3. Introduction
Monoclonal antibodies has been extensively
used from its discovery
These monoclonal antibodies are used in the
field of medicine for therapeutic applications
The research in the field of immunology has
flourished to produce human antibodies from
transgenic animals
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4. CELL CULTURE
Itis defined as the culturing up
of individual cells or by
disaggregated cell mass, either
totipotent or pleuripotent, in a
suitable culture medium
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5. THREE ROUTES FOR DESTINATION
ANIMALCELL CULTURE
TRANSGENIC ANIMALS
IMMUNOLOGY OF MAbs
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6. HISTORY
Alexis carrel(1912)-chick embryo tissue
extract increases cell proliferation in vitro
Fibrin clot plasma used as solid support
Ross harrison(1907)cultured embryonic
nerve cell of frog
Using Hanging drop method
Carcinoma cell line HE LA, myeloma cells
identified
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ANIMALS
7. Contd….
1966 Alec Issacs discovered filtrates have
secretions called interferon
Chinese hamster ovary cells were cultured
High density culturing methods were found
Stem cell technology was discovered
1996 Dolly was produce by egg enucleation
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ANIMALS
8. CHARACTERISTICS OF ANIMAL
CELL
Grow only to a limited number of
generations
Neurons cannot divide to grow
Skin cells grow only for a few generation
Process of contact inhibition
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9. SUBSTRATES
Plastics-polystyrenes, PTTES, TPS,
PVC
Glass, methyl celluloses
Metallic surface
Adhesives-d lysine, gelatin,
Feeder layer for glial cells
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ANIMALS
10. MEDIA
pH ,osmotic pressure, gas phases
NATURAL MEDIA
Plasma clots
Biological fluids
SYNTHETIC MEDIA
Serum containing
Serum free
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11. COMPOSITION
Serum
Proteins
Carbohydrates
Co factors
Examples-MEM,CMRL media
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12. CELL LINES
Finite cell lines-Epidermal
cells,
Infinite cell lines- Cancerous
cells
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13. MODEL ORGANISM
MICE
-short oestrous cycle, and gestation period
-relatively short generation time
-several litters per pregnancy
-Convenient in vitro fertilization
-successful in vitro embryo culture
-production and maintenance of ES lines
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15. Transgenic animals
Method of introducing transgene
Transfection
Transient-transgene gradually lost
from daughter cells
Stable-retained and expressed in all
the cells derived from transfected
cells
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18. Gardener et al., 2003
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19. VECTORS
Simian virus 40
Bovine papilloma virus with transforming
region of pBR322
Polyoma virus
Vaccinia virus
P element vectors
Baculovirus vectors
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20. Gene construct
Promoter/enhancer element
RSV, SV40, β-casein
Initiation codon, Gene of interest
Reporter gene
scorable marker β- galactosidase, lux
selectable marker-thymidine kinase
Termination signal, Poly A tail
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34. MONOCLONAL ANTIBODY
Derived from single cell line have identical
structure binds same epitope,
Specific antigen is repeatedly injected and
and spleen cells are extracted to obtain
MAbs
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35. Applications
Highly sensitive (magic bullets) epitopes
helps in in vitro
Immuno diagnostics (detection and
quantification)
Used in treatment of cancer, delivers drugs
specifically to target tumor cells.
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36. Negative effects
Antibodies recognized as murine
antibodies and mounts immune response
and eliminates from the body.
Causes serious allergenicity to the level
of lethality.
Repeated use of mouse antibodies
induces host to produce antibodies that
neutralizes mouse antibody called HAMA
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37. Chimerised & human antibodies
Variable region is obtained from non
human rat antibody
Constant region is obtained from human
sequence
Chimerics exhibit lower immunogenicity to
the host
Still the chimeric sequence requires
changes this is done by r-DNA by
engineering specific amino acid sequence
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38. Contd..
Humanised to make more compatible
Utilizes recombinant DNA technology
Epitopes are substituted with human gene
sequences
Constant regions are derived from human
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39. Human monoclonal antibodies
from SCID mouse by injecting human B
and T cells and grafting mouse by
transplanting spleens and lymph nodes
The transplanted mouse is immunized
with target antigens to produce human
antibodies
Phage derived combinatorial antibody
library, phage display technique-mimics in
vitro affinity maturation
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40. Hybridoma technology
First discovered by Milstein and Kohler 1975
Specific antigen is given repeatedly to the host
The spleen producing B cells are isolated
B cells are fused with myeloma cells that are
immortal and this is called hybridoma cells
This produces specific antibody indefinitely
precisely can be isolated easily
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42. CHARACTERISTICS
POLYCLONAL ANTIBODY MONOCLONAL ANTIBODY
Contains many Contains single specific
antigenic determinants determinant
Produces various Single class of antibody
classes is produced
Reproducibility and Highly reproducible
standardization is
difficult
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