigen.
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Purification of antibodies
•Some immunochemical techniques are frequently carried out using unpurified
antibodies.
•Often partial or complete purification of specific antibodies is required for use in
immunochemical assays.
Ligands for affinity chromatography:
Ligand type:
Examples(s):
Antibody purification:
Hapten
DNP
Antibodies that bind hapten
Antigen
Haemoglobin, FVIII
Polyclonal antibodies of a single specificity
Bacterial immunoglobulin
binding proteins
Protein A, Protein G
Most IgG subclasses from many species
Anti-immunoglobulin antibodies
Goat anti-human IgG Class and/or species-specific IgG fraction
Lectins
Jacalin
Mannan-binding
Human IgA
Mouse IgM
Precipitation reactions (Antibody-antigen reactions)
•The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments
•The antigen must have at least two copies of the same epitope or have different epitopes
that reacts with different antibodies present in polyclonal antisera
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Immunodiffusion methods
Radial immunodiffusion (Mancini method)
The square of the precipitin ring
diameter is proportional to the
antigen concentration
Double immunodiffusion(Ouchterlony method)
Qualitative tool for determining
the relationship between antigens
and antibody
Shared epitopes?
Agglutination
Antibody mediated precipitation of cells or particles
Agglutination can be used for blood typing by mixing red blood cells with blood type specific antibodies
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Species-specific
antibodies
Species-specific
antibodies
Important reagents in immunotechnology applications
Isolate mouse
antibodies
Inject
into goat
(total pool, specific
isotypes or fragments)
Recover
serum
Other examples:
• Rabbit anti-mouse IgG
• Rat anti-mouse IgG1
• Sheep anti-rat IgM
Purify goat-anti mouse
antibodies
Antibody labelling
E
Isotope
Fluorophore
Chromogenic
substrate
Product
•Soluble
•Precipitate
F
Horseradish Peroxidase (HRP)
Alkaline Phosphatase (AP)
β-Galactosidase (β-gal)
Fluorescein (FITC)
Rhodamine
Phycoerythrin
Cy-3
Cy-5
Alexa Fluor
125Iodine
Gold
Ferritin
Wolfram
ELISA
Immunohistochemistry
Immunoblotting
Immunohistochemistry
Immunocytochemistry
Flow cytometry
Immunoblotting
Protein arrays
Radioimmunoassays
(RIA)
Immunoelectron
microscopy
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Direct and indirect immunochemical assays
The direct labeling method:
• one step method
• a specific antibody is complexed directly
to the visible marker.
Indirect labeling method:
• two-step method
• a primary antibody specific for the antigen
• a secondary antibody coupled to a visible marker
(enzymatic, fluorescent, gold)
Immunoradioassay (RIA)
• Competitive binding radioimmunoassay
• The earliest immunoassays developed
were of this type
• Sensitive
• Difficult to automate
• Time consuming
• Hazardous
The technique was introduced in 1960 by Berson and Yalow as an assay
for the concentration of insulin in plasma. It represented the first time
that hormone levels in the blood could be detected by an in vitro assay.
Nobel prize in 1977
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ELISA – Enzyme-linked immunosorbent assay
•
•
•
•
Method for concentration determination using antibody–based technique
Solid-phase assay (polystyrene 96-well plates)
Use of enzyme-conjugated secondary antibody
Spectrophotometric measurement of the amount of coloured reaction product
formed after addition of chromogenic substrate.
• Compare result with standard curve measured with known amount of analyte
• Different formats available (indirect ELISA, competitive-ELISA and
sandwich-ELISA
Indirect ELISA; for quantitative
determination of antibody concentration
against specific antigen.
Sandwich-ELISA; for quantitative
determination of low concentration
antigen in complex solution.
Antibody-based home pregnancy test
Test for hCG: Human chorionic gonadotropin (pregnancy hormone)
Y
Y
Anti-hCG antibody no. 2
Y
Y
Y
Coloured latex beads
+
Anti-hCG antibody no. 1
Anti-Anti-hCG antibody
Y
Test line
Control line
Capillary
force
Y
Y
Y
Y
or
Y
Y
Y
Y
Y
Y
Y
Y
Y
HCG
Y
Y
Y
Y
Y
Positive
Negative
Invalid
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Y
Western blotting a.k.a. immunoblot
•
Technique for detection/analysis of either proteins or antibodies in a complex
mixture.
•
The method originated from the laboratory of George Stark at Stanford. The
name western blot was given to the technique by W. Neal Burnette and is a play
on the name Southern blot, a technique for DNA detection developed earlier by
Edwin Southern. Detection of RNA is termed northern blotting.
•
–
Complementary to ELISA techniques, but more informative.
Theoretical size of target protein available
–
–
–
Can be used to answer questions like:
Does my sample contain protein X (if antibodies to X are available)?
Does this serum sample contain antibodies to X (if protein x is available)?
How specific is my antibody?
•
•
Involves three main steps:
1.
SDS-PAGE (electrophoretic size separation of sample in gel)
2.
Transfer of size separated sample to nitrocellulose/PVDF-membrane
3.
Probing of membrane with antibodies/serum
Protein separation by SDS-PAGE
Sample loading
Sample preparation
Marker proteins
SS
Polyacrylamide gel
SDS +
Β-mercaptoethanol
Native
conditions
SS
-
- -
SS
- -
-
--
Native molecule
Negative charges introduced
No 3D-structure
Disulphides unaffected
Detect proteins
- - S
H
-
--
SDS
+
SH
Negative charges introduced
No 3D-structure
No disulphide bonds
No multimers of proteins
•Coomassie blue staining
•Silver staining
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Western blotting
Transfer proteins to membrane by:
•Capillary force
•Electrophoresis
Sample loading
Marker proteins
Membrane
Polyacrylamide gel
+
Block membrane and add
antibodies, serum etc
NB! Epitopes for mAbs can be destroyed by SDS treatment
Y
Secondary labelled
antibody
SS
Primary antibody
YY
mAbs recognizing
3D-epitope
HIV-test Western blotting
• For validation of ELISA results
• Interpretation of result is not standardized
• In general ”Three band rule”
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Immunohistochemistry
Immunocytochemistry
Immunofluorescence
Immunoelectron microscopy
Immunohistochemistry or IHC refers to the process of localizing proteins in
cells of a tissue section
Can be used to answer questions like:
• In what cells of a particular tissue is a protein expressed?
• Where in that cell is the protein localized?
Such information is important to elucidate the function of "unknown" proteins.
The functions of majority of human proteome are still unknown.
TMA – Tissue micro arrays
Biobank material
TMA production
Antibody based tissue profiling in
large set of tissues or cells
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Tissue Micro Array - Production
Microtone cut
Donor tissue
Transfer to
recipient block
Transfer to
glass slide
• Protein expression and localization data for normal tissues, cancer
tissues, cells and cell lines.
• Protein Atlas Ver 3.0 Released in October 8th, 2007 at the HUPO
meeting in Seoul, Korea (www.proteinatlas.org)
• Publically available database free of charge
• Contains >3000 antibodies corresponding to more than 2.8 million
annotated immunohistochemical images
www.proteinatlas.org
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Antibody quality assurance
1. Low similarity
2. Protein array
3. Western blot
4. Histochemistry (IHC)
Reliability score:
High
Medium
Low
Very low
Failed
Two independent antibodies with similar staining patterns
Consistent with bioinformatics and literature (if available)
At least some evidence that supports the staining patterns
No evidence that the antibody is correct
The antibody is not correct
TMAs in the Human Protein Atlas project
Normal
tissues
Cancer
tissues
Cells &
cell lines
In total 708 cores analyzed per antibody:
48 normal tissues (x 3)
20 cancer tissues (17 x 12 x 2 + 3 x 4 x 2)
58 cell samples (x 2)
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All validation data available
•Gene and protein data (Ensembl, Uniprot)
•PrEST data (size, similarity scores)
•Antibody info
•Validation summary
•Protein array validation
•Western blot
www.proteinatlas.org
In silico biomarker discovery
Prostate Breast
cancer
cancer
Colon
cancer
HPA680087
ACPP
HPA260028
HPA350021
PSA
HPA880064
HPA290101
HPA380031
HPA620019
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Tissue based search function
Advanced tissue based search
function in Protein Atlas 3.0
Immunoelectron microscopy
Label antibodies with colloidal gold,
ferritin or other electron dense labels.
Visualized as small black dots with the electron
microscope
Picture shows surface of a B cell lymphoma
stained with two antibodies:
-One against MHC Class II, labelled with 30nm gold
particles.
-One against MHC Class I molecules labelled with
15nm gold particles.
(Jenei et al, 1997)
Bar = 500nm
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Immunofluorescence
• Novel feature in Protein Atlas 3.0 provide high-resolution multi-color
images of immunofluorescently stained cells
•Increased spatial information on a fine cellular and subcellular level
•Three cell lines, U-2OS, A-431 and U-251MG currently incorporated
•Co-staining with cellular probes targeting specific compartments
(DAPI, calreticulin, tubulin, HPA-antibody)
Flow cytometry
• Analysis and sorting of cells
• Based on fluorescence and light-scattering properties of individual cells
• Cells can be labelled by:
labeled antibodies
expression of fluorescent reporter
•
•
•
•
Cells are analyzed on at the time (real-time measurement)
Several parameters can be analyzed simultaneously
Can be performed at high speed (up to 100.000 events/sec)
Cells are often viable after cell sorting
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The principles of flow cytometry
Fluorescence
FL3
detection
FL2
Side Scatter
(complexity)
104
103
FL1
102
101
Forward Light
Scatter
(object size)
Light
Source
100
0
200 400 600 800 1000
FSC-H
80
M2
Counts
60
40
20
-
0
+
100
101
102
103
104
FL1-H
Data visualisation
Sorted target
cells
Analysis of cell subpopulations
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Flow cytometry analysis of T-cell populations
One healthy and one HIV-infected person
FITC
T-helper cells
(HIV target cells)
PE
Cytotoxic T-cells
Control with labeled
but non-specific antibodies
Immunoaffinity chromatography
Sample loading
Washing
Elution
• Use of antibodies (mAbs or polyclonal) for purification of the antigen
• Simultaneous purification and concentration
• Antibodies are immobilized onto a chromatographic resin
• Stability of the antibody(ies) is important (elution, column sanitation)
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Choosing the right antibody
Every immunochemical technique presents the antigen in a different physical context
Often antibodies that work well for a particular technique
are a poor choice for another assay
Success of an immunochemical technique:
•The avidity of antibodies for the antigen
•Antibody specificity
•Damaged epitopes – How the epitope structure are altered during the technique
•Antibody access – how easily the antibody can reach the antigen
•Secondary reagents
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