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HYBRIDOMA TECHNOLOGY
Dr. A. T. Sharma
Assist. Professor
Nanded Pharmacy College, Nanded
Polyclonal & Monoclonal Antibodies
Polyclonal antibodies (Conventional):
• A collection of antibodies from different B
cells that recognize multiple epitopes on the
same antigen.
• Each antibody recognizes a unique epitope
located on an antigen.
• High potential for cross reactivity
• Variability in different batches from different
animals at different times.
Monoclonal antibodies:
• A collection of single type of antibodies recognizing a
specific epitope on an antigen
• Produced from a cell called hybridoma (Hybridoma
Technology)
• Production is continuous and uniform.
Hybridoma Technology
• Discovered in 1975 by Georges Kohler of West
Germany and Cesar Milstein of Argentina.
• Hybridomas are the hybrid cells produced by
fusing antibody forming spleen cells
(lymphocyte) with myeloma cells.
• Antibodies produced from lymphocytes is
homogeneous because they are produced
from the homogeneous clone of a cell.
(Monoclonal or M-protein)
Steps in Hybridoma Technology
(Production of Monoclonal Antibodies)
Immunization:
• A mouse is immunized (Intradermal or subcutaneous
injection) with an appropriate antigen (Antigen
emulsified with Freund’s adjuvant or other adjuvants)
for every 2-3 weeks (50µg of antigen/mouse/injection)
• Measurement of serum antibody titer by ELISA and
Flow cytometry
• After sufficient antibody titer, mice is sacrificed and
spleen is removed to use as a source of lymphocytes.
• Spleen dissociated in to large number of spleenocytes
by mechanical or enzymatic method.
Preparation of hybridoma cells:
• Spleenocytes are fused in-vitro with lymphocytic tumor
cells
• Fusing agent: A defective virus (sendai virus), chemicals
(PEG), or electrofusion
• PEG – 50% solution of PEG added to a cell pellet containing
spleen cells and myeloma cells in equal proportion –
incubated at 37⁰C for I hour
• Selection of hybrid cells by using HAT medium
(Hypoxanthine – Aminopterin – Thymidine medium)
• Spleenocytes – finite life
• Myleoma cells – Can not synthesize hypoxanthine guanine
phosphoribosyl transferase (HGPRT) from hypoxanthine in
medium.
• HGPRT is necessary for nucleotide synthesis
• Hence, only hybridomas can survive
• Hybridomas grown in HAT medium for 7-10 days
• Hybridomas are isolated, diluted in a solution and
grown individually
Screening the Products:
• Hybridomas screened for secretion of antibody of
desired specificity by ELISA and RIA
• A specific antigen coated to plastic plate is used,
only bound antibodies (monoclonal antibodies)
are retained, while unbound antibodies and
media components are washed off.
Cloning and Propagation:
• Selected hybrid cells are isolated and cloned by following methods-
- Limiting dilution method (Hybridoma suspension diluted and
transferred to micro culture wells to get single cell in a well.
- Soft agar method (Hybridoma cells cultured in soft agar to get
colonies from a single cell)
Production of Monoclonal Antibodies:
• In vitro tissue culture technique
-Hybridoma cells grown in culture medium containing
bovine serum
-Low yield
• In vivo animal method
- Hybridoma cells grown in ascitic fluid in peritoneal cavity of mouse
- High yield (20mg of Mab/ml)
- Drawbacks: Heavy risk of contamination of animal pathogens
Large number of animals have to be sacrificed
Purification of Monoclonal Antibodies:
• Product should be suitable for human being
• Impurities like host cell protein, DNA, viruses, endotoxins,
aggregates etc. are removed.
• Impurities introduced during purification like leached
protein A, extractables from resins and filters, buffers,
detergents etc. are to be removed
Characterization and Storage:
• Subjected for biochemical and biophysical characterization
for desired specificity
• Studies related to antibody class and sub-class, epitope
specificity, number of binding sites.
• Stability study of cells and MAb – withstand freezing and
thawing
• Cell lines frozen in liquid nitrogen (-196⁰C) during cloning
and culture
Production of Monoclonal Antibodies
Applications
MAb in biochemical analysis:
• MAb as a reagent in RIA and ELISA for detecting -
- Serum concentrations of hormones like insulin, growth
hormone, progesterone, thyroxine, triiodothyronine, TSH etc.
- Tissue and cell products like blood group antigens, clotting
factors, interferons, interleukins, MCH antigens, tumor
markers etc.
• In diagnostic kits for pregnancy, cancers, hormonal
disorders, infectious diseases
MAb in diagnostic imaging:
- Radiolabelled MAb used in diagnostic imaging of diseases
(Immunoscintigraphy)
E.g. Myocardial infarction, deep vein thrombosis,
atherosclerosis, cancers, bacterial infections
Therapeutic applications:
• Destruction of pathogenic organisms by opsonization and
phagocytosis
• Treatment of cancer by antibody dependent cell mediated
cytotoxicity, complement mediated cytotoxicity and
phagocytosis
• Immunosuppression during organ transplantation to prevent
transplant rejection
• Treatment of AIDS
• Treatment of auto immune disorders like rheumatoid arthritis,
multiple sclerosis
• As a targeting agents for drugs and isotopes
Miscellaneous applications:
• Catalytic MAb (Abzymes) – Antibodies with highly specific
enzymatic activity towards substrate
• Autoantibody fingerprinting – Autoantibodies are used to
detect the individual specific autoantibodies and can be used
in identification of criminals
Monoclonal Antibodies and their Indications
Thank You…!!!

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Production of Monoclonal Antibodies by Hybridoma Technology.pptx

  • 1. HYBRIDOMA TECHNOLOGY Dr. A. T. Sharma Assist. Professor Nanded Pharmacy College, Nanded
  • 2. Polyclonal & Monoclonal Antibodies Polyclonal antibodies (Conventional): • A collection of antibodies from different B cells that recognize multiple epitopes on the same antigen. • Each antibody recognizes a unique epitope located on an antigen. • High potential for cross reactivity • Variability in different batches from different animals at different times.
  • 3. Monoclonal antibodies: • A collection of single type of antibodies recognizing a specific epitope on an antigen • Produced from a cell called hybridoma (Hybridoma Technology) • Production is continuous and uniform.
  • 4.
  • 5.
  • 6. Hybridoma Technology • Discovered in 1975 by Georges Kohler of West Germany and Cesar Milstein of Argentina. • Hybridomas are the hybrid cells produced by fusing antibody forming spleen cells (lymphocyte) with myeloma cells. • Antibodies produced from lymphocytes is homogeneous because they are produced from the homogeneous clone of a cell. (Monoclonal or M-protein)
  • 7. Steps in Hybridoma Technology (Production of Monoclonal Antibodies) Immunization: • A mouse is immunized (Intradermal or subcutaneous injection) with an appropriate antigen (Antigen emulsified with Freund’s adjuvant or other adjuvants) for every 2-3 weeks (50µg of antigen/mouse/injection) • Measurement of serum antibody titer by ELISA and Flow cytometry • After sufficient antibody titer, mice is sacrificed and spleen is removed to use as a source of lymphocytes. • Spleen dissociated in to large number of spleenocytes by mechanical or enzymatic method.
  • 8. Preparation of hybridoma cells: • Spleenocytes are fused in-vitro with lymphocytic tumor cells • Fusing agent: A defective virus (sendai virus), chemicals (PEG), or electrofusion • PEG – 50% solution of PEG added to a cell pellet containing spleen cells and myeloma cells in equal proportion – incubated at 37⁰C for I hour • Selection of hybrid cells by using HAT medium (Hypoxanthine – Aminopterin – Thymidine medium) • Spleenocytes – finite life • Myleoma cells – Can not synthesize hypoxanthine guanine phosphoribosyl transferase (HGPRT) from hypoxanthine in medium. • HGPRT is necessary for nucleotide synthesis • Hence, only hybridomas can survive
  • 9. • Hybridomas grown in HAT medium for 7-10 days • Hybridomas are isolated, diluted in a solution and grown individually Screening the Products: • Hybridomas screened for secretion of antibody of desired specificity by ELISA and RIA • A specific antigen coated to plastic plate is used, only bound antibodies (monoclonal antibodies) are retained, while unbound antibodies and media components are washed off.
  • 10. Cloning and Propagation: • Selected hybrid cells are isolated and cloned by following methods- - Limiting dilution method (Hybridoma suspension diluted and transferred to micro culture wells to get single cell in a well. - Soft agar method (Hybridoma cells cultured in soft agar to get colonies from a single cell) Production of Monoclonal Antibodies: • In vitro tissue culture technique -Hybridoma cells grown in culture medium containing bovine serum -Low yield • In vivo animal method - Hybridoma cells grown in ascitic fluid in peritoneal cavity of mouse - High yield (20mg of Mab/ml) - Drawbacks: Heavy risk of contamination of animal pathogens Large number of animals have to be sacrificed
  • 11. Purification of Monoclonal Antibodies: • Product should be suitable for human being • Impurities like host cell protein, DNA, viruses, endotoxins, aggregates etc. are removed. • Impurities introduced during purification like leached protein A, extractables from resins and filters, buffers, detergents etc. are to be removed Characterization and Storage: • Subjected for biochemical and biophysical characterization for desired specificity • Studies related to antibody class and sub-class, epitope specificity, number of binding sites. • Stability study of cells and MAb – withstand freezing and thawing • Cell lines frozen in liquid nitrogen (-196⁰C) during cloning and culture
  • 13. Applications MAb in biochemical analysis: • MAb as a reagent in RIA and ELISA for detecting - - Serum concentrations of hormones like insulin, growth hormone, progesterone, thyroxine, triiodothyronine, TSH etc. - Tissue and cell products like blood group antigens, clotting factors, interferons, interleukins, MCH antigens, tumor markers etc. • In diagnostic kits for pregnancy, cancers, hormonal disorders, infectious diseases MAb in diagnostic imaging: - Radiolabelled MAb used in diagnostic imaging of diseases (Immunoscintigraphy) E.g. Myocardial infarction, deep vein thrombosis, atherosclerosis, cancers, bacterial infections
  • 14. Therapeutic applications: • Destruction of pathogenic organisms by opsonization and phagocytosis • Treatment of cancer by antibody dependent cell mediated cytotoxicity, complement mediated cytotoxicity and phagocytosis • Immunosuppression during organ transplantation to prevent transplant rejection • Treatment of AIDS • Treatment of auto immune disorders like rheumatoid arthritis, multiple sclerosis • As a targeting agents for drugs and isotopes Miscellaneous applications: • Catalytic MAb (Abzymes) – Antibodies with highly specific enzymatic activity towards substrate • Autoantibody fingerprinting – Autoantibodies are used to detect the individual specific autoantibodies and can be used in identification of criminals
  • 15. Monoclonal Antibodies and their Indications