SYNTHESIS, ISOLATION AND IMPORTANCE OF

MONOCLONAL ANTIBODIES
 ANTIBODIES
• Antibodies are glycoproteins produced by B lymphocytes
(cells of immune system) which neutralize the
pathogens/antigens

• B lymphocytes are cells of acquired immune system

• Immune system is derived from Latin term “IMMUNIS” which

refers to immunity or protection against the pathogens

• It is integrated system of organs, tissues, cells & cell

products (antibodies, cytokines, etc.) that differentiates self
from non – self & neutralizes potentially pathogenic
organisms/antigens
 RESPONSE
Foreign invaders - viruses, bacteria, allergens, toxins and
arasites- constantly bombard our body.
IMMUNE SYSTEM

• Innate immune system • Acquired immune

system

– primitive – advanced
– non specific – specific
– Various types of cells – B and T lymphocytes
(give humoral & cell
mediated immunity
respectively)
 HUMORAL IMMUNITY
• Antibodies are found in extracellular
fluids (blood plasma, lymph, mucus,
etc.) and on the surface of B cells

• There are about 10 million different

types of B lymphocytes

• B cells on stimulation will actively

secrete antibodies and are called
plasma cells

• Provide defense against bacteria,

bacterial toxins and viruses which
circulate freely in body fluids
ACIVATION AND CLONAL SELECTION OF B CELLS
 ANTIBODIES
POLYCLONAL. MONOCLONAL.

Derived from different B Derived from a single B cell

lymphocytes cell lines clone

Batch to batch variation Offer reproducible &

affecting Ab reactivity & inexhaustible supply with
titre exquisite specificity

Not powerful tools for Enable the development of

clinical diagnostic tests secure immunoassay systems
 ANTIBODIES AND THEIR STRUCTURE

• Each antibody has at least

– Two heavy chains
– Two light chains

• Variable Regions: Two regions at the

end of Y’s arms. Identical on the same
antibody, but vary in others

• Constant Regions: Stem of monomer

and lower parts of Y arms which bind
to complement or cells

Structure of antibody and antigen binding cleft

 IMMUNOTHERAPY

Treatment of the disease by Inducing, Enhancing or

Suppressing the Immune System

Active Immunotherapy Passive Immunotherapy

It stimulates body’s own It rely on the immune system
immune system to fight components (antibodies)
disease created outside the body
PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY

• Hypoxanthine guanine phosphoribosyl

transferase negative (HGPRT)¯
myeloma cells are selected

• HGPRT enzyme enables cells to

synthesize purines by salvage pathway

• Ordinarily absence of HGPRT is not a

problem because of de novo pathway

• But cells exposed to Aminopterin are

unable to use de novo pathway and are
dependent on salvage pathway only

• HGPRT+ B cells are fused with the

myeloma cells to produce hybridoma
and are grown in HAT medium
 PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: - Immunization of mice

ANTIGEN (purified
proteins, etc.) +
ADJUVANT
(emulsification)

Serum antibody titre is determined

Titre too low Titre High

BOOST BOOST
(Pure antigen) (Pure antigen)
2 weeks
PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: - Removal of spleen for antibody producing B cells

After several
weeks of
immunization

High Ab titre in Serum

Spleen is removed
(source of B cells)
PRODUCTION OF MONOCLONAL ANTIBODY

Step 3: - Formation of hybridoma cells

PEG
FUSION
SPLEEN CELLS MYELOMA CELLS
Feeder cells
Growth medium

1. Plating of cells in
HAT selective
medium
HYBRIDOMA CELLS
ELISA PLATE 2. Scanning of viable
HAT Medium hybridomas
PRODUCTION OF MONOCLONAL ANTIBODY

Step 4: - Cloning of hybridoma cell lines

A. Clone each +ve Culture

B. Test each supernatant for antibodies

C. Expand +ve clones

Tissue Mouse
culture ascites
method method
 PRODUCTION OF MONOCLONAL ANTIBODY
Step 5: - Separation & purification of monoclonal antibodies

• Cells, cell debris, lipids, and clotted material from supernatant

or ascitic fluid are removed by filtration with a 0.45 µm filter

• Antibodies are commonly separated by protein A/G affinity

chromatography

• mAbs selectively bind to Protein A/G and can be afterwards

eluted
 APPLICATIONS OF MONOCLONAL ANTIBODIES

• Therapeutic applications
Transplant rejection (Muronomab-CD3)
Cardiovascular disease (Abciximab)
Cancer (Rituximab)
Infectious Diseases (Palivizumab)
Inflammatory disease (Infliximab)

• Clinical applications
Purification of drugs, Imaging the target

• Future applications
Fight against Bioterrorism
 THERAPEUTIC APPLICATIONS
• Hematologic malignancies

• CD20 is highly expressed in B

cell malignancies (95% in
follicular lymphomas)

• Low grade non Hodgkin

lymphoma is being successfully
treated with rituximab

• Rituximab is combined with

chemotherapy for intermediate
grade or diffuse large cell non
Hodgkin lymphoma
 THERAPEUTIC APPLICATIONS

• Solid tumors

• Solid tumors do not have many specific

targets

• Edrecolomab targets 17-1A antigen (Ep-

CAM) in colon and rectal cancer by
ADCC & CDC methods

• Trastuzumab is being used to target

HER-2 (Human Epidermal growth factor
Receptor 2, CD340) antigen in breast
cancers alone or in combination with
chemotherapy
THERAPEUTIC APPLICATIONS
• Muromonab-CD3 (OKT3) binds to CD3 molecule on
surface of T cells and prevents rejection of organs

• Humanized versions of OKT3 show promise in inhibiting

autoimmune destruction of beta cells in Type 1 diabetes

• Infliximab binds to TNF-α and is used in inflammatory

diseases such as rheumatoid arthritis

• Omalizumab binds to IgE thus preventing IgE from

binding to mast cells (allergic asthma)

• Abciximab inhibits clumping of platelets by binding

receptors on their surface and helpful in preventing
clogging of coronary arteries
ANIMAL HOUSE
PRAGUE