Transcript polyclonal-and-monoclonal

Applications of
Biotechnological Processes
Antibody Production
Antibody facts
 Antibodies fight off infections in immune
systems.
 They are produced by β-lymphocyte cells (B
cells) in the spleen.
 Antibodies are made in response to antigens
(foreign bodies).
 β-lymphocyte cells divide rapidly so that they
can produce many copies of the antibodies.
The antibodies bind with the antigen to
form an antibody/antigen complex that is
engulfed by phagocytosis and destroyed.
Specificity
B cells produce specific antibodies in
response to specific antigens.
Because of the infinite number of antigens
available, a large number of different B
cells must be available.
Antibody-antigen recognition
 An antibody recognises part of an antigen’s
structure. The recognised region is called an
epitope.
 Antigens have more than one type of epitope.
This means that a number of β-lymphocyte cells
will recognise the antigen and produce
antibodies. An immune response results in the
production of a number of different antibodies.
Epitope sites
 Antibodies can be used in diagnostic tests
because they have the ability to recognise
epitope structures exactly.
 Antibodies can also be used to give a person
passive immunity against a disease.
The original method of making antibodies was
through the preparation of polyclonal sera.
The modern method involves the preparation of
monoclonal antibody sera.
Preparation of Polyclonal Sera
Polyclonal sera is a sera that contains a
mixture of antibodies. It can be injected
into the patient e.g. treatment for Hepatitis
B
Sera – clear portion of any body fluid
Procedure:
1. Inject an animal e.g. mouse, with antigen
2. The animal will elicit an immune response to raise
antibodies in the blood, against the antigen.
3. Inject an animal with antigens for a 2nd time to ensure a
high enough concentration of antibody.
4. Take blood from the animal at intervals.
5. Remove the red blood cells, to leave only the antibody
sera. The sera has a mixture of antibodies in it, due to
different β-lymphocyte cells recognising different
antigens.
Disadvantages
 The following disadvantages make polyclonal production
inconsistent:
Animal has a finite life span.
Recovery time must be left between bleeds and only a limited
volume of serum can be collected at any one time.
The animal will raise a mixture of antibodies against the antigen
(different epitopes on the antigen will raise a response from
different β-lymphocyte cells).
A new animal may not raise exactly the same antibodies as
another animal (due to different epitopes being recognised).
Monoclonal Antibody Production
 However, it is now possible to produce
antibodies with a single specificity (derived from
a single B cell).
Procedure:
1. Inject an animal e.g. mouse, with antigen.
2. The animal will elicit an immune response to
raise antibodies in the blood. The number of βlymphocyte cells in the spleen will increase.
3. After a few weeks the spleen is removed. The
spleen is then separated into individual cells.
These cells will contain a mixture of βlymphocyte cells.
4. Spleen cells are fused to myeloma cells
in tissue culture.
Myeloma cells are cancer cells that do not have
the ability to make antibodies on their own.
Therefore only the spleen cells to which the
myeloma are fused will produce antibodies,
ensuring that only one type is made.
Myeloma cells are used because they have an
almost limitless lifetime in tissue culture and
readily fuse with other cells.
Myeloma cells will have been altered (mutated)
so that they cannot utilise the nutrient
aminopterin
5. Spleen and fused myeloma cells are
grown up on aminopterin containing
medium. Only myeloma cells that are
fused to spleen cells survive (spleen
cells utilise the aminopterin).
The cells that grow are called hybridoma
cells.
They will have received the antibody
producing genes and the aminopterin utilising
genes from the spleen cells. The ability to
grow indefinitely (for many years) is derived
from the genes in the myeloma cells.
The overall process is know as immortalisation.
6. The hybridoma cells are then tested for
secretion of the desired antibody. The
cells are identified and cloned. The are
then transferred to fermenters (200-400L)
to continually produce the one type of
antibody.
7. At the end of the growth period the
antibody is extracted and purified.
Uses of Monoclonal Antibodies
The antibodies can be used to treat
disease and to diagnose it. For example:
Pregnancy testing
Food safety testing
Drug testing e.g. heroin
Breast cancer treatment e.g. herceptin
Pregnancy Testing
 A mouse is injected with HCG hormone. This
hormone is usually only present in blood and
urine of a pregnant woman.
 Antibodies are raised and monoclonal
hybridoma cells are produced.
 Hybridoma cells are screened and a suitable,
highly specific (good binding to the hormone)
line is selected.
The antibody produced by these cells are
purified. Small quanitites are coated onto a
dipstick (can be dipped into urine).
An enzyme is also attached to the dipstick.
When the antibody binds to HCG, the
enzyme is activated and reacts to produce
a blue line/product.
Blue indicates that a woman is most likely
pregnant.
http://www.sumanasinc.com/webcontent/animations/content/pregtest.html
Food Safety
 Bacteria have surface proteins that vary
between species
 If one of the surface proteins is injected into a
mouse, it will raise an antibody response to the
protein. These antibodies can be purified to
make diagnostic kits.
 Kits can be used to test for the presence of
disease causing bacteria in foods.
Drug Testing
 Consistent detection of low concentrations of
drugs e.g. heroin, can be carried out using
monoclonal antibodies.
Herceptin
 The “herceptin” antibodies that are purified are
specific to breast cancer cells. They bind to the
cancer cells, preventing further growth.
Past Paper Questions
2006 Q2
2005 Q3