Lecture 4 Antibodies

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Lecture 4- Antibodies
Discovery of antibodies Specificity Variability Pr
otein Structure of antibodies Antibody Antigen
interactions Antibody classes (isotypes) Monoclona
l antibodies (Hybridomas)
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Antibody structure and generation of B cell
diversity

• Antibodies (Ab) are circulating proteins that
  specifically bind to foreign molecules--AKA
  immunoglobulins (Ig)
• Each antibody has a specificity different from
  the others
• Antibodies are made by B cells that have
  differentiated to become plasma cells
• Each B cell makes ONE and only ONE type of
  antibody--clonal selection
• Antigens are anything that is bound by an
  antibody
• Immunogens are antigens that elicit an antibody
  response. All immunogens are antigens, but the
  converse isnt necessarily true.

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Lymphocytes have unique, clonally distributed
antigen receptors
B cells
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Brief history of antibodies
-In 1890 by von Behring and Kitasato described an
activity in serum of toxin-immunized animals that
neutralized toxin. Transfer of immune serum
could protect naïve animals from diphtheria or
tetanus. -Bordet found in 1899 that animals could
make antibodies against erythrocytes of other
species and that these could direct destruction
of the cells along with serum complement. -1901-
1920 Landsteiner demonstrates the ABO blood group
system (Rh in 1940). -1930s Heidelberger
Quantitative precipitin reactions -1930s
Landsteiners analysis of antibody
specificity -1960s Edelman, Porter, and
Hilschmanns elucidation of the primary and
secondary structure of Abs. Discovery that
Bence-Jones proteins were immunoglobulin
L-chains. -1975 Kohler and Milstein invent
monoclonal antibody technology -1976 Tonegawa
clones first antibody gene
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Early in vitro assays of antibody activity The
the end of the 19th century three assays were
developed that could measure antibodies 1)
bacteriolysis--fresh serum from immunized
individuals, which contain both antibody and the
complement system proteins, could directly lyse
bacteria in vitro. 2) precipitin reaction-
which involves the binding of antibody molecules
to antigens that allow the development of large
arrays that are poorly soluble. 3)
agglutination- for example, erythrocytes of one
species injected into another provoke antibodies
that can be detected by their ability to cause
aggregation of the cells.
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Antibody is often a major serum protein
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Landsteiner and the age of haptens
Antibodies can be quite specific and can be
raised to synthetic compounds
m-azobenzenesulfonate
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1) How does the antibody system manage to be so
specific? 2) There are many antigens, virtually
all of which can be seen specifically by
antibodies. If all of these natural antigens,
and even non-natural compounds, can be seen by
the antibodies, there must be a huge number of
different potential antibodies. How can they be
encoded in DNA?
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Hilschmann and Craigs light chain sequencing data
variable
constant
Strategy to sequence antibody light chain
proteins from patients with Bence-Jones proteins.
These are monoclonal antibody L chains secreted
into the urine of patients harboring a myeloma
(plasma cell tumor).
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How variable?
How constant?
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IgG immunoglobulin G First antibody class
discovered, it represents 80 of antibodies in
the blood
Fab fragment antigen binding Fc fragment
crystallizable
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Antibody structure (IgG)
Figure 2.2
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Figure 2.3
Fab fragment antigen binding Fc fragment crysta
llizable
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Information/ Specificity
Effector/ triage function (recruits innate
immune cells)
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Illustration of the flexible hinge of antibodies.
Antibody in blue, divalent hapten in red.
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Antibody flexibility
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Ig structure.2
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IgG complete structure
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Entire Ig structure
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Fig 2.6
V and C domains
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Ig superfamily
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Light chain CDRs contribute to part of the
combining site
Fig 2.7
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Differences between antibodies are
concentrated in hypervariable loop regions of V
regions
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The basis of antibody binding
Depending on the nature of the antigen Hydrophobi
c interactions Van der Waals forces Electrostatic
interactions Hydrogen bonds
Non-covalent, therefore reversible,
binding Equilibrium affinity Ka AgAb
AgfreeAbfree
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Example
Y
Y
Ab
Y
Y
Y
Y
Y
Y
Y
Given independence of binding sites the following
relation applies r/c Kn - Kr and plotting
our experimentally derived values of r/c vs. r
allows the determination of K and n
Slope -Ka
r/c
At x-intercept r sites/target
r Ab molecules bound/target c free Ab
concentration n number of sites/target
sites with bound ligand K free
ligand free sites
r
High and low affinity
Antibody of uniform affinity
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Typical affinity range of antibodies 105 - 109
M-1
The affinity constant K is related to the free
energy of binding as follows DGo RT
ln(Ka) where R is the gas constant (1.987
cal/mole-deg.), T is the absolute temperature,
and ln(Ka) is the natural logarithm of the
association constant. Thus a two-fold
increase in binding energy translates to an
affinity increase from Ka to (Ka)2.
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The concept of epitopes, parts of antigens bound
by antibodies
Valency
Fig 2.9
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Polio virus epitopes shown in white
VP1 protein
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Different types of binding to antigen
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Quantitative Immunoprecipitation
Constant amount of Ab
Zone of equivalence
Ab Precipitation
Zone of Ag excess
Zone of Ab excess
Ag
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Immunoprecipitation
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Human immunoglobulin Isotypes
Figure 2.4
These are monomer forms as they appear when
expressed as a B cell antigen receptor. When
secreted, the structures can be quite different.
IgM is a pentamer in serum, and IgA can be a
dimer.
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Valency
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Human Immunoglobulins
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Antibody classes have distinct and overlapping
functions
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Different antibody isotypes are found in
different parts of the body
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Reminder about gene structure and the central
dogma of molecular biology
promoter
Regular gene (eukaryote)
DNA
Exon 1
Exon 2
Exon 3
Translation on ribosomes to protein
Translation
Protein product
Alternative protein product
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Differential RNA splicing determines if an
antibody is secreted or remains as a membrane
receptor
B cell antigen receptor is a membrane bound form
of antibody
B cell
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Antibody gene
One exon is assembled from separate pieces by DNA
rearrangement in immature lymphocytes
DNA
V
Naïve B cell
DNA
Antigen stimulated B cell
C heavy
DNA
On the antibody H chain, other exons are swapped
in by a distinct DNA rearrangement
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Concepts and summary

• Antibodies are highly specific
• Can see virtually any type of molecule
• Highly variable
• Immunoglobulin domain is a conserved structure
• Antigen contact sites are in hypervariable loops
• Antibody Antigen interactions are reversible and
• characterized by affinity
• There are multiple antibody heavy chain classes
  (isotypes) that
• determine anatomical distribution and function.
• Monoclonal antibodies (Hybridomas) are useful
  tools in biology and medicine.

Next time Antibody genes and the problem of
generating diversity