1. Antigen Antibody Reaction
AlZaiem AlAzhari University
Faculty of Medical Laboratory Sciences
Department of Microbiology
Batch (19) Semester 4
Serology and clinical Immunology
U.Mahadi Hassan Mahmoud
Bsc,Msc,MIBMS-Microbiologist
18 April 2013 1Lecture No -1-
 Discuss immunoglobulin variability (ie. the
variable region)
 Describe bonds between the variable region
and the antigenic determinant
 Define antibody affinity and antibody
avidity
Specific Objectives:
THE STUDENT SHOULD BE ABLE TO:

2.  Describe a precipitin curve and discuss lattice
formation involving proteins verses carbohydrate
antigens and be able to define "zone of
equivalence".
 Understand immunodiffusion in agar gels.
(identity, nonidentity and partial identity)
 Have a conceptual understanding of
immunoelectrophoresis, Fluorescent antibody
techniques and ELISA (enzyme-linked
immunoassay)
 Define "agglutination" and understand the
functional differences between monomeric Ab (ie.
IgG) and polymeric Ab (ie. IgM and S-IgA)

3. Definitions:
The "antibody affinity" of an
antibody-antigen reaction is related to
the strength of attractiveness between an
antibody (Fab region) and its antigenic
determinant.
.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.

4.  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.

5. ANTIGENIC DETERMINANTS
INTERACT WITH
SPECIFIC ANTIBODY
CH2 CH3
CH2 CH3
IgG has a Valence of 2
TWO Identical ANTIGEN BINDING SITES

6. Movement
at the
Hinge
Region
CH2 CH3
CH2 CH3
IgG
Surface
of an
Antigen
i.e.
bacterial
cell surface
Non-Covalent Interactions
Ball in glove fit
Antigenic
Determinant
VL
VH

7. -
Gene rearrangements and
Mutational Hot Spots
Charge-Charge Interactions
Hydrophobic Interactions - And good fit !
+-
VL
VH
+
Y
Antibody Affinity
-
+-
VL
VH
+

8. Antigenic determinant 1
Antigenic determinant 2
Antigenic determinant 3
Antigenic determinant 4
PROTEIN
ANTIGEN
Y
Y
Y
MUST HAVE POLYCLONAL ANTIBODY
and at least two different antigenic determinants
TO CROSS-LINK PROTEIN ANTIGENS
Immune Complexes

9. Y
Y
Y
Y
ANTIBODY
EXCESS
NO CROSSLINKS
NO Precipitate
Y
Y
Y
Y
Y Y
Y
Y
Excess Antibody
Y
Excess Antigen = Not enough
Cross-links to cause a Precipitation

10. Y
Y
More cross-links, and higher individual affinities
= higher AVIDITY of the Immune Complexes
Y
Ab CONC
ANTIBODY
EXCESS
ANTIGEN
EXCESS
ZONE of
Equivalence
No Soluble Ag or Ab

11. Repeating Antigenic Determinants
e.g. PEPTIDOGYCAN
CHO ANTIGENS may cross-link with MONOCLONAL Ab
Y
Y Y
Y

12. Antigen Antibody
DOUBLE DIFFUSION
Immune Complex
Antigen
Antibody
Antigen
Immune Complexes
Zone of Equivalence

13. Rabbit Serum
as antigens
1:4 1:20
Goat anti-rabbit serum
(Antibodies to rabbit serum)
Non-Identity
Antigen #1 Antigen #2

14. No Shared Antigenic
Determinants
Antigen #1 Antigen #2
OUCHTERLONY ANALYSIS
Diffusion of Antigens and Polyclonal Antibodies
Antigen 1
(Molecule #1)
Antibodies to both antigens
The same Animal was injected with
antigen 1 and with antigen 2
Antigen 2
(Molecule #2)
Non-Identity

15. OUCHTERLONY ANALYSIS
Antigen 3
is a part
of antigen 4
Antibody
Antigen 4
Partial - Identity
Remember that
Protein Antigens
have different
antigenic
determinants
Also remember that this
antibody is a multi-
clonal antibody such as
an anti-serum to an
antigenic preparation
This animal
was only
injected with
Antigen #4
OUCHTERLONY ANALYSIS
Antigen 3
Antibody
Antigen 4
Partial - Identity

16. Antigen 3
Antibodies
polyclonal antibody
Antigen 4
Partial - Identity
Antibodies to determinants c and d are only on Antigen 3 and
they pass by antigen 4
OUCHTERLONY ANALYSIS
Antigen 5
Antibody
Antigen 6
is Antigen 5
Identity
These two Antigens are the Same Molecule
No spikes were formed
because:
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

17. Antigens on Cells or on Tissue Sections
UV Light
Fluorescence
Fluorescence
Double layer
Sandwich
UV Light
Antigens

18. Ag
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Microtiter ELISA
Antigens are immobilized to the plastic surface of a
Microtiter Plate
Enzyme Linked Immuno-Sorbant Assay
ELISA
Ag
Substrate that turns
from clear to green
Ag
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Microtiter ELISA
Antigens are immobilized to the plastic surface of a
Microtiter Plate
Enzyme Linked Immuno-Sorbant Assay
ELISA
Ag
Substrate that turns
from clear to green

19. 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
IgM >>IgG