HYBRIDOMA TECHNOLOGY & ITS

APPLICATION

Represent by: Sankar Jana

M.Sc. Biotechnology
Institute Of Genetic Engineering
Hybridoma Technology

A hybrid cell resulting from the

fusion of a lymphocyte & a tumor
cell - used to in vitro cultured, a
specific - this
is called
 Discovery of Hybridoma Technology
 1964- Littlefield developed a way to isolate hybrid cells from 2 parent cell lines
using the hypoxanthine-aminopterin-thymidine (HAT) selection media

 1975- Hybridoma technology was first discovered by Georges Kohler and Cesar
Milstein They were also awarded Nobel Prize along with Niels Kaj Jerne in
Physiology & Medicine field during 1984

 1990- Milstein produced the first Monoclonal antibodies

G.Kohler

C.Milstein
Niels K Jerne
Basic protocol for production of monoclonal antibodies from
hybrid myelomas

1) Immunisation of a mouse

(2) Isolation of B cells from the

spleen

(3) Cultivation of myeloma cells

(4) Fusion of myeloma and B cells

(5) Separation of cell lines

(6) Screening of suitable cell lines

(7) Multiplication (a) in vitro (b)

or in vivo

(8) Harvesting
 PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 1: - Immunization & Selection Of Mouse Donor For
Generation Of Hybridoma cells
ANTIGEN ( Whole cell
membrane/ micro-organisms ) +
ADJUVANT

 Mouse is immunized by giving

antigen injection along with an
adjuvant via subcutaneously or Ab titre reached in Serum
by peritoneal cavity; this is
followed by booster doses of the
antigen. Spleen removed
 Adjuvant is non-antigenic in (source of cells)
nature but they stimulate the
immune system.
 PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 2: - Screening Of Mice For Antibody Production
After several
weeks of
 Blood sample of mice for immunization
measurement of serum
antibodies. Serum Antibody Titre Determined
 Titre is too low mice can be (Technique: - ELISA / Flow cytometery)
boosted until an adequate
response is achieved.
 Titre is high mice are commonly
boosted by injecting antigen Titre too low
Titre High
with out adjuvant.
 Then mice spleens removed for
BOOST 2 weeks
in vitro hybridoma cell (Pure antigen) BOOST
production. (Pure antigen)
 PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

Step 3: - Preparation of Myeloma Cells

Myeloma Cells
+ 8 - Azaguanine

Immortal Tumor Of
Lymphocytes
 Myeloma cells are
immortalized cells
that are cultured with
8 Azaguanine to their
sensitivity to HAT Myeloma Cells
medium used after HGPRT-
High Viability & Rapid Growth
cell fusion
 PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 3: - Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells
PEG
FUSION
SPLEEN CELLS
MYELOMA CELLS
Growth Medium
o Spleen cells harvested from Feeder Cells
mice are fused with myeloma
cells 1. Plating of Cells in
HAT selective
Medium
o Fission is done through co- HYBRIDOMA CELLS
centrifusing in polyethylene ELISA PLATE 2. Scanning of
glycol Viable
HAT Medium
Hybridomas
o Only fused cells with grow on
HAT
 PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 5: - Cloning of Hybridoma Cell Lines by “ Limitin Dilution” or Expansion
 Cloning by “Limiting
Dilution” at this time
a majority of wells 1. Clone Each +ve Culture
each contain a single
clone 2. Test Each Supernatant for Antibodies
 Once the desired
hybridoma clone-it
is multiplied either
in vivo or in vitro C. Expand +ve Clones

Tissue Mouse
Culture Ascites
Method Method
FLOW-DIAGRAM
 ENGINEERED ANTIBODIES

 MAbs derived from mouse

they are not human origin,
they show HAMA (Human
Anti-Mouse Antibody)
response

 Antibody made of Fc and

Fab fragments

 Fab facilitate penetration

into tumor tissue and result
in a longer half-life

 Engineering is needed to
reduce the immunogenicity
Engineered antibodies
EVOLUTION OF MONOCLONAL ANTIBODY
Applications of Monoclonal Antibodies
 Diagnostic Applications
Biosensors & Microarrays

 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
A breakthrough in Diagnostics

 Antibodies are used in several

diagnostic tests to detect small
amounts of drugs, toxins or
hormones

 Human Monoclonal antibodies

to Human chorionic
Gonadotropin (HCG) are used in
pregnancy test kits

 Another diagnostic uses of

antibodies is the diagnosis of
AIDS by the ELISA test
 Helps in critical diagnostic decisions

 Once monoclonal antibodies for a

given substance have been produced,
they can be used to detect the
presence of this substance

 The Western blot test and Immuno

dot blot tests detect the protein on a
membrane

 Useful in immunohistochemistry,
which detect antigen in fixed tissue
sections and

 Immunofluorescence test, which

detect the substance in a frozen tissue
section or in live cells.
 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.
 Diagnosis of HIV infection

The test of HIV infection is based on

detecting the presence of HIV antibody in
the patient’s blood serum.

a) HIV antigen is attached 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.
 FAD Currently Approved Therapeutic Monoclonal Antibodies
Indication (Targeted Antibody Antibody Type Target Approved
Disease) Name

C Colorectal Cetuximab Chimeric epidermal 2004

A Cancer Bevacizumab Humanized growth
N factor
C receptor
E Non-Hodgkin’s Tositumomab Murine CD20 2003
R Lymphoma Rituximab Chimeric 1997
T
H
Metastatic Breast Cancer Trastuzumab Humanized ErbB2 1998
E
R
A Acute Myeloid Leukemia Gemtuzumab Humanized CD33 2000
P
E
Y Chronic Lymphocytic Alemtuzumab Humanized CD52 2001
Leukemia
 CARDIO VASCULAR DISORDERS
Indication Antibody Antibody Target Approved
(Targeted Name Type
Disease)

Coronary Abcixima chimeric inhibition ofglycoproteinIIb/IIIa 1994

intervention b platelets

Inflammatory (Auto immune Diseases)

Indication Antibody Antibody Type Target Approve
(Targeted Name d
Disease
Allergic Asthma Omalizumab Humanized immunoglobulin 2002
E (IgE
Crohn’s Disease Infliximab Chimeric Inhibition of TNF-α 1998
signaling
Rheumatoid Adalimumab Fully Human Inhibition of TNF-α 2002
Arthritis signaling
 Infectious Diseases
Indication (Targeted Antibody Name Antibody Type Target Approved
Disease)

Bronchial Asthma Omalizumab immunoglobulin 2004

Humanized E (IgE)
Respiratory Syncytial Palivizumab Humanized An epitope of 1998
Virus the RSV F protein

Transplant rejection
Indication (Targeted Antibody Antibody Target Approved
Disease Name Type

Organ transplant Muromonab Murine T cell CD3Receptor 1986

Rejection
Kidney transplant Basiliximab Chimeric IL-2Rα receptor 1998
Rejection Daclizumab Humanized (CD25) 1997
 Clinical Applications of MAbs
Imaging the target organ
 Monoclonal antibodies directed
against tumour-associated antigens
labelled with radioisotopes localize
specifically into tumour after
intravenous injection. This property is
used for diagnostic tumour imaging by
immunoscintigraphy.
 The radio-labeled antibody-isotope
conjugate is injected into the patient
and allowed to localize to the target
over a 2- to 7-day period. The patient
then undergoes imaging with a
nuclear medicine gamma camera, and
radioisotope counts are analyzed.
 used in colorectal & prostate cancer

COLORECTAL CANCER -- RADIO IMAGING

 ADEPT (Antibody Directed Enzyme Pro-drug Therapy)

 Involves the application of

cancer associated
monoclonal antibodies
which are linked to a drug-
activating enzyme
 Subsequent systemic
administration of a non-
toxic agent results in its
conversion to a toxic drug,
and resulting in a cytotoxic
effect which can be
targeted at malignant cells
 Future use in Bioterrorism
Raxibacumab
 It is a human monoclonal
antibody
 antibody against Bacillus
anthracis protective antigen

 Intended for the prophylaxis and

treatment of inhaled anthrax.
 Its efficacy has been proved in
rabbits and monkeys.

 As of January 2011, the drug has

reached stage three clinical testing
in humans

 On December 14th 2012 the FDA

approved Raxibacumab for the
treatment of inhalation anthrax,
 References
 Goodman & Gilman’s The Pharmacological
Basis of Therapeutics 12th edn
 Katzung Basic & clinical Pharmacology 11th
edn
 Harrison’s Internal Medicine 17th edn

 Dipiro’s pathophysiologic Approach 8th edn

 Jancie, M Recheit, etal. Nature

biotechnology, 2005, Sep,Vol. 23,
No.9Stamatis-Nick C. J Allergy Clin.
Immunol, Oct. 2005
 www.wikipedia.com
 www.medscape.com
Acknowledgements

I am very thankful to our

respected principal sir
Dr Amit Chakrabarty,vice
principal mam,all our
respected faculty
members and all my
friend.