human antibodies from transgenic animals
TRANSCRIPT
CENTRE FOR PLANT MOLECULAR BIOLOGY
HUMAN ANTIBODIES FROM TRANSGENIC
ANIMALS
S. Kumarakurubaran,
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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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CELL CULTURE
It is 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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THREE ROUTES FOR DESTINATION
ANIMAL CELL CULTURETRANSGENIC ANIMALS IMMUNOLOGY OF MAbs
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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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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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CHARACTERISTICS OF ANIMAL CELL
Grow only to a limited number of generations
Neurons cannot divide to grow Skin cells grow only for a few generationProcess of contact inhibition
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SUBSTRATES
Plastics-polystyrenes, PTTES, TPS, PVC
Glass, methyl cellulosesMetallic surfaceAdhesives-d lysine, gelatin,Feeder layer for glial cells
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MEDIApH ,osmotic pressure, gas phasesNATURAL MEDIANATURAL MEDIAPlasma clotsBiological fluids
SYNTHETIC MEDIASYNTHETIC MEDIASerum containingSerum free
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COMPOSITION
SerumProteinsCarbohydratesCo factorsExamples-MEM,CMRL media
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CELL LINES
Finite cell lines-Epidermal cells,
Infinite cell lines- Cancerous cells
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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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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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Transfection methods
Calcium phosphate precipitation DEAE-dextran mediated transfection Lipofection Retroviral vector infection Microinjection Embryonic stem cell transfer
Mark.M.Davis (2004)
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Gardener et al., 2003
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Gardener et al., 2003
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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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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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Milstein and Kohler 1975
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HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 31Nil Lonberg (2005)
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Nil Lonberg (2005
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Nil Lonberg (2005)
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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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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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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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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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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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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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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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William Fodor 2004
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CHARACTERISTICS
POLYCLONAL ANTIBODY
Contains many antigenic determinants
Produces various classes
Reproducibility and standardization is difficult
MONOCLONAL ANTIBODY
Contains single specific determinant
Single class of antibody is produced
Highly reproducible
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CONCLUSION
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