Immunology Lecture Robert J. Boackle, Ph.D.

Antigen-Antibody ReactionsSpecific Objectives: THE STUDENT SHOULD BE ABLE TO1. Discuss immunoglobulin variability (ie. the variable region)2. Describe bonds between the variable region and the antigenic determinant3. Define antibody affinity and antibody avidity4. 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

CH2 CH3

CH2 CH3

IgG has a Valence of 2

TWO Identical ANTIGEN BINDING SITES

Movement at the Hinge

Region

CH2 CH3

CH2 CH3

IgGSurface

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

PROTEINANTIGEN

Y

YY

MUST HAVE POLYCLONAL ANTIBODYand at least two different antigenic determinants

TO CROSS-LINK PROTEIN ANTIGENS

Immune Complexes

YYY

YANTIBODYEXCESS

NO CROSSLINKSNO Precipitate

YYY

Y

Y YY

Y

Excess Antibody

Y

Excess Antigen = Not enough Cross-links to cause a Precipitation

Y

YY

More cross-links, and higher individual affinities

= higher AVIDITY of the Immune Complexes

YY

Y

Am

ou

nt

of

Pre

cip

itat

e

Ab CONC

ANTIBODYEXCESS

ANTIGENEXCESS

ZONE ofEquivalence

No Soluble Ag or Ab

Repeating Antigenic Determinants

e.g. PEPTIDOGYCAN

CHO ANTIGENS may cross-link with MONOCLONAL Ab

YY Y

Y

Antigen Antibody

DOUBLE DIFFUSION

Immune Complex

AntigenAntibody 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 ANALYSISDiffusion of Antigens and Polyclonal Antibodies

Antigen 1(Molecule #1)

Antibodies to both antigensThe same Animal was injected with

antigen 1 and with antigen 2

Antigen 2(Molecule #2)

Non-Identity

OUCHTERLONY ANALYSIS

Antigen 3is 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

Antibodiespolyclonal antibody

Antigen 4

Partial - IdentityAntibodies 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

FluorescenceFluorescenceDouble layer Sandwich

UV Light

Antigens

Ag

Peroxidase Enzyme is permanently attached to the Antibody Probe

Microtiter ELISAAntigens 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 ELISAAntigens 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

Y Y

Y

IgM >>IgG