EXMD 610 Immunochemistry February 2005

Immunochemical Techniques

! 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

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Antibodies – agents of immunochemical specificity

! Antibodies
 What are they?
 Immunoglobulins, Receptors, Antigens and
Antibodies
 Structure
 Polypeptide subunits
 Folding
 Classes and isotypes
 Functions
 In natural and artificial systems
 Definitions

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Antibodies in immunochemistry - 1

Antibodies are proteins called immunoglobulins which have

binding sites for other molecules.

They are produced in animals as a part of the immune response to

foreign material entering the body tissues (e.g. microbes and
viruses).

Their functions in the animal include:

• neutralizing toxins and viruses by blocking their interaction
with cells
• binding to surfaces of the foreign material to improve uptake
by phagocytic cells
• triggering activation of the complement cascade on the
surface of a pathogen to damage the cell and improve its
clearance by phagocytes.

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Antibodies in immunochemistry - 2

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 immunized with the molecule of interest.

To the extent that proteins other than antibodies bind molecules

with high specificity and affinity, they can be used in many ways
like antibodies in the laboratory.

Recombinant techniques allow us to fuse the specific binding

domains of antibodies (or other proteins) to any other protein
domain and expand the range of uses for them.

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EXMD 610 Immunochemistry February 2005

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.

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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

The constant Variable regions of

regions of the heavy and light chains
heavy chains provide a range of
(CH) have different specificities
binding sites for antigen
for cell surface
receptors When some types of antibody bind antigen,
they can activate the complement cascade
through binding of complement C1 to CH sites
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IgG structure

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Antibody Structure

Hinge VL
Different types of CL
immunoglobulin
differ in the number
of CH domains,
CH1 VH
disulfide bond
number and position, CH2
flexibility of the so-
called hinge region, CH3
and differences in
sites for glycosylation
Different classes are illustrated in
the next two slides
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Schematic representations of human IgG subclasses

IgG1 IgG2 IgG4

IgG3

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Three other human Ig classes

IgA IgE
monomer
IgM + J chain
IgAdimer includes a J chain.
IgA dimer on mucosal surface incldes a secretory component
– a fragment of the receptor which mediates the transcytosis.
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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 cell

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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Properties of Immunoglobulin Classes

Class IgG1 IgG2 IgG3 IgG4 IgM IgA IgD IgE

Molecular
146 146 165 146 970 160 184 188
Weight
Serum Level
9 3 1 0.5 1.5 3.5 0.03 5x10-5
mg/ml
Activates
++ + +++ - +++ - - -
Complement
Placental
+++ + ++ +/- - - - -
Transfer
Binding to
Mast cells & - - - - - - - +++
eosinophils

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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, respectively, V, D, J and V, J variable
region gene segments together.

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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Basis for Antibody Diversity (2)

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Basis for Antibody Diversity (3)

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Basis for Antibody Diversity (4)

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.
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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 (2)

After activation, B cells proliferate in secondary lymphoid tissues

(e.g. lymph nodes, spleen), during which there is a relatively high
frequency of mutation in the CDR encoding parts of its Ig gene
(somatic cell hypermutation).

Daughter cells which no longer bind antigen with high affinity do

not receive survival signals. Those which have gained a higher
affinity for antigen have a selective advantage.

Early antibody is low-affinity IgM, later, especially after a boost

immunization, higher affinity antibody is being made, and class
switching has produced other classes (e.g. IgG) bearing the
same antigen specificities.

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B cell activation and class switching (3)

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Polyclonal Antibody

Most B cells will not respond to a particular antigen.

Each B cell which is activated by an antigen is selected

because its antibody binds an epitope on the antigen.

There are usually many epitopes on an antigen.

Each epitope stimulates a different B cell. Thus, there is

clonal expansion of several different B cells.

It is said to be a polyclonal antibody response, and the

serum containing the mixture of antibodies is called a
polyclonal antiserum.

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Polyclonal Antibody Preparation

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).

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.

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

IgG
schedule will produce several
batches of high titre antiserum. IgM
(see notes). Mainly IgM after
primary. IgG after boost.

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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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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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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.

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Antibodies as Antigens

Antibodies from one animal may become antigens when

injected into another animal.

One may raise antisera specific to a particular class of Ig

heavy chain or light chain.

When injected into the same species, an antibody may

elicit an antibody response against only the part of the
antibody which binds antigen.

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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

Antibody Antigen
excess excess
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.
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The Ouchterlony double diffusion method

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Polyclonal and Monoclonal

Below: Monoclonal
antibody preparations
require cloning of
individual antigen-specific
B cell lines.

Above: Polyclonal
antibody in serum results
from activation of many B
cells with different
specificities for the
antigen

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Comparison of different types of Ab preparation

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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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Before starting to generate hybridomas

Be sure you have a good assay for the antibody you want
You will need to assay media from several hundred clones in a day

Immunization
Antigen need not be pure.
If antigen is a native protein, antibody may not bind denatured form.
If antigen is a denatured protein, antibody may not bind native form.

Getting to know your antibody

Determine what class it is and what light chain it has ( 6 or 8 ).
Does it bind native &/or denatured antigen?
Does it cross-react with related molecules?

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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.
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Monoclonal antibodies may exhibit different degrees of

cross-reactivity

Selective, intermediate and (common) 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.

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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.

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Purification of Antibodies

Here are a few affinity purification products which can facillitate Ab

purification:

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Purification of Antibodies

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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Detection of antibody

Tags:

The antibody may be labelled with a radioactive isotope.

Label biosynthetically by growing hybridoma in the presence
of 35S-methionine & cysteine.
Label with 125I by iodinating tyrosine residues

Fluorescent groups, biotin, or enzymes may be coupled to

the antibody. Often this is through linking to Lysine residues.

Biotinylated antibody will bind avidin or streptavidin. The

latter is modified to carry a tag (e.g. enzyme of fluorochrome)

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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.

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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 popular

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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Enzymes and substrates commonly used in ELISA

Enzyme Common Substrates for Colour- Abs

Change Reaction nm

Alkaline Phosphatase p-nitrophenyl phosphate pNPP 405

Horseradish Peroxidase o-phenylenediamine OPD 492

3,3’,5,5’ tetramethylbenzidine TMBZ 450
o-dianisidine 403

Beta-Galactosidase p-nitrophenyl-$-D-galactopyranoside 405

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Types of ELISA

Direct ELISA, two step.

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Competitive ELISA

Subsequent steps as in direct ELISA

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Sandwich ELISA.

Two antibodies (to different epitopes) are required. One is adsorbed

in plastic well. It picks up Ag. The other detects the bound antigen.

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Considerations for Sandwich ELISA

In the sandwich ELISA, it is important to consider how you

will detect the second antibody without background signal
from the imobilised antibody.

The second antibody could be tagged with enzyme,

radioactive label, or fluorochrome.

It could be tagged with biotin and another step would be

incubation with enzyme coupled streptavidin.

The first antibody could be a mouse monoclonal antibody,

and the second could be polyclonal rabbit antibody. In that
case the additional step could be with an enzyme coupled
goat anti-rabbit Ig as long as the goat antibody did not cross-
react with mouse Ig.

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Be aware that interference can occur - an example:

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.

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A simple solution to some types of interference

Sterically blocked antigen binding - false positive.

Mouse Ab Labelled Antigen

Antigen Detected by competition

with labelled Ag
Something
which binds
to mouse Ig

Irrelevant Mouse Ig “Neutralized” interference

One common precaution is to include an irrelevant antibody from

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

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Radio-Immuno-Assay

RIA

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Western Blot

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Surface Plasmon Resonance for detecting & measuring binding

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Spot Assays
Coherent Membrane
Supports for Parallel
Microsynthesis and
Screening of
Bioactive Peptides

Holger Wenschuh,
Rudolf Volkmer-Engert,
Margit Schmidt, Marco
Schulz, Jens Schneider-
Mergener, Ulrich
Reineke.

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

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Peptide Array Epitope Mapping

In investigating ligand binding sites or epitopes within a protein,

spot screening can be performed on a series of overlapping
peptides which cover the whole polypeptide chain, or a part of it
which corresponds to the domain of interest.

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From: M. Valle et al. (1999) Protein Science 8: 883-889

A Bacillus subtilis
A. Preimmune bacteriophage connector
protein p10 was the
antigen used to raise
polyclonal antibody.

B. Antisera Overlapping synthetic

peptide array covering the
P10 sequence.

Antibodies in the antiserum

recognized clusters of
C. peptide 10 specific overlapping peptides.

These spots cut out of a

similar array were used to
affinity purify the reactive
antibodies.
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Averaged electron microscope

images of connector-antibody
complexes.

Three of the monospecific Ab

preparations shown.

E. Location of the different epitopes

in the 3D map of the connector based
on the two-dimensional projection
averages of the immune
complexes and their comparison with
the side view of the 3D connector
map. The diameter of the widest
domain of the connector
side view represents 13.5 nm.

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