1. Lecture 4- Antibodies Discovery of antibodies Specificity Variability
Protein Structure of antibodies
Antibody: Antigen interactions
Antibody classes (isotypes)
Monoclonal antibodies (Hybridomas)

2. 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.

3. Lymphocytes have unique, clonally distributed antigen receptors
B cells 13 14 15 16 17 18 19 20 2 3 4 5 6 7 8 9 1 10 11 12 10
Antibodies 10 10 10

4. Day 0 2 4 6 8
Resting B cell
Antibody
forming cell
(plasma cell) Y

5. 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”.
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

6. 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.

7. Antibody is often a major serum protein

8. Landsteiner and the age of haptens
Antibodies can be quite specific and can be raised to synthetic compounds
m-azobenzenesulfonate

9. 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?

10. 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).

11. How variable?
Millions of different variable regions
~10 different
constant regions
How constant?

12. IgG
immunoglobulin G
First antibody class discovered, it represents ~80% of antibodies in the blood
Fab fragment antigen binding
Fc fragment crystallizable

13. Antibody structure (IgG)
Figure 2.2
Antibody structure (IgG)

14. Figure 2.3
“Fab”
fragment antigen binding Fc
fragment
crystallizable

15. Effector/ triage function (recruits innate immune cells)
Information/
Specificity
Effector/ triage function
(recruits innate immune cells)

16. Illustration of the flexible hinge of antibodies
Illustration of the flexible hinge of antibodies. Antibody in blue, divalent hapten in red.

17. Antibody flexibility

18. Ig structure.2

19. IgG complete structure

20. Entire Ig structure

21. Fig 2.6
V and C domains

22. Ig superfamily

23. Light chain CDRs contribute to part of the combining site
Fig 2.7

24. Differences between antibodies are concentrated
in hypervariable loop regions of V regions

25. 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]

26. Example Y Y + Ab* Y Y Y Y Y Y Y r/c r Bound Ab [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:
Bound Ab
[Ab incubated]
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
Antibody of uniform affinity
High and low affinity

27. 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.

29. The concept of epitopes, parts of antigens bound by antibodies
Valency
Fig 2.9

30. epitopes shown in white
Polio virus
epitopes shown in white
VP1 protein

31. Different types of binding to antigen

33. Quantitative Immunoprecipitation
Constant amount of Ab
Zone of equivalence
Ab Precipitation
Zone of Ag excess
Zone of Ab excess
[Ag]

34. Immunoprecipitation

35. Low affinity
IgM class
High affinity
IgG class
IgM

36. 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.

37. Immunoglobulin classes

38. Valency

39. Human Immunoglobulins

40. Antibody classes have distinct and overlapping functions

41. Different antibody isotypes are found in different parts of the body

46. Regular gene: (eukaryote)
Reminder about gene structure and the central dogma of molecular biology
RNA
AAAA
Regular gene:
(eukaryote)
promoter
DNA
Exon 1
Exon 2
Exon 3
RNA splicing
AAA mRNA
AAA
Many genes generate
alternative splicing isoforms
Translation on ribosomes
to protein
Translation
Protein product
Alternative protein product

47. B cell antigen receptor
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

48. 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

49. Antibody genes and the problem of generating diversity
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