Transcript Antigens and Antigen Receptors (lecture notes pages 19-24)

Antigens and
Antibodies
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
crosslinking
B cell
B cell
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 Bcell to start secreting antibody.
Molecules that are too small to cross-link surface immunoglobulin,
are not immunogenic
haptens
carrier
Hapten:
An antigen that can be bound and recognized by
the immune system but cannot induce an immune
response in the host
Carrier:
A substance that can help induce an immune response
against a chemically linked hapten
Model Antibody (Immunoglobulin) Structure
L
H
Constant
VH
CH2
Variable
CH1
Hyper variable
Hinge
CL
VL
CH3
Immunoglobulin: quaternary structure
Proteolytic Fragments of Immunoglobulin
Pepsin treatment
F(ab') 2
pepsin
papain
Papain treatment
Fab
+
Fc
Immunoglobulin heavy chain constant regions in
humans fall into five major classes. These are:
IgM (m), IgA (a), IgG(g), IgD (d) and IgE (e)
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:
Igk and Igl
Structural differences among Immunoglobulin
isotypes
monomer
dimer
pentamer
J chain
J chain
IgG, IgD, IgE
IgA
IgM
Valence: number of antigen binding sites
Avidity: Strength of binding based on valence
Antibodies bind to antigens based on the
‘complementarity’ of the epitope on the antigen and
the
antigen-binding site of the antibody.
Complementarity can be thought of as ‘goodness of fit’.
The better the fit, the stronger the binding. The strength
of the reaction is referred to as the affinity of the antibody.
Generally speaking, a high affinity antibody is more protective
than a low affinity antibody because it will bind antigens at
lower 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 different
groups create partial opposite charges.
Van der Waals forces: Fluctuations in electron clouds around
Molecules oppositely polarize neighboring atoms.
Hydrophobic forces: hydrophobic amino acids attract .
Structural differences among Immunoglobulin
isotypes
monomer
dimer
pentamer
J chain
J chain
IgG, IgD, IgE
IgA
IgM
Valence: number of antigen binding sites
Avidity: Strength of binding based on valence
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
IgA:
Major subclass of Ig in external secretions and mucosa
occurs in serum as a monomer; in secretions as a dimer
IgE:
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
Property
IgG
g
IgA
a
IgM
m
IgD
d
IgE
e
molecular weight
(Daltons)
150,000
150,000 600,000
900,000
150,000
190,000
serum concentration
(mg/ml)
12-14
3
1.5
0.03
0.0003
complement activation
++
-
++++
-
-
mast cell degranulation
-
-
-
-
++++
presence in secretions
-
+++
-
-
-
transplacental transfer
+++
-
-
-
-
DO NOT MEMORIZE!!!!!
Property
IgG1
IgG2
IgG3
IgG4
IgA1
IgA2
Serum
concentration
(mg/ml)
9
3
1
0.5
3
0.5
serum half life
(days)
23
23
8
23
6
6
complement
activation
++
+/-
+++
+/-
-
-
transplacental
transfer
++
+/-
++
++
-
-
presence in
secretions
-
-
-
-
++
++
Immune Response
Hallmarks of the adaptive immune response:
Specificity for antigen
Induction of memory
1o anti-A
Mainly IgM
Antigen A
0
2o anti-A
Mainly IgG
Antigen A + Antigen B
14
0
Time (Days)
6
1o anti-B
IgM
14
L
H
CH2
Variable
COOH
CH1
hv
Hinge
CL
NH2
VL
CH3
Idiotypic
Allotypic
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 Bcells to Ig with only 1 specificity.
1. Heavy chain and light chain combination
Essentially 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 genes
The 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
Gene Sequence
Ig Protein
Liver Cell
B Cell
During B-cell development (in the bone marrow), genes
that encode antibody heavy and light chains undergo breakage
deletion, and repair to form ‘new’ genes. This process is called
SOMATIC RECOMBINATION
Germline DNA--------------
V--J Joined DNA----------Primary RNA Transcript-
Somatic
Recombination
Transcription
Mature RNA Transcript--
mRNA Splicing
(Processing)
Polypeptide chain----------
Translation
Genomic Structure
Heavy Chain
3. VJ and VDJ Recombination
See animation of rearrangement
4. Recombination Inaccuracies
See Animation
5. Somatic mutations
Mutations 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.
Antigen-independent B-cell development
pre B
pro B
stem cell
D-Jh
Vh-DJh
immature B
Vl-Jl
IgM
IgM
Exit bone
marrow
mature B
IgD
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
Therapy
Diagnostics
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
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 affinities
against multiple epitopes found on the immunogen.
A polyclonal response is the sum of all of the clonal
responses (or monoclonal responses)
Monoclonal Antibody Production
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 v. Polyclonal Antibodies
Monoclonal: Limitless supply, defined specificity and subclass,
typically less ‘background reactivity’ and
less expensive, but sometimes application is limited
because 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