antibody its stucture and function

1. SUBMITTED TO:
Dr. NARAYAN MOGER
DEPARTMENT OF
BIOTECHNOLOGY
SUBMITTED BY:
Balaji Rathod
ID NO. PGS16AGR7165

2. Principles
of Antibody
Production
1900 *Paul Erlich, Antibody formation theory

3. The blood contains two types of white blood
cell or leukocyte
Phagocytes ingest bacteria by endocytosis
Lymphocytes produce antibodies

4. Antibodies
• Antibodies are proteins secreted by B cells
that specifically bind to a foreign substance-
Antigen
• Antigens are foreign substances that
stimulate the production of antibodies
• Many of the molecules on the surface of
viruses and bacteria are antigens

5. Antibodies are specific – they usually bind to
only one specific antigen.
Antigen
Antibodies
Microbe

6. Production of Antibodies by Lymphocytes
A lymphocyte can produce only one type of
antibody so a huge number of different types are
needed
Each lymphocyte has some of its antibody on its
surface…

7. The antigens of a pathogen bind to the antibodies
in the surface membrane of a lymphocyte…
…This activates the lymphocyte.

8. The active lymphocyte divides by mitosis to
produce a clone of many identical cells
MITOSIS
The clone of cells
starts to produce large
quantities of the same
antibody…
… the same antibody
needed to defend
against the pathogen!

9. Most microbes have more
than one antigen on their
surface, so…
…they stimulate more than
one type of lymphocyte…
…resulting in the production
of many different antibodies.
These are called
polyclonal antibodies.

10. Structure of an antibody

11. What is an antibody?
• Protein secreted by B-cells that
specifically bind a foreign
substance (antigen)
• Immunoglobulin domains
• Complementarity-determining
Regions (CDRs)
• Fab= Fragment antigen binding
• Hinge
• Fc= Fragment crystalline
• F(ab)’2= Protease digestion still
useful to bind antigen

12. Stages in Antibody Production
5.Production of memory cells
4.Production of plasma cells
3.Activation of B-cells
2.Activation of helper T-cells
1.Antigen presentation

13. Antibody Production: The Primary Response
Macrophages take in antigen
by endocytosis
Antigen
Macrophage
The macrophage processes the
antigen and attaches it to a
membrane protein called a
MHC protein
The MHC protein is moved to the
cell surface membrane by
exocytosis so that the antigen is
displayed on its surface.
MHC protein
Step 1: Antigen Presentation

14. Helper T-cell binds to macrophage presenting
the antigen
Step 2: Activation of Helper T-cell
Helper T-cells have receptors on
their cell surface membranes which
can bind to antigens presented by
macrophages.
receptor
Helper T-cell
Macrophage sends a signal to activate the helper T-cell

15. Step 3: Activation of B-lymphocytes
B-cells have antibodies in their cell surface
membranes
Inactive B-cell
Antibody
Antigens bind to the antibodies in the surface
membranes of B-cells
Antigen

16. An activated helper T-cell with
receptors for the same antigen
binds to the B-cell
SIGNAL
The helper T-cell sends a signal to the B-cell,
activating the B-cell.

17. Step 4: Proliferation
Plasma cells are activated B-cells
with a very extensive network of
rough endoplasmic reticulum.
Plasma cells synthesis large
amounts of antibody, which they
excrete by exocytosis.
The activated B-cell starts to divide
by mitosis to form a clone of plasma
cells.

18. The Secondary Response: Memory Cells
• If an antigen invades your body a second time, a
much faster response occurs which produces
much larger quantity of the required antibody.
•When activated B-cells are dividing during the
primary response, some cells stop dividing and
secreting antibody and become memory cells.
• Large numbers of memory cells remain in the
body for a long time…
• …they are capable of producing large amounts of
antibody very quickly when stimulated.

19. B-cell
Macrophage
Antigen
Activate
Antigen
Helper
T-cell
Activate
Clone
Memory
Cell Plasma Cell Antibodies
Antibody Production:
Summary

20. Principles of antibody production
• Clonal selection
– > 1015 antibodies
– B-cells clone themselves
• Challenge and response
– Immunity developed only when disease
challenges the immune system

21. Antigen dependent
maturation of B cells

22. Classes of antibodies
Isotype Structure Placenta
transfert
Activates
complement
Additional features
IgM
No Yes First Ab in development and response
IgD
No No B-cell receptor
IgG
Yes Yes Involved in opsonization and ADCC.
Four subclasses; IgG1, IgG2, IgG3,
IgG4
IgE
No No Involved in allergic responses
IgA
No No Two subclasses; IgA1, IgA2. Also found
as dimer (sIgA) in secretions.

23. Production of monoclonal antibodies
1. Antigens injected to an animal
2. B-cells extracted from the animal
3. Tumour cells obtained
4. B-cells fused with tumour cells
5. Hybridoma cells- produce antibody
6. Antibodies are extracted and purified
http://highered.mcgraw-hill.com/olc/dl/120110/micro43.swf

24. Monoclonal Antibody Production
• Monoclonal Antibody Production technology was developed in 1975. Since its
development it has been very important in the modern medical science with the
diagnosis, therapy, research and even basic science today. It is still largely
dependent upon animal testing however. Because it requires immunization of mice
in order for them to create the antibodies to be grown.
• Monoclonal Antibody Production or mAb is produced by cell lines or clones
obtained from the immunized animals with the substance to be studied. Cell lines
are produced by fusing B cells from the immunized animal with myeloma cells. To
produce the desired mAb, the cells must be grown in either of two ways: by
injection into the peritoneal cavity of a suitably prepared mouse (the in vivo, or
mouse ascites, method) or by in vitro tissue culture.
• The vitro tissue culture is the method used when the cells are places in culture
outside the mouse's body in a flask.

26. Why this method is used!!
• This method is used because antibodies must be formed from
the immunization of the substance being studied. So
antibodies must be produced. Once the antibodies are
produced the animal aspect of the study can be eliminated
and tissue culture can then be used.
• When using live mice researchers have found that it is the
better option because in vitro doesn’t always produce
adequate cell lines that are adaptive to tissue culture. Protein
denaturation can occur from purification techniques and
antibody activity is decreased with normal activity not
represented. Also cell lines could possibly become
contaminated when using in vitro technique.

27. Polyclonal antibodies:
If an animal is immunized with a protein, a wide array of B
cells will be stimulated to produce anti-protein antibodies.
Antibodies may be made to a number of different epitopes
of the protein.
Even antibodies that bind to the same epitope may have
different antigen-binding sites and bind the epitope with
different affinity.
The mixture of antibodies produced in response to an
antigen are referred to as polyclonal antibodies (they are
produced by many different clones of B cells).

28. Producing polyclonal antibodies

29. MONOCLONAL VS POLYCLONAL
ANTIBODIES
MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES
Expensive to produce Inexpensive to produce
Training is required for the
technology used
Skills required are low
Time scale is long for hybridization. Time scale is short
Can produce large amount of
specific antibodies.
Produces large amount of non
specific antibodies.
Recognizes only 1 epitope on an
antigen
Recognizes multiple epitopes on
anyone antigen
Once hybridoma is made it is a
constant and renewable source.
No or low batch to batch variability.
Can have batch to batch variability.

30. Active and passive immunity
• Active immunity: antibodies by the organism
itself
• Passive immunity: antibodies received from
another organism
– During pregnancy antibodies passed to the fetus

31. Antigenic determinants
• An antibody will recognize
– Epitope: defined segment of an antigen
– Immunoreactivity of epitopes may depend on primary,
secondary, tertiary or quaternary structure of an
antigen
– Define the possible applications
– Variability of epitopes depends on the species
• Antibodies are antigen themselves

32. Commercial production of antibodies:
polyclonal vs monoclonal
• Slected clones from a polyclonal each recognizing a
single epitope can be fused to a tumor cell
(hybridoma) to proliferate indefinitely
• Host animals ca be used to raise antibodies against
a given antigen

33. Laboratory use of antibodies
• Quantitation of an antigen
– RIA, Elisa
• Identification and characterization of protein antigens
– Immunoprecipitation
– Western blotting
• Cell surface labelling and separation
• Localisation of antigens within tissues or cells
• Expression librairies
• Phage display

34. Elisa: Enzyme-linked immunosorbent assay

35. Western blotting

36. Phage display

37. Clinical use of antibodies
• Diagnostic
– Detection of peptides and other molecules in various diseases
• Endocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidism
• Tumor antigens (p53 tumor suppressor, PSA, a-foetoprotein)
• Antibodies against viral proteins (AIDS, hepatitis)
• Therapeutic
– Neutralizing antibodies
• Anti-ErbB2 for breast and ovarian cancer
• Anti-CD20 for B-cell non-Hodgkin's lymphoma
• Antisera and antidotes (viruses and venoms)
• Drug discovery
– Identification of therapeutic targets (phage display)

38. Physiological roles of antibodies
• Protect against
– Viral infections
– Bacterial infections
– Foreign bodies
• Antigens
• Deleterious in
– Autoimmune diseases
• Reumathoid arthritis Lupus
• Type 1 diabetes Croh’n disease
– Graft rejection and hypersensitivity
responses

39. Lacking an antibody for your
protein or antigen of interest is
limiting the progression of your
research!