The presentation includes the information about the production of monoclonal antibodies by hybridoma technology. The slides focus on the points like monoclonal and polyclonal antibodies, steps involved in hybridoma technology and its analytical, diagnostic, therapeutic and some miscellaneous applications. It also includes some marketed products of monoclonal antibodies.
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
