This document discusses monoclonal antibodies, including their preparation and applications. It begins with an introduction to antibodies and defines monoclonal antibodies as artificial antibodies produced from a single clone. The document then summarizes the key steps in producing monoclonal antibodies, which involves immunizing an animal, fusing B lymphocytes with myeloma cells to create immortal hybridomas, selecting antibody-producing hybridomas, culturing the cells, and extracting and purifying the monoclonal antibodies. The applications section notes that monoclonal antibodies are used for disease diagnosis, passive immunization, detecting and purifying biomolecules, and some have been authorized to treat COVID-19.

1. MONOCLONA
L
IN E.COLI

2. CONTENT
S
• Basic concept and introduction.
• Advantages and disadvantages of using
monoclonal antibodies.
• Preparation of monoclonal antibodies.
• Applications of monoclonal antibodies.
MONOCLONAL IN E.COLI 2

3. BASIC CONCEPT AND INTRODUCTION
MONOCLONAL ANTIBODIES

4. WHAT ARE ANTIBODIES?
“ Every body has a specialized search and destroy army.
Antibodies are key players in that fight ”
Antibodies are protein made by plasma cells ( type of WBC’s) in
response to an antigen.
It is a Y shaped protein .
Every body naturally produces antibodies.
Are elements of the immune system produced by B lymphocytes.
Bind to foreign proteins in the body known as antigens, with the
aim of eliminating them.
Naturally circulate in the body searching for foreign bodies
(antigens).
MONOCLONAL IN E.COLI 4

5. MONOCLONAL IN E.COLI 5
Each tip of the “Y” of an antibody
contains a paratope (analogous to a
lock) that is specific for one particular
epitope (analogous to a key) on an
antigen, allowing these two structures
to bind together with precision.
They attach to the antigen and destroy
it by using various immune
mechanisms.
Classes- IgA, IgD, IgE, IgG, or IgM.

6. ANTIBODY
MONOCLONAL IN E.COLI 6

7. MONOCLONAL ANTIBODIES
Monoclonal: forming a clone which is derived asexually from a
single individual or cell.
Monoclonal antibodies are artificial antibodies that are produced
from a single clone of cells by fusing B lymphocytes to myeloma
cells.
The fusion of B-lymphocytes with myeloma cells by somatic cell
hybridization secretes desired antibody-producing elements which
are immortalized cell-lines known as a hybridoma.
These hybridomas produce homogenous monoclonal
antibodies.
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8. MONOCLONAL IN E.COLI 8
Monoclonal antibodies (mAbs) have the ability to recognize unique
binding sites (epitopes) found on the specific antigens.
This differentiates monoclonal antibodies from polyclonal
antibodies i.e. monoclonal antibodies are derived from a single B-
cell clone to target single epitopes, unlike polyclonal antibodies
that target multiple epitopes.
Monoclonal antibodies (mAbs) have been produced to target
receptors or other foreign proteins that are present on the surface
of normal cells and cancer cells.
mAbs have given researchers the ability to study biological
processes reliably and with unprecedented accuracy

9. HISTORY
•In the early 1900s, immunologist, Paul
Ehrlich proposed the idea of a Zauberkugel – “magic
bullet", conceived of as a compound which selectively
targeted a disease-causing organism, and could
deliver a toxin for that organism.
•This underpinned the concept of monoclonal
antibodies and monoclonal drug conjugates
•By the 1970s, lymphocytes producing a single
antibody were known, in the form of multiple
myeloma – a cancer affecting B-cells
MONOCLONAL IN E.COLI 9
PAUL EHRLICH
(1854-1915)

10.  In 1973, Jerrold Schwaber described the production of
monoclonal antibodies using human–mouse hybrid cells
 In 1975, Georges kohler and Cesar Milstein succeeded in
making fusions of myeloma cell lines with B cells to create
hybridomas that could produce antibodies, specific to known
antigens and that were immortalized.
 In 1988, Greg and his team pioneered the techniques
to humanize monoclonal antibodies,
 By the 1990s research made progress in using monoclonal
antibodies therapeutically,
MONOCLONAL IN E.COLI 10

11. MONOCLONAL ANTIBODIES
MONOCLONAL IN E.COLI 11

12. TYPES OF MONOCLONAL ANTIBODIES
Accordingly, mAbs are of four broad types.
Murine: Made from mouse proteins, names of drugs based on this end in –
omab.
Chimeric: A combination of mouse and human proteins, names of drugs
based on this end in –ximab.
Humanized: Here small doses of mouse proteins are attached to human
proteins, names of drugs based on this end in –zumab.
Human: These are fully human proteins, names of drugs based on this end
in –umab.
MONOCLONAL IN E.COLI 12

13. ADVANTAGES AND DISADVANTAGES
MONOCLONAL ANTIBODIES

14. ADVANTAGES OF USING MONOCLONAL
ANTIBODIES
Monoclonal antibodies are one of the most successful
biotherapeutic drugs used in the treatment of many types of
cancer and autoimmune conditions.
They are also proven to reduce side-effects and improve patient
survival and well-being.
 As side effects can be treated and reduced by using mice-human
hybrid cells or by using fractions of antibodies.
It is highly scalable, unlimited production source
It can produce antibodies when needed.
Antigen or immunogen need not be pure.
MONOCLONAL IN E.COLI 14

15. DISADVANTAGES OF USING MONOCLONAL
ANTIBODIES
It is a time consuming project, it may take time between 6 months
to 9 months.
It is very expensive.
It needs considerable effort to produce them.
System is only well developed for mouse and rat and not for other
animals.
More than 99% of the cells do not survive during the fusion
process that reduces the range of useful antibodies that can be
produced against an antigen.
There is also a possibility of generating immunogenicity (the ability
of a molecule or substance to provoke an immune response).
MONOCLONAL IN E.COLI 15

16. PREPARATION
MONOCLONAL ANTIBODIES

17. STEPS IN PRODUCTION OF MONOCLONAL
ANTIBODIES
MONOCLONAL IN E.COLI 17
Step 1-Immunization of rabbit or rat and extraction of B-
lymphocytes
 In order to isolate B-lymphocyte producing certain antibodies,
rabbit or lab rat is immunized through repeated injection of specific
antigen (sheep RBCs)
 A sample of B-cells is extracted from spleen of rabbit or rat.

18. MONOCLONAL IN E.COLI 18
Step 2- Fusion of myeloma cell with B- Lymphocytes
• The extracted B-lymphocytes is added to a culture of myeloma cell from bone
marrow.
Multiple myeloma cells are abnormal plasma cells (a type of white blood cell)
that build up in the bone marrow and form tumors in many bones of the body.
• Hybridoma cells formed by fusion of B-cell and myeloma cell.
• The fusion is done by using Polyethylene glycol (PEG) or by electroporation or
by using phages.

19. MONOCLONAL IN E.COLI 19
Step 3- Selection of hybridoma cell
The B-lymphocytes contains HPRT1 gene which codes for enzyme
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
(HGPRT) is one of the central enzymes that recycle the building blocks of RNA and DNA
B- cells can grow in medium containing Hypoxanthine amonopterin thymine
(HAT media).
HAT Medium is a selection medium for mammalian cell culture.
Myeloma cell lack HPRT1 gene so, it does not produce HGPTR enzyme and it
does not grow in HAT medium.
The myeloma cell do not utilize Hypoxanthine.
Only hybridoma cell i.e.. fused cell between myeloma and B-cell can
survive and divide in HAT medium.
Screening is done to select hybridoma cells which are the desired cell for
monoclonal antibodies production

20. MONOCLONAL IN E.COLI 20
Step 4- Culture of Hybridoma cell.
The selected hybridoma cells are cultured in suitable medium like insulin,
transferon, ethanol, amine and other additional hormones.
Some commonly used culture media for hybridoma cell for production of
monoclonal antibodies are:
• DMEM (Dulbecco’s modified eagle medium)
• IMDM (Iscove’s Modified Dulbecco’s Medium)
• Ham’s F12
• RPMI 1640 medium (Roswell Park Memorial Institute
• 1640 medium)

21. MONOCLONAL IN E.COLI 21
Step 5-Inoculation of hybridoma cell into suitable host.
These hybridoma cells are then injected into lab animal so that they starts
to produce monoclonal antibodies.
These hybridoma cells may be frozen and store for future use.

22. MONOCLONAL IN E.COLI 22
Step 6-Extraction and purification of Monoclonal antibodies.
Monoclonal antibodies from host animal can be extracted and purified by the
following methods:
Ion exchange chromatography
Antigen affinity chromatography
Radial immunoassay
Immune precipitation

23. MONOCLONAL IN E.COLI 23

24. MONOCLONAL ANTIBODIES IN E.COLI

25. MONOCLONAL IN E.COLI 25
Escherichia coli
 Escherichia coli, also known as E. coli, is a Gram-
negative, facultative anaerobic, rod-shaped bacteria.
 It is commonly found in the animal feces, lower intestines
of mammals, and even on the edge of hot springs.
 The simplicity and ease of fermentation has made E. coli
an ideal host for antibody fragment production.
 E. coli advantages include:
• Well characterized genetics
• Short process development timeline
• Simple fermentation
• Scalability
• Less safety issues from viral contaminants

26. MONOCLONAL ANTIBODIES IN E.COLI
MONOCLONAL IN E.COLI 26
There are situations where E. coli may become the preferred production host
over the presently used mammalian cells.
A monoclonal antibody (mAb) was obtained from a mouse immunized with
solubilized outer membrane proteins extracted from a bovine
enterohemorrhagic strain of Escherichia coli (EHEC), O26.
EHEC is a pathogenic group of strains.

27. MONOCLONAL IN E.COLI 27
 The mAb produced a strong immunoblot reaction
for E.coli 026.
 This mAb was used in a sandwich enzyme-linked
immunosorbent assay (ELISA) format to screen
strains from animal and human sources, and all
reactive strains.
 The antigen was detected in a group of strains
containing a high proportion of O26.
 The association of the antigen detected by the MAb
with significant enteropathogenic E. coli and EHEC
virulence factors in isolates from both animal and
human enteric infections indicates a diagnostic
potential for the assay developed.

28. MONOCLONAL IN E.COLI 28
OBJECTIVE
 The object was to produce monoclonal antibodies (mAbs) to EHEC surface
adhesion antigens, and to investigate their diagnostic application for the
detection of EHEC in animal and human enteric infections.

29. APPLICATIONS
MONOCLONAL ANTIBODIES

30. Disease diagnosis
• ELISA to test HIV, hepatitis, Herpes etc.
• RIA- to test viral infection.
• MAbs to Human chorionic gonadotropin.
Passive immunization or disease prevention
• Monoclonal antibodies based drugs can be used
to treat septic shock
• Used as vaccine
Detection and purification of biomolecules
• MAbs are very useful in determining the presence
and absence of specific proteins through western
blotting technique.
• Besides that, it can be used to classify strains of a
single pathogen. E.g. Neisseria gonorrhea can be
typed using Monoclonal antibodies.
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31. COVID -19
 In 2021, the monoclonal antibody therapies bamlanivimab /etesevimab and
casirivimab/imdevimab have been found to reduce the number of
hospitalizations, emergency room visits, and deaths. Both combination drugs
were granted emergency use authorization by the US Food and Drug
Administration (FDA).
 In September 2021, the Biden administration purchased US$2.9 billion worth
of Regeneron monoclonal antibodies at $2,100 a dose to help curb the
shortage
 As of December 2021, in vitro neutralization tests indicate monoclonal
antibody therapies (with the exception of sotrovimab and tixagevimab
/cilgavimab ) are likely not active against the Omicron variant.
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32. SIDE-EFFECTS OF MONOCLONAL ANTIBODIES
MONOCLONAL IN E.COLI 32
Several monoclonal antibodies, such as bevacizumab and
cetuximab, can cause different kinds of side effects.
These side effects can be categorized into common and serious
side effects.
Common side effects include:
• Dizziness
• Headaches
• Allergies
• Fever
• Itching
• Insomnia
• Constipation etc.

33. 33
Serious side effects are
• Anaphylaxis
• Bleeding
• Arterial and venous blood clots
• Autoimmune thyroiditis
• Hypothyroidism
• Hepatitis
• Heart failure
• Cancer

34. PRESENTED
BY
Thank You
Kashish Imran
-Basic concept and
introduction
 Sadqua Urooj
-Advantages and
disadvantages of using
monoclonal antibodies.
 Khadeeja Yasmeen
-Preparation of monoclonal
antibodies
 Draksha
-Applications of monoclonal
antibodies.
MONOCLONAL IN E.COLI 34
-BSc Biotechnology, Sem II
PRESENTED TO
Dr. Saima Wajid|Professor
(Assistant)|Jamia Hamdard