Antigen-Antibody Reactions 2011

• Features

• Examples in vivo in vitro

John F Kearney410 Shelby Building

4-6557jfk@uab.edu

Learning Objectives

• To understand– The features of the antigen-antibody interaction

• Reversible• Affinity• Avidity

– What makes a good antigen– Examples of in vivo antigen-antibody interactions– Examples of in vitro antigen-antibody interactions

Structure of an IgG Antibody

Features of the Antigen-Antibody Interaction

A reversible bimolecular association

involving various non-covalent interactions

Is similar to an enzyme-substrate interactions,

but not lead to an irreversible chemical alteration

However catalytic antibodies have been

described

Non-covalent binding

- Four types of non-covalent forces operates over a very short distance

( generally 1 angstrom )

Nature of Ag/Ab Reactions

Specificity• The ability of an individual antibody combining site to

react with only one antigenic determinant.

Cross Reactivity• The ability of an individual Ab combining site

to react with more than one antigenic determinant.

• The ability of a population of Ab molecules to react with more than one Ag

Anti-A Ab

Ag A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

K.R. Deepthi

Affinity = attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity

• Strength of the reaction between a single antigenic determinant and a single Ab combining site

Calculation of Affinity

Ag + Ab Ag-Ab

Keq = [Ag-Ab]

[Ag] x [Ab]

Applying the Law of Mass Action:

Avidity• The overall strength of binding between an Ag

with many determinants and multivalent Abs

Keq = 104

Affinity106

Avidity1010

Avidity

Factors Affecting Measurement of Ag/Ab Reactions

• Affinity

• Avidity

• Ag:Ab ratio

• Physical form of Ag

Ab excess Ag excess

Equivalence – Lattice formation

Precipitation reactions in fluids yield a precipitin curve. FIGURE 6-4

( Lattices or large aggregates )

( no precipitate is formed if an Ag contains only a single copy of each epitope )

Precipitation Reactions

Antigen-Antibody Binding

Epitope

Features of the Antigen-Antibody Interaction

• ReversibleNon-covalent Interactions

• AffinityMeasure of the strength of the bindingEase of association or dissociation

• AvidityIncrease in affinity due to

multivalent bindingThe summation of multiple affinities

Antigen-Antibody Binding

Terminology

Antigen Original - Substance able to generate antibodyMore general - Substance that can be recognizedby antibody or T cells

Immunogen Substance able to generate antibodyor T cell responses

What Makes a Good Antigen?

Parameter ImmunogenicitySize Large > SmallChemicalComposition

Proteins>Carbohydrates>Lipids

Similarity to self-antigens

Multiple differences > Fewdifferences

Dosage and routeof administration

Subcutaneous > Oral

Use of adjuvants Increase titer and affinityGeneticcomposition ofrecipient

Major HistocompatibilityComplex (HLA)

For T cell responses antigens should be degradable

Terminology

Antigen Original - Substance able to generate antibodyMore general – able to bind antibody

Immunogen Substance able to generate antibody or T cellresponses

Hapten Non-immunogenic substanceUsually low molecular weightCan induce antibody formation when coupled to a larger “carrier” molecule

Protein CarrierBovine Serum Albumin

Immunize with: Antibodies formed:DNP NoneBSA Anti-BSA

DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA

Hapten, DNP

Examples of Haptens

CortisolEstradiol Testosterone

Thyroid hormonesPenicillin

Antigen-Antibody Interactionsin vivo

Direct Inhibition

© 1998 Gold Standard Multimedia Inc.

Toxin

Inhibit attachment of bacteria to cells

Pseudo colored EM image of Helicobacter pylori attached to a gastric epithelial cell

Would antibodies be effective at preventing H. pylori attachment?

Yes N

o

0%0%

1. Yes

2. No

Antibodies Enhance Phagocytosis

Opsonization

© 1998 Gold Standard Multimedia Inc.

Fc Receptor

Complement Lysis

Complement activation can be initiated by antigen-antibody complexes

© 1998 Gold Standard Multimedia Inc.

Antigen-Antibody Reactions In Vivo

• Neutralization of viruses and toxins• Opsonization of pathogens• Complement lysis of bacteria• Prevention of bacterial adherence

The Bad

• Autoimmunity - myasthenia gravis• Transfusion reactions

The Ugly

• Allergy

The Good

Antigen-Antibody Interactionsin vitro

Techniques

Hemagglutination

Used to identify blood group antigens or antibodies to them

Agglutination/Hemagglutination

• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin

+

• Qualitative agglutination test– Ag or Ab

FIGURE 6-7

Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs).

Agglutination Reactions

+ + + (control)

- visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads.-routinely performed to type RBCs for blood transfusion.

Hemagglutination

Used to identify blood group antigens or antibodies to them

ELISAEnzyme Linked Immunosorbent Assay

HIV Ag HumanSerum

Enz-GoatAnti-Human IgG

Colorless →Yellow

HIV Testing

1. ELISA, determine if patient sera is + or – for antibodies to HIV

2. Western Blot, determine which viral antigens the patient has made antibodies to.

Immunofluorescence

mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

FIGURE 6-14

Fluorochromes- Fluorescein (490→517nm)- Rhodamine (515→546nm)- Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.

Bacillus anthracis B. thuringiensis B. subtilis

Examples of markers to dissect heterogeneity of immune cells

Optical Design

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser

Flow cell

PMT 3

Scatter

Sensor

Sample

Hematoxlin eosin staining

Staining of spleen with antibodies to distinguish cell types

Marginal zone macrophages

Metallophilic macrophages

IgM +B cells

CD4/CD8 T ells

Pregnancy Testing

1. Bioassay (Rabbit Test)

2. Agglutination inhibition

Assay for human chorionic gonadotrophin

Summary of Antigen-Antibody Interactions

Specificity depends on the variable regions of the immunoglobulin heavy and light chains

In vivo• After antibody binds antigen, the outcome

depends on the constant region of the heavy chain

• Effector functions are determined by antibody isotype

Summary of Antigen-Antibody Interactions

In vitro• All assays depend on exquisite specificity

of the antibody• Assays differ in the means used to detect

the bound antibody or bound antigen• Sensitivity: For example, ELISA and

Western blot>>Agglutination

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1. is due to covalent bonds between antigen and antibody.

2. is always the same as avidity.

3. is an expensive imported automobile.

4. is a measure of the strength of antigen-antibody binding.

5. Answers 2 and 4 are correct.

Pregnancy Testing

1. Bioassay (Rabbit Test)

Assay for human chorionic gonadotrophin

3. ELISA

2. Agglutination inhibition

Evolution of Flow Instruments

WA Bonner, HR Hulett, RG Sweet and LA Herzenberg, Fluorescence Activated Cell Sorting, Review of Scientific Instruments 43, 1972

Slide Kindly Supplied by Compucyte