Lecture:

Monoclonal Antibodies
Prepared by :
Dr. Doaa Safwat
 Introduction to Immune System

 Antibodies are highly specific, extremely sensitive molecules of

humoral immune system.
 They are soluble proteins that help to defend against foreign entities
such as microorganisms and transformed (malignant) cells.
 Higher animals posses a sophisticated immune system which
recognizes harmful molecules.
 When the immune system is activated, it destroys or eliminates such
harmful molecules.
 Two mechanisms mediate the immune response to the presence of
antigens.
 Cell mediated immunity
 Humoral immunity.
 Antigens "Ags" are substances that elicit an immune response and
react with the products of that response.
 Requirements for a chemical substance to be antigenic:
Foreignness, Large molecular size, Chemical complexity &
specificity.
 Each antigen can have several antigenic determinant sites or
epitopes (region or sites in the antigen that bind to the antigen-
binding site of a specific antibody or with T cell receptors).
 Valence: the number of antigen determinant sites on the surface
of antigen.
 One antigen determinant sites: monovalent , two: bivalent,
more: multivalent.
 Humoral immune response
Involves production of specific antibodies against specific antigens.

Immunoglobulins or Antibodies: The large Y shaped protein

produced by the body’s immune system when it detects harmful
substances, called antigens like bacteria and viruses. The basic structure
of all antibodies are same. One molecule of antibody is split into three
parts one Fc and two Fab pieces. Fc fragment is responsible for
biological properties of the immunoglobulin such as complement
fixation and placental transfer while Fab fragment is responsible for
antigen binding.

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• Also as shown from figure (1), each molecule consisting of two pairs of
polypeptide chains of different sizes held together by disulfide bonds. The smaller
chains are called light (L) chains and the larger ones heavy (H) chains. The L
chain is attached to H chain by a disulfide bond.
• Serum containing antigen-specific antibodies is called antiserum.
• Each antibody has five isomers (isotypes) of immunoglobulins which are called -
IgG, IgM, IgA, IgD, IgE - and are classified according to the type of heavy
chain constant region, and are distributed and function differently in the body.

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Clonal Selection:
Each individual has a large pool of B lymphocytes, each of which is
programmed to make only one antibody and it expresses this IgM on its
outer surface to act as an antigen receptor. When the antigen enters the
body, it selects the B lymphocyte that has the specific IgM receptor,
binds to it, and stimulates its proliferation to give a clone of B cells
which differentiate to plasma cells that secrete IgM antibody specific to
that antigen.

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Figure ( ): Clonal selection and generation of plasma cells and memory
cells after primary contact with the antigen.
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Functions of Immunoglobulin
 Monoclonal antibodies
Definition of Monoclonal Antibodies:
Identical immunoglobulins generated from single B cell clone and
designed to target unique epitope of a single antigen.
Production of Monoclonal Antibodies:
• Hybridomas are cells that have been engineered to produce a
desired antibody in large amounts, to produce monoclonal
antibodies.
• Hybridoma technology was discovered in 1975 by two scientists,
Georges Kohler of West Germany and Cesar Milstein of
Argentina, who jointly with Niels Jerne of Denmark, were
awarded the 1984 Noble prize for physiology and medicine.
Characteristics of Monoclonal Antibodies
• MAbs produced from a single clone of B cells.
• Monoclonal antibodies all have identical antigen-binding sites. Thus,
they all bind to the same epitope with the same affinity.
• Mostly produced by fusing a B cell secreting the desired antibody
with a myeloma cell capable of growing indefinitely in tissue
culture forming hybridomas.
• So that, hybridomas have the advantages of both cells (B
cells & myeloma cells):
• Specificity of normal plasma cells.
• Proliferative capacity of malignant plasma cells which permit
continuous production of monoclonal antibodies.
• Fusion of both cells is done by sendai virus but later,
with polyethylene glycol (more efficient).
Steps for production of monoclonal antibodies
1- Immunization of an animal
2- Cell fusion

3- Selection of hybridomas

4- Isolation of hybridomas of single specificity

5- Screening of products, cloning and propagation

6- Characterization and storage

1- Immunization of an animal
• Immunization of mice and selection of mouse donors for
generation of Hybridoma Cells

• Mice are immunized with an antigen that is prepared for injection

either by emulsifying the antigen with Freund’s adjuvant or other
adjuvants. In general, mice are immunized every 2-3 weeks.

• Screening of mice for antibody production. And after several weeks

of immunization, blood samples are obtained from mice for
measurement of serum antibodies.

• Serum antibody titer is determined with various techniques, such as

enzyme-linked immunosorbent assay (ELISA).

• When the antibody titer is high enough, the mice are sacrificed and
their spleens removed for in vitro hybridoma cell production.
1- Immunization of an animal
 2- Cell fusion
• Fusion of myeloma cells with immune spleen cells.

• Spleen cells from the immunized mouse are fused with the previously
prepared myeloma cells.

• Fusion is accomplished by a technique called somatic cell

hybridization.

• This is achieved by co-centrifuging freshly harvested spleen cells and

myeloma cells in polyethylene glycol, a substance that causes cell
membranes to fuse.
2- Cell fusion
Continue to Cell fusion
Continue to Cell fusion
 3- Selection of hybridomas
• Those myeloma cells that have lost the ability to synthesize any
antibody molecules of their own and lack enzyme hypoxanthine-
guanine-phosphoribosyltransferase (HGPRT) and thymidine
kinase (TK) are selected.

• They are negative for HGPRT-, TK- and Ig- :

-These enzymes enable cells to synthesize purines using an
extracellular source of hypoxanthine and thymidine as a
precursor (salvage pathway, use degraded parts of DNA ) .

• Ordinarily, the absence of HGPRT is not a problem for the cell

because cells have an alternate pathway (de novo pathway which
use sugars and amino acids as precursors for purines)) that they
can use to synthesize purines.
 • When cells are exposed to aminopterin, which block dihydro
folate reductase enzyme. So, they are unable to use this other
pathway and are now fully dependent on HGPRT for
survival. The mouse derived B cells have this enzyme and can
produce antibodies too. They are HGPRT+ , TK+ and Ig+.
• The cells are then placed in HAT (hypoxanthine, aminopterin,
thymidine) medium.
- Since the unfused myeloma cells lack HGPRT and
aminopterin disallows alternative pathway, they die. Unfused
B Cells (short life span) are mortal and cannot proliferate,
so they die too .
• Only the hybridoma cells that have the ability to multiply
immortally and possess HGPRT will survive.
• The HAT medium allows only the fused Hybridoma cells to
survive in culture
3- Selection of hybridomas
Continue to Selection of hybridomas
Continue to Selection of hybridomas
Continue to Selection of hybridomas
Continue to Selection of hybridomas
Continue to Selection of hybridomas
4- Isolation of hybridomas of single specificity
 The supernatants from each culture are tested to find those
producing the desired antibody. Since there may be more than
one hybridoma cell in the original cultures, single cells from
each antibody-positive culture must be isolated and
subcultured. The supernatant of each must be tested for the
desired antibodies using ELISA or RIA .
4- Isolation of hybridomas of single specificity
Continue to Isolation of hybridomas of single specificity
5- Screening of products, cloning and propagation

• There are two methods for growing these cells:

- By injecting them into the peritoneal cavity of a mouse
 When injected into a mouse, the hybridoma cells multiply
and produce fluid (ascites) in its abdomen; this fluid
contains a high concentration of antibody.
• The other alternative is to grow hybridoma cells in a tissue-
culture medium. Further processing of the mouse ascitic fluid
and of the tissue-culture supernatant might be required to
obtain mAb with the required purity and concentration.
5- Screening of products, cloning and propagation
6- Characterization and storage
Polyclonal antibodies:
Polyclonal antibodies are heterogeneous mixture of antibodies
each recognizing a different epitope on the same antigen.

Polyclonal antibodies arise from many B-cell clones and have a

heterogeneous collection of binding sites.
Production of polyclonal antibodies
Production of Polyclonal antibodies
Each B cell binds to an epitope on the antigen through unique
B cell receptors (BCRs).
Proliferation of each B cell and secretion of antibodies (antibodies
from each B cell clone are specific to one epitope on the same antigen)
Antibodies derived from multiple clone of B cells
Applications of Monoclonal antibodies
Ex. Pregnancy test
Pregnancy test (Diagnostic application)

Monoclonal antibodies can be used to test for pregnancy via the

presence of human chorionic gonadotrophin (hCG) in urine
• hCG is a hormone produced by women during foetal development
and thus its presence in urine is indicative of pregnancy
Pregnancy tests use a process called ELISA (enzyme-linked
immunosorbent assay) to identify a substance via a colour change

• Free monoclonal antibodies specific to hCG are conjugated to

an enzyme that changes the colour of a dye

• A second set of monoclonal antibodies specific to hCG are

immobilised to the dye substrate

• If hCG is present in urine, it will interact with both sets of

monoclonal antibody (forming an antibody ‘sandwich’)

• When both sets of antibody are bound to hCG, the enzyme is

brought into physicial proximity with the dye, changing its colour

• A third set of monoclonal antibodies will bind any unattached

enzyme-linked antibodies, functioning as a control
How a Pregnancy Detection Kit Works