EXMD610 Biomedical Methods - McGill University

EXMD610: Immunochemistry

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[email protected] EXMD610 - February 2008 1

EXMD610 Biomedical Methods — Immunochemistry

This class will deal with the following:

Antibodies

The generation of antibody diversity

Polyclonal verses Monoclonal preparations

How to produce Polyclonal or Monoclonal antibody

Recombinant “antibodies”

Purification and characterization of antibodies

Modifying antibodies (& proteins) for specific uses

Antigen detection, quantitation, characterization

Epitope mapping example


Antibodies – agents of immunochemical specificity

Antibodies

What are they?

Glycoprotiens, Immunoglobulins, Receptors,

Antigens and Antibodies

Structure

Polypeptide subunits

Folding

Classes and isotypes

Functions

In natural and artificial systems

Definitions

epitope, paratope, idiotope ...


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Antibodies

Antibodies are proteins called immunoglobulins which

have high-affinity binding sites for other molecules

They are produced in animals as part of the immune

response to foreign material entering the body tissues.

Normally the immune response leads to high level

production only of antibodies which specifically bind to

sites (epitopes) on the surface of any of the foreign

molecules or their degradation products.

Autoimmune disease may result when antibodies to “self”

molecules are produced.


Antibodies

Normal functions of antibodies include:

• Neutralizing toxins and viruses by blocking their

interaction with cells.

• Binding to surfaces of the foreign material to

improve its uptake, inactivation, degradation and

clearance by phagocytic cells.

• Triggering activation of the complement cascade

on the surface of a pathogen to damage the cell

and improve its clearance by phagocytic cells.


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Immunoglobulins: Receptors and Antibodies

Immunoglobulins are glycoproteins produced by B lymphocytes

and the plasma cells which differentiate from them.

B lymphocytes start out expressing cell surface immunoglobulin

in the form of a transmembrane “antigen” receptor. It is the

specificity-determining part of a receptor complex.

Upon activation a B lymphocyte proliferates, and many of its

progeny become plasma cells which secrete large amounts of a

soluble form of immunoglobulin called antibodies.

Some progeny become memory B cells, ready to respond more

quickly to the same antigen.


Antigens and Antibodies

An immune response is an activated process which

can neutralize, degrade and clear potentially damaging

foreign material (e.g. parasites, microbes, viruses, some

chemicals) which has entered the body’s tissues, or

endogenous material (e.g. mutant cells) recognized as

aberrant by the immune system.

Broadly speaking, an antigen is anything which

stimulates an immune response.

In immunochemistry we focus on antigens which

stimulate B lymphocyte responses, and use the

antibodies thus produced.


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Antibody Structure and Function

The structure of a simple immunoglobulin:

Four polypeptide chains

2 identical heavy chains, 2 identical light chains

Variable regions of

heavy and light chains

provide a range of

different specificities

for antigen

The constant

regions of the

heavy chains

(CH) have

binding sites

for cell surface

receptors When some types of antibody bind antigen,

they can activate the complement cascade

through binding of C1 to CH sites


IgG structure


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Antibody Structure

Different types of

immunoglobulin

differ in the number

of CH domains,

disulfide bond

number an position,

flexibility of the so-

called hinge region,

and differences in

sites for glycosylation

VL

VH

CL

CH1

CH2

CH3

Hinge

DIfferent classes are illustrated in

the next two slides


IgG1

IgG3

IgG4IgG2

Schematic representations of human IgG subclasses


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IgA

monomer

IgM + J chainIgAdimer includes a J chain.

IgA dimer on mucosal surface incldes a secretory component

– a fragment of the receptor which mediates the transcytosis.

IgE

Three other human Ig classes


Antibody Class Properties

IgA Minor serum component

IgA dimer Major secretory component

IgD B Cell surface Immunoglobulin

IgE Binds to IgE receptors. Ag triggers mast cells

degranulation. Very low level in serum.

IgG Predominant circulating antibody after booster

immunization. Activates complement.

IgM Predominant circulating antibody early after first

immunization. Efficiently activates complement.


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Functional

Fragments of

IgG Antibodies

Fragment

Fab = antigen binding

Fc = crystalizing

The prime in F(ab’)2

reminds us that the

polypeptide chain is cut

at a site different from

that which generates

the Fab.


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In immunochemistry, antibodies are used as tools in the

detection, quantitation and characterization of the

molecules they bind.

Certain classes of antibody (IgM and IgG) can be obtained

easily in useful quantities from blood taken from an animal

which has been inoculated with the molecule of interest.

Generalization: Any protein which, like an antibody, binds

another molecule with high specificity can be adapted for

the detection, quantitation, and characterization of that

molecule.

Antibodies in Immunochemistry


Most proteins are modular. In other words, the polypeptide chain

is folded into several distinct and more-or-less functionally autonomous

domains.

Ligand-binding domains of proteins are often isolated and adapted for

uses similar to those of antibodies. This usually involves genetic

engineering and expression of recombinant protein.

One strategy is to make a construct in which the ligand-binding domain

is coupled to the Ig heavy chain constant domains which form the Fc

part of the Ig. This allows the use of many techniques worked out for

antibodies which use Fc characteristics.

LBDIgG

Fc

Antibody fusion

protein

“receptor-

globulin”


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Recombinant forms which bind antigen Fv, scFv

CDR = complementarity

determining regions

FR = Framework regions

Recombinant forms:

Fv = Fragment variable

(VH + VL)

scFv = single chain Fv

Phage display can be

used to screen for scFv

with desired specificity.


Basis for Antibody Diversity (1)

As B lymphocytes differentiate in the bone marrow, the

germline immunoglobulin heavy and light chain genes are

rearranged to bring V, D, J and V, J variable region gene

segments together respectively.

There are many possible random selections from multiple

diverse copies of these segments. There is also random

additions of nucleotides when these segments are joined.

The most variable (hypervariable) parts of the resulting gene

encode parts of the variable region which form the potential

antigen binding site.


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Hypervariable regions

coincide with the parts

of the gene encoding the

loops of the so-called

complementarity

determining regions

(CDR) of the antibody.

It is illustrated for the

light chain, but is similar

for the heavy chain.


B cell activation and class switching (1)

Antigen binding to a B cell receptor (surface Ig) provides one

activating signal. The antigen is engulfed, protein components are

broken down, and protein fragments (peptides) are picked up by

MHC-class II molecules and presented on the B cell surface.

In order to become fully active and driven to proliferate, the B cell

must also receive signals (cytokine) from a “helper” T cell which

recognises the peptide-MHC combination, and has already been

activated through such recognition during interaction with an

antigen presenting cell.

Later, T cells help active B cells switch the class of Ab they make.


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B cell activation and class switching (3)


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Polyclonal Antibody Preparation

Adjuvant is used to

• reduce the rate of clearance of the antigen from the body

• keep it fairly localized

• provide a low non-specific stimulus to the immune system to

improve antigen presentation and B cell activation.

Because T lymphocytes must also be activated in order to help a B

lymphocyte response, simple molecules, and molecules which do

not have a protein component are coupled to protein carriers like

Keyhole Limpet Hemocyanin (KLH).

Common adjuvants use an oil-aqueous emulsion to trap antigen

for slow release.


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Raising Polyclonal Antiserum

Syringe method for emulsifying

antigen with adjuvant

Boosting and bleeding

schedule will produce several

batches of high titre antiserum.

(see notes). Mainly IgM after

primary. IgG after boost.

IgM

IgG


Bleeding mice for antiserum

To test for antibody response, a

small quantity of blood may be

taken from behind the eyeball into

a capillary, or from a cut on the tail.

When bleeding out, the armpit can

make a convenient basin for blood

collection.

With a little practice one may

obtain about 2 ml blood by cardiac

puncture.

Consult institutes animal facility for

approved practice and training.


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Epitope, Paratope, Idiotope.

Epitope = the surface of the Ag

which makes contact with the Ab

Paratope = the surface of the Ab

which makes contact with the Ag

Idiotope = surfaces in the

variable regions which

distinguish one Ig from

another of the same

isotype. The idiotopes on

an Ig define its Idiotype.


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The Precipitin Reaction

If you have polyclonal

antibody and a few tens of

micrograms of antigen, you

can mix dilutions of them and

achieve a ratio, the

equivalence point, where the

maximum precipitate forms

Single radial

immunodiffusion has

the antibody in agarose

on a slide. Antigen

solutions are put into

small wells. Precipitate

forms as it diffuses out.


Schild, G.C., J. gen. Virol. (1970), 9: I9I

Immunodiffusion and Immunoelectrophoresis

Kaufman and DiRienzo, Oral Microbiol Immunol 1988: 3: 145

Antiserum in centre, antigen

preparations from different flu virus

strains in surrounding wells.

Antiserum

Antigen preparation

Monoclonal Ab


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The Ouchterlony double diffusion method


Polyclonal and Monoclonal

Above: Polyclonal

antibody in serum results

from activation of many B

cells with different

specificities for the

antigen

Below: Monoclonal

antibody preparations

require cloning of

individual antigen-specific

B cell lines.


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Comparison of different types of Ab preparation


Monoclonal Antibodies - Hybridomas

Since normal B cells do not grow

well in culture, they are fused with

an immortal B cell like tumour cell

called a myeloma. The Balb/c

mouse has been the source of

such lines.

Some myeloma lines secrete a

light chain, but the best myeloma

lines for fusion do not produce

endogenous Ig chains.

The hybrid produced is called a

hybridoma.

See notes for method.


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HAT selection against unfused myeloma

If the main biosynthetic pathway for nucleic acid precursors is

blocked with the drug Aminopterin, normal lymphocytes can

survive by means of the salvage pathway if Hypoxanthine and

Thymidine are supplied. Myeloma lines have been selected with

mutations affecting thymidine kinase or hypoxanthine guanine

phosphoribosyltransferase. They die in the presence of HAT.

H

A

T



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Crossreactivity of antibody

The left shows how a hapten specific antibody (raised against the

meta isomer of aminobenzenesulfonate) may crossreact with related

haptens.

The right shows how a conformation specific antibody will not

recognize unfolded polypeptide.


Selective, intermediate and group specific antibodies.

Each Ab raised against hapten 1

Haptens 2-5 are chemically closely releated, having very similar

structures but differing from each other in some chemical groups.

Monoclonal antibodies may exhibit different degrees of

cross-reactivity


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Purification of Antibodies

Purified antibody is necessary for tagging of the

antibody (e.g. enzyme, biotin, fluorochrome, radioactive

group).

High purity is needed when making immuno-affinity

matrices for antigen purification.

It is preferred for clean detection and quantitation

assays.



Here are a few affinity purification products which can facillitate Ab

purification:


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If one has plenty of antigen, one may perform affinity

chromatography of antibody on immobilized antigen.

In all cases, buffer conditions can greatly affect the success of

antibody purification. The buffer conditions can improve binding

capacity of the affinity column and preserve its ability to bind

antibody so it can be reused.

Elution buffers should be as mild as possible while being effective.

Polyclonal antibody can lose some activity, yet continue to be

useful.

Some monoclonal antibodies can be susceptible to partial

denaturing at low-pH or in other elution conditions and may lose

antigen-binding activity during purification. This must be tested.



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Coupling tags to antibodies – E.g. Biotin

Biotin is the ligand for

Avidin (an egg protein).

Many tags like biotin

are made convenient

for coupling to NH2 by

adding succinimidyl

groups.

Coupling to epsilon

amino groups of lysine

residues is efficient.


Crosslinking protein to protein – e.g. Ab to enzyme

There are many crosslinking

agents. They may be useful in

coupling peptide to carrier protein

for immunization, or enzyme to

antibody for use in ELISA or

immunoblot.

The reagent depicted here is

SPDP.

It reacts with –NH2 groups to yield the product on the left, which

can be attacked by free sulfhydryl groups to form disulfide links.

Thus Ab and enzyme (e.g. Horse Raddish Peroxidase, HRP) can

each be reacted with SPDP. The group on HRP can be reduced,

then the two proteins mixed to conjugate.


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ELISA (enzyme-linked immunosorbent assay)

A convenient efficient assay

is essential for screening

hybrids (see notes).

The ELISA is a popupar

screening method as well as

being a quantitative assay for

antigen once Ab is available.

The type of ELISA illustrated

here has antigen adsorbed to

plastic wells, unlabelled

primary antibody, and

enzyme-coupled second Ab

(e.g. goat anti-mouse IgG)



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Types of ELISA:— Direct ELISA

Antigen adsorbed

to surfaceAdd Antibody Wash away excess Ab

Measure amount of retained antibody by enzyme reaction (colour)

Single-Step

Primary Ab

is tagged

Two-Step

Primary Ab is treated

as antigen as above.

Secondary Ab is

tagged


Types of ELISA:— Competitive ELISA

Antigen adsorbed to

surface

(controlled amount)

Add unknown solution of Ag mixed with

controlled amount of Ab — incubate

Wash away fluid-phase

Ab-Ag complexes

Measure retained antibody

(single-step or two-step)


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Types of ELISA:— Sandwich ELISA

Antibody adsorbed

to surface — will

capture antigen

Add crude

solution

containing Ag

Wash away other

molecules

Measure retained antibody

(single-step or two-step)

Add antibody to a

different epitope

on the Ag


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An example of interference in immunoassays:

Macromolecules or aggregates of molecules in sample which bind

to Fc of antibody may sterically inhibit antigen binding to

neighbouring antibodies. Sample preparation tricks can overcome

or reduce this type of problem.

Be aware that interference can occur - an example:


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One common precaution is to include an irrelevant antibody from

the same species as the immobilized Ab in the fluid phase.

A simple solution to some types of interference

Mouse Ab

Sterically blocked antigen binding - false positive.

Antigen

Labelled Antigen

Something

which binds

to mouse Ig

Detected by competition

with labelled Ag

“Neutralized” interferenceIrrelevant Mouse Ig


Radioimmunoassay — RIA

Immobilized Ab to

capture antigen

Add crude solution containing unlabelled

Ag together with labelled radioactive Ag

May sample and measure

radioactivity in supernatant

Measure

retained

radioactivity

May wash away unbound

radioactivity and ...


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Radioimmunoassay (RIA)

A technique for quantitating hormone levels in physiological

samples

Basic Requirements for RIA

ANTIBODY (polyclonal or monoclonal)

- high specificity

- high affinity

- high titer LABELED ANTIGEN (Hormone)

- high specific activity

- low radiolabel damage

ANTIGEN STANDARDS

- High purity

[email protected] 57EXMD610 - February 2008

Separation of Bound and Free Antigen

Dextran-coated charcoal treatment

will adsorb the “free” antigen

the antigen-antibody complex will remain in the supernatent

The radioactivity bound to the antibody and present in the suprenatent

is measured.

Second Antibody Treatment

will precipitate antigen bound to the primary antibody

The radioactivity present in the (immune-complex) precipitate is measured.

RIA: 3H or 125I-labelled antigen is mixed with test sample and antibody.

The unlabelled Ag competes with the labelled for binding to Ab.



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The amount of labeled antigen bound to the antibody will decrease with

increasing concentrations of the unlabeled antigen.

Allows you to generate a standard curve using serial dilutions of pure

antigen in the pM range. The antigen concentration in unknown samples

can then be read off the standard curve.

Validation of RIA

Precision - Intra-assay variation

Sensitivity - Smallest amount that can be measured

Reproducibility - Inter-assay variance

and quality control

RIA: Data Analysis



From: Phillip D.K. et al, 2007,

Journal of Clinical Endocrinology and Metabolism 81:2002-5

An RIA example with standard and controls

In this case, radioactive

and cold competing

antigen samples were

mixed with Ab in solution.

Antibody-bound Ag was

precipitated for counting by

adding a second antibody

and polyethylene glycol

sufficient to precipitate Ig

but not the IGF molecules.


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Competitive Chromatographic Immunoassay


Example of insulin assayH.-J. Liu, R. E. Strong, I. S. Krull and S. A. CohenAnalytical Biochemistry 298, 103–111 (2001)

Labelled insulin with AQC

6-aminoquinolyl-N-hydroxysuccinimidylcarbamate

Used an AQC:insulin molar ratio of 20:1 in order to saturate all 3 amino groups

and have a homogeneous preparation. Dialyzed away non-coupled products.

Excitation and emission wavelengths used for detection: 250nm and 395 nm

A small column of immobilized protein G was used to bind Ab. Then sample plus

labelled insulin was pumped through.

Free labelled insulin passing through the column at pH7

Ab and (bound) insulin eluted at pH2

Labelled insulin mixed with unlabelled insulin sample

Labelled insulin only

Unlabelled insulin in the sample competes for binding to the Ab, so there is more free labelled insulin passing through during loading in the top chromatogram.



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Electrophoretic Transfer Immuno-staining the blot


Ab for affinity purification of molecule

� Ab which recognizes an epitope on the target

molecule is coupled to a solid phase such as small

or microscopic beads, or membrane.

� The molecule is typically obtained in a crude extract

from tissue or cell culture.

� The extract is incubated with the immobilized

abtibody.

� Other molecules are washed away

� Target molecule is eluted with eluant solution


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When using antibody for affinity purification of its ligand:

� Have the antibody as pure as conveniently possible.

� Attach it to a solid phase material which does not

appreciably bind any other component in the crude ligand

preparation.

� Preferably attach antibody to solid phase via covalent

bonds to reduce elution of antibody with the ligand.

� In the crude ligand solution and in wash solutions, include

salt and surfactants which will reduce non-specific

binding without disrupting specific binding of the antibody

with the epitope.

� Preclear the crude ligand preparation by incubating it with

an irrelevant affinity matrix as much like the specific

affinity matrix as possible.


Test for ligand binding and elution:Try a range of pH, salt concentrations, detergents,

chaotropic agents, solvent mixtures etc.

Conditions sought:� Efficient binding of ligand to antibody with minimal

contamination from binding of other components of the

crude preparation.

� Efficient elution of the ligand. Preferably:

� Without denaturing or chemically modifying the ligand

� Without denaturing the antibody (so affinity matrix is

reusable)

� With ligand eluted in solution which is compatible with

the next step, or easily exchanged.


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Surface Plasmon Resonance for detecting & measuring binding



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Holger Wenschuh, et al

Biopolymers (Peptide Science), Vol. 55, 188-206 (2000)

Coherent Membrane Supports for Parallel Microsynthesis and

Screening of Bioactive Peptides



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