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B io Factsheet

September 2002

www.curriculumpress.co.uk

Number 112

Monoclonal Antibodies
This Factsheet describes:
the production of monoclonal antibodies.
the uses of monoclonal antibodies in pregnancy testing and the targeting of drugs to specific substances or cells.
Production of monoclonal antibodies
Until 1975 B-lymphocytes would only transform into plasma cells and manufacture antibodies when inside the body. Plasma cells are unable to divide
and so cannot be cultured outside the body. In 1975 Georges Kohler and Cesar Milstein fused mouse spleen lymphocytes (capable of making antibodies)
with myeloma cells (capable of dividing) to form a hybridoma. The hybridoma cells possessed both useful characteristics - they could make antibodies
and they could divide.

Fig 1. Production of monoclonal antibodies


2. The substance is recognised as foreign by a few
B-lymphocytes in the mouse spleen and these
lymphocytes multiply greatly to form a clone of
plasma cells which produce the required antibody.
Samples of spleen cells, which should contain
large numbers of the required plasma cells (active
B-lymphocytes) are extracted from the mice.

1. The antigen is injected into a mouse

lymphocytes activated by
specific antigen

culture of mouse myeloma cells


culture vessel

other lymphocytes
culture vessel

3. The extracted lymphocytes are mixed with mouse myeloma cells.


They fuse, producing hybridoma cells.

required hybridoma cells


4. Non-hybridized lymphocytes cannot
divide so die. Non-hybridized myeloma
cells are killed by growing the cell culture
in a medium lacking a vital metabolite only
required by myeloma cells. Lymphocyte
cells possess the enzyme required to
synthesize the missing metabolite and so
this enzyme is also present in hybridoma
cells, allowing their survival.

5. The remaining cells are subcultured. When large


numbers of cells are formed, a small sample of
each culture is tested against the specific antigen.
If the cells are producing the specific monoclonal
antibody agglutination (clumping) will occur.

other hybridoma cells


specific hybridoma cells
Monoclonal antibodies have also been produced by
genetic engineering. Monoclonal antibody genes from
hybridoma cells can be transplanted into the bacterium,
E. coli, using its -bacteriophage as vector. The bacteria
then rapidly and cheaply produce large quantities of the
monoclonal antibody. Attempts are also being made to
introduce the antibody genes into plants.
A problem of using monoclonal antibodies of mouse
origin in human medical treatments, is that the mouse
antibody (protein) provokes an immune response against
itself, so that the monoclonal antibody is destroyed.
This problem is avoided when genetically engineered
human monoclonal antibodies are used.

6. Hybridoma cell cultures which cause agglutination of the


specific antigen are then mass cultured in large industrial
fermenters. The cultures will continually produce large
quantities of specific monoclonal antibodies.

........
....
.........
..

Hybridoma cells produce the monoclonal antibody in large amounts.


The antibody is collected and labelled as required

Bio Factsheet

Monoclonal antibodies

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Uses of monoclonal antibodies

Fig 2. Urine dipstick for pregnancy testing

Monoclonal antibodies can be produced against antigens of specific cells,


antigens of cellular components or antigenic chemicals. They can then be
used to tag these antigens in the cell or body, by labelling the antibody.
There are three ways of labelling the antibody:
1. Using a fluorescent dye. The location of the antigen can then be found
using a light microscope on sections of the tissue.

Control window

2. Using an electron-dense substance. The location of the antigen can be


found using an electron microscope on sections of the tissue.
3. Using a radioactive marker. The location of the antigen can then be
located by a variety of radioisotope analyses, such as a gamma scan of
a whole body to locate labelled cancer cells.

Test window

Some uses of monoclonal antibodies are:


in cytological studies to locate the position of all the tubulin
(cytoskeleton) within the cell, to locate the sodium-potassium pumps,
to locate the ATP-synthesizing complexes of the mitochondria and to
locate the positions of many enzymes, for example, RNA polymerases
within the cell.

to locate the positions of cancerous cells and tumours within the body.

to carry specific cancer-treating drugs directly to the cancer cells so


that only the cancer cells receive the drug and other normal body cells
are not affected by the drug thus reducing side effects and making the
treatment more efficient.

as diagnostic tools for AIDS/HIV infection and Chlamydia infection (a


sexually transmitted disease).

in the industrial preparation of interferon (a drug used to treat cancer


and hepatitis).

in a diagnostic test for pregnancy.

a blue line always


appears in the
control window to
show that the test
has been used
correctly

with row of immobilised


anti-anti-hCG antibodies
which combine with
latex-tagged anti-hCG antibodies
in the absence of hCG.

with row of immobilised


anti-anti-hCG antibodies
which combine with hCG
bound to latex-tagged
anti-hCG antibodies

a blue line in the


test window
indicates a
positive result.
No blue line in
the test window
indicates a
negative result

blue latex - tagged


anti-hCG antibody
bound to an hCG molecule
free blue latex - tagged
anti-hCG antibody

free hCG molecule

urine

The tip of the test strip is dipped into the urine sample from the woman
with the suspected pregnancy. hCG molecules will combine with the antihCG antibodies in the tip of the strip and the antigen-antibody complexes
formed move up the strip. When they reach the test window they combine
with the immobilised anti-anti-hCG antibodies and the accumulated blue
latex gives the appearance of a blue line. Any anti-hCG antibodies that are
not carrying hCG move higher up the strip and combine with the anti-antihCG antibodies in the control window forming a blue line. Thus:

Magic bullets to treat cancer


Monoclonal antibodies, referred to as magic bullets, are used against
cancer cells in a method called antibody-directed enzyme pro-drug therapy
or ADEPT.
1. The patient is injected with a monoclonal antibody against antigens on
the cancer cells. The monoclonal antibody is attached to an enzyme
which becomes bound to the cancer cells when the antigen-antibody
complexes form.
2. The patient is then injected with an inactive pro-drug. This only
becomes activated when it comes in contact with the enzyme attached
to the cancer cells.

a positive test is indicated by a blue line in both test and control


windows.

a negative test is indicated by the presence of a blue line in the control


window but an absence of a blue line in the test window.

Exam Hint you should be able to refer to a wide range of applications


of monoclonal antibodies but you should know the details of their use
in pregnancy testing and drug targeting.

3. The pro-drug is activated into a cytotoxic drug which destroys the


cancer cells. Since the pro-drug is not activated by normal body cells,
these cells should not be affected.

Make sure that you are clear about the three types of monoclonal
antibodies used in the pregnancy test.

Pregnancy testing
During the early weeks of pregnancy a hormone called human chorionic
gonadotrophin (hCG) is released from the placenta and accumulates in the
mothers blood stream and is excreted in the urine. Detection of this hormone
in the urine is the basis of some pregnancy home testing kits. Three types
of monoclonal antibody are used in the test.
Because hCG is a glycoprotein, monoclonal antibodies can be prepared
against it (anti-hCG antibodies). Monoclonal antibodies (anti-anti-hCG
antibodies) can also be prepared against the anti-hCG antibodies with
attached hCG and against anti-hCG antibodies without attached hCG.
The anti-hCG antibodies are tagged with a blue latex so that their position
can be clearly seen.

Acknowledgements:
This Factsheet was researched and written by Martin Griffin.
Curriculum Press, Unit 305B, The Big Peg, 120 Vyse Street, Birmingham. B18 6NF
Bio Factsheets may be copied free of charge by teaching staff or students,
provided that their school is a registered subscriber. No part of these Factsheets
may be reproduced, stored in a retrieval system, or transmitted, in any other
form or by any other means, without the prior permission of the publisher.
ISSN 1351-5136

The antibodies are immobilised on a urine dipstick in the arrangement


illustrated in Fig 2.

Bio Factsheet

Monoclonal antibodies

www.curriculumpress.co.uk
Practice Questions
1. (a) What is meant by the term monoclonal antibody?
3
(b) In human medicine why are genetically engineered monoclonal
antibodies preferred for use rather than mouse-origin monoclonal
antibodies?
4
(c) Lymphocytes and plasma cells cannot be used directly for the
commercial production of monoclonal antibodies. Explain why.3
Total 10

Answers
1. (a) a highly specific, pure antibody;
produced in large quantities by a specific clone of cells;
called a hybridoma;
3
(b) antibodies are protein and so could induce an immune response;
human monoclonal antibodies are not foreign to the body and so
are unlikely to cause an immune response;
mouse monoclonal antibodies are foreign to the body and so cause
an immune response;
this is only likely to be serious if the mouse antibodies are used
more than once so that the human has become sensitized to them;
4
(c) lymphocytes/plasma cells can only undergo a specified number of
mitoses in their life cycle;
they have already divided and cloned before extraction from the
mouse:
thus they are unlikely to divide again/live for long, and so are
unsuitable for long-term culture;
3
Total 10
2. antigen; plasma cells/B-lymphocytes; myeloma cells; hybridomas;
clone; cancer/HIV/AIDS/Chlamydia; pregnancy;
Total 7

2. Read through the following passage about monoclonal antibodies and


then fill in the spaces with the most appropriate word or words.
Monoclonal antibodies are produced by cells called hybridomas.
A mouse is exposed to a particular ................................ which stimulates
the production of large numbers of ............................. cells which secrete
a specific antibody. These mouse cells are then fused with
.................................to produce cells called .................... .
These divide to produce a culture of identical cells thus forming a
........................ .The monoclonal antibodies produced by these cells are
collected and concentrated before being used, for example, in diagnosing
the disease ............................... or testing for .................. .
Total 7

3. (a) lymphocytes/plasma cells cannot be cultured because they have a


limited life span/
cannot divide/have a limited number of mitoses in their life span;
myeloma cells can divide continuously and so perpetuate themselves
for years;
by combining lymphocytes and myeloma cells into hybridomas
the cells would divide
perpetuating the culture and continually produce the monoclonal
antibody;
3
(b) (i) negative test;
presence of blue line in upper control window and absence of
blue line in lower/test window;
2
(ii) no hCG in urine;
so anti-hCG antibodies remain uncoupled;
when they move up the strip they are not trapped in the test
window;
but become trapped by the anti-anti-hCG antibodies in the
control window (forming a blue line due to the blue latex label);
4
(iii) hCG in urine combines with some of the anti-hCG antibodies;
these move up the strip and become trapped by the anti-antihCG antibodies in the test window;
anti-hCG antibodies which do not combine with hCG move
up to the control window and become trapped;
thus blue lines (due to the latex labelling) appear in both
windows indicating a positive test;
4
Total 13

3. Polyethylene-glycol (PEG) has the property of making cells fuse


together and can be used in producing cell lines to synthesize monoclonal
antibodies.
(a) Explain why it was so important to be able to fuse cells together
when producing monoclonal antibodies.
3
(b) The diagram below shows a urine dipstick for testing for pregnancy.

thin blue line in this window

no blue line in this window

end dipped in urine


(i) Does the dipstick represent a positive or a negative test for
pregnancy? Explain your answer.
2
(ii) Describe what has happened on the dipstick to give this result.
4
(iii) Describe what would have happened on the dipstick if the
alternative other possible result had occurred.
4
Total 13

Glossary - Basic definitions

Immunity: The ability to be resistant to injury by poisons, foreign


proteins and invading parasites, due to the presence of antibodies.
Antigen: A substance, that when introduced into the tissues or blood,
activates the immune system to form antibodies. The antibodies are
specific to the antigen and react with it to make it harmless.
Antibody: A protein produced by certain cells in the body in response
to the presence of a specific antigen. The antibody reacts with that
antigen to neutralise, inhibit or destroy it.
Immunoglobulin: An antibody synthesised by plasma cells formed
from B-lymphocytes in response to the presence of a specific antigen
The immune system: This consists of a number of lymphoid organs
linked by lymphatic vessels and capillaries. Examples of lymphoid
organs are the thymus, spleen, tonsils, bone marrow and the lymph
nodes. These organs contain billions of lymphocytes which are
responsible for identifying and eliminating infectious parasites.

Lymphocytes: These are formed from stem cells in the bone marrow.
The B-lymphocytes make antibodies (humoral response) and are found
in all lymphoid organs except the thymus. The T-lymphocytes develop
in the thymus, and give cellular immunity (by destroying foreign cells)
and also regulate antibody secretion by the B-Lymphocytes.
The clonal selection theory: At birth the lymphoid system contains
as many as one hundred million different clones of lymphocytes, each
one committed by the shape of its antigen receptors to recognise just
one antigen grouping. Each clone consists of less than a hundred cells
but there are more than enough clones to recognise all the different
antigen groupings that might be encountered throughout life. (A clone is
a population of genetically identical cells formed by mitosis). Thus, if
the lymphocytes encounter a particular antigen only a few cells (one
clone) will recognise it and respond.
Monoclonal antibody: This is a highly specific, pure antibody,
produced in large quantities by a specific clone of cells called a hybridoma.
Hybridoma: A clone of cells produced by fusing specific B-lymphocytes
with myeloma (cancer) cells.

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