lecture 4- antibodies discovery of antibodies specificity variability protein structure of...
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Lecture 4- Antibodies
Discovery of antibodiesSpecificityVariabilityProtein Structure of antibodiesAntibody: Antigen interactionsAntibody classes (isotypes)Monoclonal antibodies (Hybridomas)
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 isn’t necessarily true.
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Lymphocytes have unique, clonally distributed antigen receptors
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Antibodies
B cells
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Resting B cell
Antibodyforming cell
(plasma cell)Y
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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 Landsteiner’s analysis of antibody specificity-1960s Edelman, Porter, and Hilschmann’s 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
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.
Antibody is often a major serum protein
Landsteiner and the age of haptens
m-azobenzenesulfonate
Antibodies can be quite specific and can be raised to synthetic compounds
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?
Hilschmann and Craig’s 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).
~10 different constant regions How constant?
Millions of different variable regions
How variable?
IgGimmunoglobulin GFirst antibody class discovered, it represents ~80% of antibodies in the blood
Fab fragment antigen bindingFc fragment crystallizable
Antibody structure (IgG)
Figure 2.2
“Fab”fragment antigen binding
Fcfragmentcrystallizable
Figure 2.3
Information/Specificity
Effector/ triage function
(recruits innate immune cells)
Illustration of the flexible hinge of antibodies. Antibody in blue, divalent hapten in red.
Antibody flexibility
Ig structure.2
IgG complete structure
Entire Ig structure
Fig 2.6V and C domains
Ig superfamily
Fig 2.7
Light chain CDRs
contribute to part of the combining
site
Differences between antibodies are concentratedin hypervariable loop regions of V regions
The basis of antibody binding
Depending on the nature of the antigen:
Hydrophobic interactions
Van der Waals forces
Electrostatic interactions
Hydrogen bonds
Non-covalent, therefore reversible, binding
Equilibrium affinity Ka= [Ag:Ab] [Agfree][Abfree]
r = Ab molecules bound/targetc = free Ab concentrationn = number of sites/target
[sites with bound ligand]K = [free ligand] [free sites]
BoundAb
[Ab incubated]
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:
Example + Ab*Y
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Y Y YY
r/c
r
Slope = -Ka
At x-interceptr= # sites/target
Antibody of uniform affinity High and low affinity
The affinity constant K is related to the free energy of binding as follows:
Go = 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.
Typical affinity range of antibodies 105 - 109 M-1
Type of bond G (kcal/mole) Comments
Ionic bond (salt bridge) 3-7 Not directional, G 1/R2
Hydrogen bond 3-7 Strongly directionalVan der Waal's force 1-2 Requires close contact, G 1/R6
Hydrophobic forces 2-5 Indirectly driven by water's H-bondsCovalent bond 50-100Thermal energy (25oC) 0.6
Numerical relationship between the equilibrium association constant and G at 25o.
K G (kcal/mole)
1 0103 4.1106 8.2109 12.3
Fig 2.9
The concept of epitopes, parts of antigens bound by antibodies
Valency
Polio virus
epitopes shown in
white
VP1 protein
Different types of binding to antigen
Quantitative Immunoprecipitation
Ab Precipitation
[Ag]
Constant amount of Ab
Zone of Ag excess
Zone of Ab excess
Zone of equivalence
Immunoprecipitation
Low affinityIgM class High
affinityIgG class
IgM
Human immunoglobulin Isotypes
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.
Figure 2.4
Immunoglobulin classes
Valency
Human Immunoglobulins
Antibody classes have distinct and overlapping functions
Different antibody isotypes are found in different parts of the body
Reminder about gene structure and the central dogma of molecular biology
Regular gene:(eukaryote)
DNAExon 1 Exon 2 Exon 3promoterRNAAAAA
RNA splicing
AAA mRNAAAAMany genes generate
alternative splicing isoforms Translation on ribosomesto protein
Translation
Protein product
Alternative protein product
B cell antigen receptoris a membrane bound
form of antibody
B cell
Differential RNA splicingdetermines if an antibody is
secreted or remains as a membrane receptor
Antibody gene
DNA
One exon is assembled from separate pieces by DNA rearrangementin immature lymphocytes
DNA
DNA
On the antibody H chain, other exons are swapped in by a distinct DNA rearrangement
V
C heavy
Naïve B cell
Antigen stimulated B cell
•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.
Concepts and summary
Next time:Antibody genes and the problem of generating diversity