Download MONOCLONA L ANTIBODIES What is?

By Andrew Kliewer, Jeff Siswanto, and Rony Eshaque
CHE 100 LAB
Due: November 15, 2004.
SMALLPOX VACCINES
• Small pox is caused by 2 strains of the virus name Variola, Variola major which is
more severe and Variola minor
• There is no treatment for this, but vaccines can help protect against the disease.
• The earliest form of vaccination was inoculation which was to take fluid from
lesions of infected person. This is known to be effective because the symptoms in
persons given the inoculation had less sever symptoms than those that didn’t
receive the inoculation.
•Edward Jenner who invented this method is known as a father of small pox
vaccination
• The vaccine Dryvax is licensed in U.S.A., although it is not regularly used since
small pox is declared as eradicated. The vaccine is however given to people who
work with live vaccines is places for disease control.
•With the wake of terrorism, there is a strong possibility that it might be used as
biological weapon
POLIO VACCINES
• In early 1900, vaccine development for polio begun
• Polio is caused by three strains of viruses which have RNA.
• In 1947, Dr. Jonas Salk began his research on Polio virus
• He needed to process the viruses in a way that they will be less infectious
• In 1952, he developed a vaccine using the mixtures of three types of viruses that
grow in monkey kidney cultures
• He invented a chemical called formalin that inactivated the whole virus
• The results of the vaccines were dramatic and government granted permission for
the vaccines to be used in the country
• In the field of recombinant biotechnology, scientists are attempting to alter the
gene of Polio virus
INSULIN
• In the late 1900, Banting and Best discovered Insulin
• Insulin is hormone produced by the pancreas in the body
• Diabetic people can’t produce insulin naturally
• Pancreatic islets and the insulin protein were isolated from animals
• Bovine made from the cow’s pancreatic cells and porcine made from the pig’s
pancreatic cells work very well
• In 1980, technology allowed scientists to make human insulin
• The human gene which codes for the insulin was copied and then put inside a
bacteria
• By using this technique, pharmaceutical companies can isolate pure human insulin
• The advantage of human insulin is that there is lower chance of inducing an
allergic reaction because all human beings have the same insulin
MONOCLONAL ANTIBODIES
What is?
• Antibodies are natural defenses that act to fight infectious agents (antigens)
such as viruses and disease-causing bacteria entering through the body
• Production of antibodies can be stimulated by vaccines
• Certain antibodies are “activated” by diseases to prevent future invasion of
the same disease
Uses of antibodies
•Antibodies are extremely specific, meaning that they bind and destroy
certain antigens
• Antibodies are used to protect diseases, diagnose illnesses, detect drugs
and any blood abnormalities
• Antibodies are potentially safer than drugs since they do not attack the
body’s own cells, hence the patient experiences no side effects
MONOCLONAL ANTIBODIES
History
• Nobel prize winners Köhler and Milstein devised a technique in 1975 which
allowed them to produce antibodies
Past Objective
• To mass produce antibody of a single specificity
• Produce an antibody that can reproduce itself indefinitely
Procedure
• Köhler and Milstein used a technique called somatic cell hybridization by
fusing myeloma cells (found in tumor of bone marrow) with cells from an
immunized mouse
• “Hybrid” cells containing one selective drug are produced and cultured in a
medium
Animal produces antibodies
Once a significant amount of antibodies
are produced, the spleen of the animal
is removed and its cells are cultivated
Antigen of choice injected into one animal
Drugs are injected into the medium
which will kill remaining myolema cells.
The remaining spleen cells will die in 1
to 2 weeks. “Hybrid” cells called
“hybridomas” that are inherited from
cells immune to the antigen survive.
Polyethylene glycol is added to promote
fusion between spleen and myeloma
cells, producing “hybrids”
Hybridomas continue to replicate
and inherit their characteristics
Unfused
Fused
spleen cells
spleen cells
Unfused
myeloma cells
Clonal Expansion
Flow chart adapted from Johns Hopkins Arthritis [5]
…
…
…
…
PROLIFERATION
…
Cell death
Hybridomas are analyzed and
determined whether the desired
antibody is secreted. Selected
hybridomas are cloned and
cultivated in great quantities.
…
Cell death
Fused
hybridomas
myeloma cells
MONOCLONAL ANTIBODIES
Production
• The conventional method of injecting an antigen to a number of animals
were inefficient because the animals often yield unwanted substances and
very little usable antibody (the procedure is referred to as polyclonal antibody
technology)
• Since monoclonal antibody technology was developed, high demands lead
to mass production of natural antibodies
• Cultures are used to cultivate cells that produce natural antibodies
• Tumor cells are known to be fused with cells to produce “hybridomas”
• These particular cells have the ability to endlessly replicate themselves thus
it is possible to produce great quantities of antibodies
Advantages of the monoclonal antibody technology
• An impure antigen may be injected to one animal since the monoclonal
antibody of interest is cultivated by individual selection strategy
• Hybridomas are immortal hence there is an unlimited source of antibody
CYTOKINES
What are they?
•
Cytokines are proteins that act as communicators between cells. They are
created by one cell and act upon another, relaying messages that tell the
cell to grow, stop growing, move to a trouble spot, or otherwise somehow
modify its function
•
Some uses include activation of hormones that both promote and
discourage growth, begin immunal activities against harmful bacteria and
viruses, and promote wound healing
Uses
•
Promote growth and healing: they have possible uses in patients who do
not produce sufficient cytokines on their own
•
Cytokines activate killer T cells, which are vital in fighting against deformed
or corrupted cells such as tumors
•
Cytokines activate hematopoiesis which can be used to treat patients with
anemia or decreased blood cell counts
CYTOKINES
History
• Relatively new idea. Originally appeared in the 1980’s when a cytokine therapy was
developed in the 1980’s, originating from the need of leukocyte
IFN, and cloning of HuIFN(alpha) subtypes.
•
Medicines such as Insulin are included in this field despite the fact that insulin is not
actually a cytokine. It does however promote DNA synthesis and cell proliferation.
Recent Uses/Discoveries
• Hematopoiesis: boost production of mature blood cells of various lineages by using
lineage specific cytokines as replacement therapy (has been used to increase red
blood cell counts, neutrophils, eosinophils, and monocytes in people with aids).
•
One recent cytokine to be licensed is an interferon. In 1993, FDA approved interferon
beta (Betaseron) for multiple sclerosis. Studies showed it helped prevent flare-ups of
the disease.
Way Produced:
• The DNA strand that produces the cytokine desired is removed from a cell. It is
then inserted into an E.coli
• The E-coli then produce the cytokine in high concentration, and when the
desired amount is reached, inserted into people where it activates (or deactivates)
certain cells, and creates the desired effects
C Y T O K I N E S : Bone Remodeling Cycle
Current Research
• Nerve growth for use in Alzheimer's disease and brain-derived neurotrophic factor
(BDNF) and for amyotrophic lateral sclerosis
•
Wound Healing: Insertion of the wound healing cytokines (transforming growth factor
beta (TGF- beta) and others) has sped up the healing rate of rats when given the
TGF up to 24 hours before wounding
Example of Bone Remodeling Cycle
Quiescence
Mineralization
Activation
Formation
‘Coupling’
Resorption
CYTOKINES
Problems with Cytokines
•
The cytokines have the correct DNA structure, but because they aren’t
synthesized by the body itself, they wont be identical in their ‘post-translational
modifications’. This could affect how the cytokines react to their target cells
when inserted into the body
•
If mammalian cells are used to create the cytokines (yeast etc) then there is a
possibility of other bacteria or viruses being present, therefore chemical
cleansing is required, which may introduce some undesired chemicals into the
body
•
Over exposure to cytokines results in a toxicity reaction with symptoms similar to
those of the flu, such as headache and malaise
•
For patients who received greater amounts of cytokines (30 million units), they
experience disorientation, confusion and eventually comatose. This means that
cytokines, while not lethal, cannot be taken for extended periods of time
•
The biggest problem with cytokines is that different cytokines may act upon the
same cell with the same results, meaning that finding the specific cytokine
desired is very difficult
WORKS CITED
[1] Vaccines. Available Online:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vaccines.html,
[2004,Nov. 09].
[2] Perez, Arely., 2003. Small Pox. Available Online:
http://www.goshen.edu/bio/Biol206/Biol206LabProject/smallpox/Smallpox.html,
[2004,Nov. 05].
[3] Morganstein, Linda., 2002. Development of Polio Vaccines. Available Online:
http://www.accessexcellence.org/AE/AEC/CC/polio.html, [2004,Nov. 05].
[4] Insulin. Available Online:
http://www.endocrineweb.com/diabetes/2insulin.html, [2004,Nov. 05].
[5] Soloski, Mark J., 2004. What on Earth is Monoclonal Antibody? Available
Online: http://www.hopkins-arthritis.som.jhmi.edu/edu/mono_anti.html [2004,
Nov. 7].
[6] Meager, Anthony. 1990. Cytokines. Buckingham: Open University Press
WORKS CITED
[7] Access Excelence, 2004. Monoclonal Antibody Technology - The Basics.
Available Online:
http://www.accessexcellence.org/RC/AB/IE/Monoclonal_Antibody.html [2004,
Nov. 5].
[8] Kimball, John W., 2004. Monoclonal Antibodies. Available Online:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html
[2004, Nov. 8].
[9] Cotner, J. and T. Johengen. 1999. The Impact of a Recurrent Coastal
Plume on Phosphorus Dynamics and Production in Lake Michigan. Available
Online: http://www.glerl.noaa.gov/eegle/projects/p09/p09.html [1999, Sept.
15].
[10] Marian Segal 1995 Cytokines: Putting Body Mechanics to Work.
Available Online: http://www.fda.gov/bbs/topics/CONSUMER/CON0291f.html
[11] Clemens, M.J. 1991. Cytokines. Oxford: BIOS Scientific Publishers
Limited