1. Isolate B cells from recovered COVID-19 patients that produce antibodies against the virus. 2. Fuse the B cells with myeloma cells to generate immortalized hybridoma cell lines. 3. Select a hybridoma that produces the desired anti-COVID-19 antibody and sequence the antibody genes. 4. Insert the antibody genes into a mammalian expression vector and transfect CHO cells for recombinant antibody production.

1. Monoclonal Antibodies Production
Technology

2. What are antibodies?
• An antibody is a protein used by the immune system
to identify and neutralize foreign objects like bacteria
and viruses.
• Each antibody recognizes a specific antigen unique to
its target.
• Monoclonal antibodies (mAb) are antibodies that are
identical because they were produced by one type of
immune cell, all clones of a single parent cell.
• Polyclonal antibodies are antibodies that are derived
from different cell lines.
• According to differences in their heavy chain constant domains,
immunoglobulins are grouped into five classes, or isotypes: IgG,
IgA, IgM, IgD, and IgE.

3. Monoclonal antibodies
• Monoclonal antibodies (mAbs)
are produced by introducing an antigen to
a mouse and then fusing polyclonal B cells
from the mouse's spleen to myeloma cells.
• The resulting hybridoma cells are cultured
and continue to produce antibodies to the
antigen.

6. • Antibodies have important uses beyond
fighting infections in the body.
• Production of long-lasting monoclonal
antibodies is a recent invention and it is used
in both medicine and research.
• Monoclonal Antibody: a stable antibody
which can be used over a period of time

7. Producing Monoclonal Antibodies
1. Inject a mouse with a specific antigen to
stimulate its immune system to produce
necessary antibodies.
2. Extract mouse spleen cells (containing B-
lymphocytes) and culture them in the lab.
3. Extract mouse tumour cells, which grow
continuously, and culture them in the lab.
4. Mix spleen cells and tumour cells on the
same plate and culture.

8. Producing Monoclonal Antibodies
5. Add polyethylene glycol – this causes some B-
lymphocytes to fuse with tumour cells to
produce a hybrid cell called a hybridoma.
6. Grow the cells under conditions that allow only
hybridoma cells to survive.
7. Extract the cells, culture them separately and
test the medium around each cell for the
specific antibody of interest.
8. Culture the cells making the desired antibody
and use as needed.

10. http://youtu.be/7ymKofaHCoY

11. mAb’s uses

12. Pregnancy Tests
• A pregnant woman has the hormone human
chorionic gonadotrophin (HCG) in her urine.
• Monoclonal antibodies to HCG have been
produced.
• These have been attached to enzymes which
can later interact with a dye molecule and
produce a colour change.

13. Pregnancy Tests
• Pregnancy test contains three regions – reaction
region, test region and control region
– Reaction region: contains monoclonal anti-HCG
antibodies linked to enzyme
– Test region: contains polyclonal anti-HCG antibodies,
which will bind to HCG molecules bound to
monoclonal anti-HCG antibodies. Also contains dye
molecules which will be activated if monoclonal
antibodies bind to polyclonal anti-HCG antibodies
– Control region: contains anti-mouse antibodies and
dye molecules which will be activated if monoclonal
antibodies bind here

14. Pregnancy Tests
• When pregnant woman’s urine travels up the
pregnancy test, HCG will bind to monoclonal
antibodies in reaction region
• Movement of the urine will move the monoclonal
antibodies up to the test region
• Monoclonal antibodies with bound HCG will bind to
antibodies in test region and activate dye molecules,
producing a colour change
• Any unbound monoclonal antibodies will continue to
travel to control region and will bind to anti-mouse
antibodies, activating dye molecules and producing a
colour change

15. Pregnancy Tests

16. Diagnosis of HIV Infection
• The test of HIV
infection is based on
detecting the
presence of HIV
antibody in the
patient’s blood
serum.

17. a) HIV antigen is attached to the plate.
b) Patients serum passed over the plate. Any HIV
antibody in the patients serum will attached to
the antigen already on the plate.
c) A second antibody which is specific to the HIV
antibody is passed over the plate. This
antibody will attach to the concentrated HIV
antibody on the plate. This second antibody
has an enzyme attached to its structure.
d) Chromagen dye is passed over the complex of
concentrated HIV antibody/conjugated
antibody.
e) The enzyme will turn the chromagen to a more
intense colour. The more intense the colour,
the greater the HIV antibody level. This would
be the a positive result for a HIV test.

18. Monoclonal antibodies to treat cancer
• The immune system attacks foreign substances in
the body is by making large numbers of antibodies.
• An antibody is a protein that sticks to a specific
protein called an antigen.
• Antibodies circulate throughout the body until they
find and attach to the antigen.
• Once attached, they can recruit other parts of the
immune system to destroy the cells containing the
antigen

19. Treatment of Cancer
• Cancer cells carry specific tumour-associated
antigens (TAA) on their plasma membrane.
• Monoclonal anti-TAA antibodies have been
produced.
• Drugs which kill tumour cells or inhibit key
proteins in tumour cells are attached to
monoclonal anti-TAA antibodies.
• Cancer cells are specifically targeted, avoiding
damage to healthy host cells.

20. Treatment of cancer

21. Types of monoclonal antibodies
 Naked monoclonal antibodies
Naked mAbs are antibodies that work by themselves. There is no
drug or radioactive material attached to them. These are the most
common type of mAbs used to treat cancer.
 Conjugated monoclonal antibodies
Monoclonal antibodies (mAbs) joined to a chemotherapy drug or to
a radioactive particle are called conjugated monoclonal antibodies.

22. Monoclonal antibody therapy as a therapeutic
approach and prevention of covid-19
infection.???
Coronavirus disease 2019 (COVID-19) is expanding rapidly,
which made it as one of top priorities for scientists to develop
novel treatment strategies. Researchers are racing to develop
treatments based on antibodies to block and/or neutralize the
coronavirus in affected patients.

24. Mode of action and proposed
mechanism
 Human neutralizing antibodies can block COVID-19
infection.
 Most monoclonal antibodies inhibit binding S protein
to ACE2 receptor.
Researchers are trying to develop treatments based on
antibodies to block and/or neutralize SARS-CoV-2.
 The genetic and structural similarity of the virus to
SARS-CoV helps for designing new methods for
treatment of COVID-19.

25. Problems…
• Many patients develop immune response to
monoclonal antibodies produced in mice, as
these are foreign proteins.
• Genetically engineered antibodies are being
perfected to avoid triggering immune
response.

26. Assignment
• SO.., based on your biotechnology or genetic
engineering technology procedures..
• Can you produce recombinant mAb’S using
isolating DNA from human cell??
• A. What is the most suitable cell?
• B. Vector ?
• C. Recombinant cell or expression system?
• D. Main procedures or techniques?