Antigens and Antibodies. Antibodies Antigens Antigen: A substance that can be recognized by the adaptive arm of the immune system. Immunogen: Those antigens.

Antigens andAntibodies

Antibodies

Antigens

Antigen:

A substance that can be recognized by the adaptive arm of the immune system.

Immunogen:

Those antigens that induce a specific adaptive immune response in a host.

Factors that influence the immunogenicity of an

antigen • Size

– the larger the better

• Chemical composition and heterogeneity

– the more complex the better

• Uptake and degradability

– the less soluble the better

• Foreignness

– self molecules do not elicit immune

responses

Other Factors

1. Host Genotype (responder v. non-responder)

2. Route of Antigen Exposure

3. Dose of Antigen

Adjuvant

A substance that when mixed with an antigen can enhance the immune response to that antigen.

An adjuvant can

Render the antigen insoluble

Prolong persistence of antigen at the site of entry

Activate inflammatory cells (source of PAMP)

Examples: Bacterial components from Mycobacteria(Freund’s complete adjuvant), Cholera toxin, Bordetella, or insoluble aluminum salts.

Linear and Conformational Epitopes (or Antigenic Determinants) formed by

1o-4o Protein structure

Model Protein with several potential epitopes

B-cells produce antibodies following contact with antigen. The antigen ‘cross-links’ antibody molecules on the surface of B-cells and sends signals to the B-cell to start secreting antibody.

B cell B cell

crosslinking

haptens

Carrier: A substance that can help induce an immune

response against a chemically linked hapten

carrier

Molecules that are too small to cross-link surface immunoglobulin, are not immunogenic

Hapten:An antigen that can be bound and

recognized by the immune system but cannot induce an immune response in the host

Variable

ConstantH

CH1

CH2 CH3VH

VL

CL

Hinge

L

Model Antibody (Immunoglobulin) Structure

Hyper variable

Immunoglobulin: quaternary structure

Proteolytic Fragments of

Immunoglobulin

+Fab

Fc

pepsinpapain

F(ab') 2Pepsin treatment

Papain treatment

Immunoglobulin heavy chain constant regions in humans fall into five major classes. These are:

IgM (), IgA (), IgG(), IgD () and IgE ()

IgG is further subdivided into four subclasses:

IgG1, IgG2, IgG3, IgG4 (human)

IgG1, IgG2a, IgG2b, IgG3 (mouse)

IgA is subdivided into two subclasses

IgA1 and IgA2

Immunoglobulin light chain constant regions are:

Ig and Ig

Structural differences among Immunoglobulin isotypes

Valence: number of antigen binding sites

Avidity: Strength of binding based on valence

IgG, IgD, IgE

monomer

J chain

dimer

IgA

pentamer

IgMJ chain

Antibodies bind to antigens based on the ‘complementarity’ of the epitope on the antigen and theantigen-binding site of the antibody.

Complementarity can be thought of as ‘goodness of fit’.

The better the fit, the stronger the binding. The strengthof the reaction is referred to as the affinity of the antibody.

Generally speaking, a high affinity antibody is more protectivethan a low affinity antibody because it will bind antigens atlower concentrations.

Ag + Ab AgAb

Antibodies bind antigens through reversible non-covalent interactions

These include:

Electrostatic forces: Attraction between opposite charges.

Hydrogen bonds: hydrogen ion shared between differentgroups create partial opposite charges.

Van der Waals forces: Fluctuations in electron clouds aroundMolecules oppositely polarize neighboring atoms.

Hydrophobic forces: hydrophobic amino acids attract .

Structural differences among Immunoglobulin isotypes

Valence: number of antigen binding sites

Avidity: Strength of binding based on valence

IgG, IgD, IgE

monomer

J chain

dimer

IgA

pentamer

IgMJ chain

IgA: Major subclass of Ig in external secretions and mucosa

occurs in serum as a monomer; in secretions as a dimerIgE: occurs at very low concentrations in serum

can bind to mast cells and basophils and mediates allergy

IgD: occurs primarily as surface Ig, co-expressed with IgM

function unknown

IgM: the first isotype to be expressed in every B cell

occurs in serum as a pentamer (valence = 10)

can inhibit attachment of microbes

can activate complement cascade

IgG: the most abundant Ig subclass found in human serum

occurs as a monomer (valence=2)

can opsonize antigen for uptake by phagocytic cells

can activate complement cascade

cross placenta and protect offspring

DO NOT MEMORIZE!!!!!

Imm

un

e R

esp

on

se

Time (Days)

0 14 = 0 6 14

Antigen A

1o anti-AMainly IgM

Antigen A + Antigen B

2o anti-AMainly IgG

1o anti-BIgM

Hallmarks of the adaptive immune response:Specificity for antigenInduction of memory

Idiotypic

Allotypic

Variable CH1

CH2 CH3

H

VL

CL

Hinge

NH2

hv

COOH

L

III. The Genetic Basis for Antibody Diversity  It is estimated that a mammal can generate antibodies with as many as 108 different antibody specificities.

Since antibodies are proteins and proteins are encoded by genes, it stands to reason that the diversity of antibodies must arise from diversity in the genome.

The problem is that the genome does not contain enough genes to encode all of those different Ig specificities.

In addition, each B-cell produces Ig of only 1 specificity so some mechanism must exist to limit the production of Ig by B-cells to Ig with only 1 specificity.  

1. Heavy chain and light chain combinationEssentially any light chain can associate with any heavy chain. If there were 104 different light chains and 104 different heavy chains, we could derive 108 different specificities (104 104 = 108).

Light chains

Heavy chains

2. Multiple V genesThe variable portion of light chains and heavy chains are encoded by gene segments called variable gene segments or V gene segments that are found in the respective gene complexes of the Ig light chain and Ig heavy chain. In different species, there are different numbers of V gene segments. The DNA between the V and C regions is not deleted, but the RNA transcript from the gene complex is processed so the final mRNA contains only the complete light chain transcript without the intervening sequences. 

VL1---VL2----VLn--------CL

VH1---VH2----VHn--------CH

B Cell

Liver Cell

Gene Sequence

Ig Protein

During B-cell development (in the bone marrow), genesthat encode antibody heavy and light chains undergo breakagedeletion, and repair to form ‘new’ genes. This process is calledSOMATIC RECOMBINATION

Germline DNA--------------

V--J Joined DNA-----------

Primary RNA Transcript-

Mature RNA Transcript--

Polypeptide chain----------

Somatic Recombination

Transcription

mRNA Splicing(Processing)

Translation

Genomic StructureHeavy Chain

3. VJ and VDJ Recombination

See animation of rearrangement

4. Recombination Inaccuracies

See Animation

5. Somatic mutationsMutations occur within the gene complexes during the lifetime of the cell, resulting in the formation of antibodies that are slightly different that the original. Sometimes the resulting antibodies will bind to an antigen stronger than the original antibody. The new antibody molecule is said to have a higher affinity for the antigen. When antigen is present, these cells will preferentially bind antigen, proliferate, and secrete antibody of a higher affinity. This process is referred to as affinity maturation. 

Vh-DJh Vl-JlD-Jh

IgD

IgM

mature B

stem cell pro B pre B

IgM

immature B

Exit bone marrow

Antigen-independent B-cell development

Specificity of Antibodies Make them Ideal Reagents for Many Applications

Therapy – Treatment against infection or intoxication

Diagnostics – Appearance of pathogen-specific antibodies in serum is indicative of exposure/infection

Research Reagents – Molecular probes for (protein) expression

http://www.talecrisusa.com/products.asp

Therapy

A 20 year old diabetic college student returning from Spring Break was concerned that he became infected with HIV. How does his clinician help him?

Examine his serum by ELISA for the resence of HIV-specific antibodies as a marker for infection

Diagnostics

Research: Immunohistochemistry

Specific staining of pancreatic cells in murine pancreas using low-power antigen-retrieval (LAR) protocol. Insulin+ ß cells are shown in green and pancytokeratin (CK+) pancreatic duct epithelial cells in red (A1). Immunohistochemical (IHC) staining of insulin was exclusively specific for ß cells but not for duct epithelial cells (A2). Insulin+ ß cells are the dominant cell population in the islet (green), and cells are scattered around the periphery of the islet (red) (B1). Low magnification showing the highly specific staining in the large area of tissue (B2); pancreatic ß cells are stained with insulin in cytoplasm (green) and PDX-1 in nuclei (red) (C1). There was no unspecific binding in exocrine area (C2) and specific staining of pancreatic duct with CK (red) and vascular endothelial cells with PECAM (CD31) (green) (D1). Ab against PECAM stained only vessel but not duct (D2). Bars: A–C,D2 = 50 µm; D1 = 25 µm.

From: Ge et al. Journal of Histochemistry and Cytochemistry 54: 843-847, 2006

Antibody responses are generally polyclonal:Many individual ‘clones’ make antibodies of varying affinitiesagainst multiple epitopes found on the immunogen.

A polyclonal response is the sum of all of the clonal responses (or monoclonal responses)

The selection of hybridoma cells is accomplished by culturing the fused cell mixture in hypoxanthine-aminopterin-thymidine (HAT) medium. Aminopterin blocks the De Novo biosynthesis of thymine, purines and pyrimidines. Myeloma cells do not survive because they are incapable of growing when the De Novo nucleotide synthesis is blocked with HAT because they lack functional hypoxanthine-guanine phosphoribosyl-transferase (HGPRT-). B cells die in vitro within two weeks. The resulting hybridoma cells survive because they have received hypoxanthine-guanine phosphoribosyl transferase (HGPRT+)from the B cells and immortality from the myeloma cells.

Monoclonal Antibody Production

Monoclonal v. Polyclonal Antibodies

Monoclonal: Limitless supply, defined specificity and subclass,

typically less ‘background reactivity’ andless expensive, but sometimes application is

limitedbecause only 1 epitope is recognized.

Polyclonal: Initially easier to produce, greater reactivity with antigen (usually more applications), but not a

limitless supply. Specificity is less well-defined and

background reactivity is more of problem