lecture 4- antibodies discovery of antibodies specificity variability protein structure of...

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.

1 2

13 14 15 16 17 18 19 20

3 4 5 6 7 8 911 1210

Lymphocytes have unique, clonally distributed antigen receptors

1010

10101010

10101010 1010

1010 10101010

Antibodies

B cells

Day 0

2

4

6

8

Resting B cell

Antibodyforming cell

(plasma cell)Y

Y

Y

Y

Y Y

Y Y

Y YY Y

YY

YY

YY

Y

Y

Y

YY

Y

Y

Y

YYY

Y

Y

Y

Y

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

YY

YY

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