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Immunology Review
Part Two
Antibodies and Antigens
Immunoglobulins
Glycoprotein molecules produced by
plasma cells in response to an antigen
and capable of binding to that antigen.
AKA Ig or antibody
BASIC STRUCTURE OF
IMMUNOGLOBULIN


Four polypeptide
chains
2 heavy chains



NH3+
these define the class of
Ig
2 light chains
The chains are held
together by disulfide
bonds.
COO-
REGIONS
NH3+


Each Ig molecule has
a constant region and
a variable region.
It is the variable
region that confers
antibody specificity.
COO-
DOMAINS
Within the regions, are
domains.

These are loops of
compatible amino acids
that determine the biologic
properties of the
immunoglobulin molecule.
C
C
C
C

THE EFFECT OF ENZYMES

Papain cleaves Ig at the
hinge region, above the
disulfide bonds. This
leaves 3 pieces.


2 Fab pieces (fragment
antigen binding)
containing the antigen
binding sites.
1 Fc piece (fragment
crystalizable). The Fc
piece plays a role in
complement activation,
monocyte binding, and
transfer of IgG across the
placenta.
THE IMMUNOGLOBULIN
CLASSES
IgG




Gamma heavy chain.
Comprises 80 % of
Ig.
Exists as a monomer.
 160,000 Daltons
 7s
Reacts best at 37oC
and the AHG phase
NH3+
COO-
IgG





May activate complement
Enhances opsonization
Neutralizes toxins and viruses
Crosses the placenta - cause of HDFN
Considered an “incomplete” antibody
IgM



Mu heavy chain.
Comprises 4-6 % of Ig.
Exists as pentamer.



900,000 Daltons
19s
React best in cold
temperatures (4oC to
room temp.)
Y
IgM





Surface receptor on B lymphocytes
Activates complement - can cause
intravascular hemolysis
Increases opsonization
Neutralizes toxins
Considered a “complete” antibody
IgA



Alpha heavy chain.
Comprises 13% of Ig.
Exists as a monomer or a dimer.




360,000 Daltons
7s
Main Ig in secretions such as saliva and
tears.
Prevents bacteria from adhering to mucosa,
neutralizes toxins, and opsonization.
IgD


IgD - Delta heavy chain.
Comprises 1% of Ig.


Exists as monomer.



Greatest quantity is found attached to
immunocompetent, unstimulated B lymphocytes.
160,00 Daltons
7s
Probably involved in B cell maturation into
plasma cells.
IgE



IgE - Epsilon heavy chain.
Present in trace amounts.
Exists as monomer.




200,000 Daltons
8s
Almost all IgE is bound to circulating basophils
or tissue mast cells.
Enhances allergic reactions, and promotes
inflammation.
Antigens
Antigen


A chemical substance, seen by the
body as foreign, that is capable of
eliciting an immune response when
introduced into an individual (host)
who lacks that substance.
In Immunohematology, the antigens
of interest are usually on red blood
cells.
Lock and Key Fit

Antigen combines with antibody based on
complementary structure. The better the “fit”
the more tight the bond between the two.
AG
AB
AG
AB
Epitope



The portion of the antigen that reacts
with the antibody.
A single antigen may have multiple
epitopes.
May be linear or conformational
SPECIFICITY



The antigen that is the “target” of the
antibody produced.
A single antigen may possess many
different epitopes, each capable of eliciting
a specific response.
Each epitope is the target of an antibody
produced by the response, yet all of these
antibodies are said to have the same
specificity (the antigen).
For Example:



The D antigen is a
mosaic, comprised of 4
epitopes : Da, Db, Dc,
and Dd.
Each epitope may
stimulate antibody
directed at that portion
of the molecule.
All 4 antibodies would
have anti-D specificity.
Da
Db
Dc
Dd
Hapten



A small molecule that alone cannot
stimulate antibody production.
When a hapten is coupled with another
molecule, it may stimulate production of an
antibody that will bind to the hapten.
Once the antibody has been produced, the
hapten no longer needs the carrier
molecule in order to bind with the antibody.
IMMUNOGENICITY


The ability of an antigen to elicit an immune
response (i.e. stimulate antibody production)
Dependant on the following factors:





Antigen composition & size
Route of entry
Dose
Degree of foreignness
Host factors
ANTIGENICITY


The ability of the antigen to bind
with its antibody.
Affected by :





location
size
shape
charge
biologic properties
Antigen/Antibody Reactions
Immunology vs. Serology

The study of
host reactions
to foreign
substances.

The study of
serum; in
particular, the
study of
antibodies in
serum and other
body fluids.
Definitions



Sensitivity: How well a test is able
to detect minute quantities of an
analyte.
Specificity: The ability of a test to
detect one unique analyte.
Cross reactivity: Reactions with
substances that are structurally
similar to an analyte. (False positive
reaction)
Antigen / Antibody Reactions



Antigen and antibody bind with “Lock and Key” fit.
Affinity – the total attractive force that draws
antibody to antigen.
Avidity – how “tightly” antigen and antibody bind.
AG
AB
Antigen / Antibody Reactions


Antigen / antibody reactions are
readily reversible.
Free Ag + Free Ab
Ag-Ab
complex
Antigen / Antibody Reactions
Visualized when lattice structures form.


Sensitization: Antibody attaches to antigen
Agglutination or precipitation: Antibody crosslinks form between antigens from different
cells, causing cells to “clump” together.
Y

Zone of Equivalence
Y
Y
Y
Y
Y Y
Prozone –
antibody excess
Y
Postzoneantigen excess
Ag/Ab binding is influenced by:



Ionic strength- shielding and zeta
potential
Shielding – charges that surround
the Fab portion of an antibody.
Zeta potential – the difference in
electrical charge between the
surface of a cell and the outer
layer of the ionic cloud that
surrounds the cell in an electrolyte
solution.
When the zeta potential is lowered, the red cells can more
closely approach each other, allowing antibodies to crosslink antigens between cells.
-------------
Ag/Ab binding is influenced by:





pH- optimal 6.5 to 7.5
Reaction time- depends on the
immunoglobulin class and test
medium
Temperature- depends on the
immunoglobulin class
Ag/Ab ratio – Zone of equivalence
Number and location of antigens
Dosage

An antibody that reacts stronger
with a cell that has homozygous
expression of an antigen than it
does with a cell showing
heterozygous antigen expression is
said to show dosage.
If there is an anti- antibody, which of these
cells will yield the stronger reaction with it?
Homozygous
Heterozygous
Visualizing Antigen/Antibody
Reactions
Test Methods
Agglutination Based Methods
Antibodies cause the cross-linking of
particulate antigen, usually found on
a cell.
Direct Agglutination




The antigen is a
natural part of the
solid’s surface.
Often performed at
room temperature.
May use centrifugation
to bring antigen and
antibody into closer
proximity.
Can be used to detect
antigen or antibody
Inhibition of Agglutination


Y
Y
Y

Y

Antibody reagent is combined
with patient’s specimen.
If patient’s specimen contains
the antigen for that antibody,
they will react.
Reagent antigen is added.
A positive reaction will show
no agglutination, because the
antibodies were bound to the
patient antigen before the
reagent antigen was added.
Neutralization
Positive Test
Negative Test
The presence of an antibody prevents the
antigen from functioning correctly.
Precipitation Based Methods
Soluble antigen combines with
antibody to form aggregates which
precipitate out of solution.
One example of a precipitation based
method: Flocculation
Uses fine particles of antigen to detect
antibody in patient’s serum.
Negative test
Positive test
“Labeled” Methods
Attaches a “tag” to either the
antigen or antibody. This “tag”
can be detected and measured.
One example of a “labeled” method:
Competitive EIA



Enzyme labeled
antigen competes
with unlabeled
patient antigen for
binding sites on
fixed antibodies.
A chromogen is
added that reacts
with the enzyme.
The level of color
development is
inversely
proportional to the
level of patient
antigen.
This concludes “Immunology
Review, Part Two: Antibodies and
Antigens”
Please complete the exercise
“Comparison of Immunoglobulins”