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 1 Lecture 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:

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)

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.

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

CH2 CH3

CH2 CH3

IgG has a Valence of 2

TWO Identical ANTIGEN BINDING SITES

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

-

Gene rearrangements and

Mutational Hot Spots

Charge-Charge Interactions

Hydrophobic Interactions - And good fit !

+ -

VL

VH

+

Y

Antibody Affinity

-

+ -

VL

VH

+

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

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

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

Repeating Antigenic Determinants

e.g. PEPTIDOGYCAN

CHO ANTIGENS may cross-link with MONOCLONAL Ab

Y

Y Y Y

Antigen Antibody

DOUBLE DIFFUSION

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

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

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

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

Antigens on Cells or on Tissue Sections

UV Light

Fluorescence

Fluorescence Double layer Sandwich

UV Light

Antigens

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

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