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ImmunologyLecture

RobertJ.Boackle,Ph.D.

AntigenAntibodyReactions

Specific Objectives: THE STUDENT SHOULD BE ABLE TO


1. Discuss immunoglobulin variability (ie. the variable region)
2. Describe bonds between the variable region and the antigenic
determinant
3. Define antibody affinity and antibody avidity
4. Describe a precipitin curve and discuss lattice formation involving
proteins verses carbohydrate antigens and be able to define "zone of
equivalence".
5. Understand immunodiffusion in agar gels.
(identity, nonidentity and partial identity)
6. Have a conceptual understanding of immunoelectrophoresis,
Fluorescent antibody techniques and ELISA (enzyme-linked
immunoassay)
7. Define "agglutination" and understand the functional differences
between monomeric Ab (ie. IgG) and polymeric Ab (ie. IgM and S-IgA)

Definitions:
1. The "antibody affinity" of an antibody-antigen reaction is related to the
strength of attractiveness between an antibody (Fab region) and its antigenic
determinant.
2. The "antibody avidity" is the total strength of binding of the Fab regions of the
population of antibodies evoked to an antigen, and involves the reaction with all
the antigenic determinates. Thus it is the total strength of the binding of
antibodies to antigens.
3. Immune Complex = Antigen-Antibody Complex [the size depends on the ratio
of antigen to antibody].
Also the student should be prepared to answer and discuss the following:
1. List and describe the possible bonds between the immunoglobulin variable region and
an antigenic determinant. Then draw and explain a precipitin curve and "lattice
formation" involving protein antigens and polyclonal Ab.
2. What is meant by "hypervariable regions" on immunoglobulins? How do B cell
clones differ in regard to the hypervariable regions of the immunoglobulins on their
surface? At the level of the gene, explain what is believed to account for these clonal
diversities.
3. Can two different classes of immunoglobulins have identical variable regions? In your
answer include a discussion of the switch mechanism.

ANTIGENIC DETERMINANTS
INTERACT WITH
SPECIFIC ANTIBODY

IgG has a Valence of 2


TWO Identical ANTIGEN BINDING SITES

CH2

CH3

CH2

CH3

Surface
of an
Antigen
i.e.
bacterial
cell surface

Movement
at the
Hinge
Region

IgG

CH2

CH3

CH2

CH3

Non-Covalent Interactions
Ball in glove fit

VL
Antigenic
Determinant

VH

Charge-Charge Interactions
Hydrophobic Interactions - And good fit !
VL

-+
VH
Gene rearrangements and
Mutational Hot Spots

+
-

VL

-+
VH

+
-

Antibody Affinity

Antigenic determinant 1
Antigenic determinant 2
PROTEIN
ANTIGEN

Antigenic determinant 3

Antigenic determinant 4

MUST HAVE POLYCLONAL ANTIBODY


and at least two different antigenic determinants
TO CROSS-LINK PROTEIN ANTIGENS

Y
Y

Y
Immune Complexes

Excess Antibody

Y
Y Y Y
Y
Y
Y
Y Y
Y
Y

ANTIBODY
EXCESS

NO CROSSLINKS
NO Precipitate

Excess Antigen = Not enough


Cross-links to cause a Precipitation

More cross-links, and higher individual affinities


= higher AVIDITY of the Immune Complexes

Y
Y

Y
Y

Y
Y

Amount of Precipitate

ZONE of
Equivalence
No Soluble Ag or Ab
ANTIGEN
EXCESS

Ab CONC

ANTIBODY
EXCESS

Repeating Antigenic Determinants


e.g. PEPTIDOGYCAN

CHO ANTIGENS may cross-link with MONOCLONAL Ab

Y
Y

DOUBLE DIFFUSION

Antigen

Antibody

Immune Complex

Antigen

Antibody

Antigen

Immune Complexes
Zone of Equivalence

Rabbit Serum
as antigens

1:4

1:20

Goat anti-rabbit serum


(Antibodies to rabbit serum)

Non-Identity
Antigen #1

Antigen #2

No Shared Antigenic
Determinants
Antigen #1

Antigen #2

OUCHTERLONY ANALYSIS
Diffusion of Antigens and Polyclonal Antibodies

Non-Identity
Antigen 1

(Molecule #1)

Antigen 2

(Molecule #2)

Antibodies to both antigens


The same Animal was injected with

antigen 1 and with antigen 2

OUCHTERLONY ANALYSIS

Partial - Identity

Antigen 3

Antigen 4

is a part
of antigen 4

Also remember that this


antibody is a multiclonal antibody such as
an anti-serum to an
antigenic preparation

This animal
was only
injected with
Antigen #4

Antibody

Remember that
Protein Antigens
have different
antigenic
determinants

OUCHTERLONY ANALYSIS

Partial - Identity

Antigen 3

Antigen 4

Antibody

Partial - Identity
Antibodies to determinants c and d are only on Antigen 3 and
they pass by antigen 4

Antigen 3

Antibodies
polyclonal antibody

Antigen 4

OUCHTERLONY ANALYSIS

Identity

These two Antigens are the Same Molecule


Antigen 6
is Antigen 5

Antigen 5

No spikes were formed


because:

Antibody

Antigenic determinants on
Antigen 5 captured all the
antibodies to Antigen 6 and
antigenic determinants on
Antigen 6 captured all the
antibodies to Antigen 5

Fluorescence

UV Light

Antigens on Cells or on Tissue Sections

Fluorescence

Double layer
Sandwich

UV Light

Antigens

ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Substrate that turns
from clear to green

Ag

Ag

Microtiter ELISA

Antigens are immobilized to the plastic surface of a


Microtiter Plate

ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Substrate that turns
from clear to green

Ag

Ag

Microtiter ELISA

Antigens are immobilized to the plastic surface of a


Microtiter Plate

Capture ELISA

-- using pre-immobilized
mouse monoclonal Ab to capture the Specific
Antigen and a second Probe monoclonal
Antibody against a different antigenic
determinant

Ag

Ag

Agglutination

Y
Y

IgM >>IgG

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