•Most antigens offer multiple epitopesand
therefore induce proliferation and differentiation of a variety of B-cell
clones, each derived from a B cell that recognizes a particular epitope. The
resulting serum antibodies are heterogeneous, comprising a mixture of
antibodies, each specific for one epitope. Such a polyclonal antibody has clear
advantages for the organism in vivo.
•the antibody heterogeneity reduces the
efficacy of an antiserum for various in vitro uses.
For most research, diagnostic, and therapeutic purposes, monoclonal antibodies are preferable.
For most research, diagnostic, and therapeutic purposes, monoclonal antibodies are preferable.
•Monoclonal antibodies:antibodies derived from
a single clone and thus specific for a single epitope.
Principle
•Fusion of lymphocytes cells from an
immunized animal with cells from cultured myelomacell (cancer cells) line to
produce hyberidoma.
Procedure
•Animals are immunized with antigen.
•Spleen is removed and cell suspension is
prepared.
•Myelomacells (have metabolic defect) are
selected and prepared in cell line.
•Fusion is carried out using Polyethylene
glycol to promote fusion.
•Small portion fuse successfully to form
hyberidoma.
•Selection of the hyberidomais done using
culture media: HAT medium (hypoxanthin, aminopterin, thymidine).
•Aminopterinis a powerful toxin which block
certain metabolic pathway.
This metabolic pathway can be by-passed if the cell is provided with hypoxanthinand thymidine.
This metabolic pathway can be by-passed if the cell is provided with hypoxanthinand thymidine.
•Spleen cells (unfusedto myelomacells, may
fuse to each other) can use this by-pass pathway, so HAT is not toxic to them
but due to their normal short life span they die.
•Myeloma cells (unfused with spleen cells, may
fuse to each other) can’t use this by-pass, so they die in HAT medium.
•Only fused cells: hybridoma survive because
they have the bypass of spleen cells and the immortality of myeloma cells and
they are antibody producers.
•Culture is distributed in wells.
•Wells showing growing cells are tested for
the production of the desired specific antibody by gel electrophoresis.
•Positive cultures are serially diluted to
obtain single cell in each well.
•Each cell will produce a clone of specific
antibody derived from a single progenitor: Monoclonal antibody.
•Cloning may be propagated in vitro in cell
line or in vivo in mouse.
Genetic engineering
-Genetic engineering= Recombinant DNA technology
= genetic modification / manipulation (GM) and gene splicing are terms that
apply to the direct manipulation of an organism's genes.
-There are a number of
ways through which genetic engineering is accomplished. Essentially, the
process has five main steps.
1.Isolation of the genes of interest.
2.Insertion of the genes into a transfer vector.
3.Transfer of the vector to the organism to be modified.
4.Transformation of the cells of the organism.
5.Selection of the genetically modified organism (GMO) from those that have not been successfully modified.
1.Isolation of the genes of interest.
2.Insertion of the genes into a transfer vector.
3.Transfer of the vector to the organism to be modified.
4.Transformation of the cells of the organism.
5.Selection of the genetically modified organism (GMO) from those that have not been successfully modified.
Genetic engineering
Impact of genetics on fermentation technology.
-The main impact of classic genetics on biotechnology was the improvement of the industrial strains which resulted in increased yield and reduction in costs.
-In genetic engineer the genes could move from one cell type to another e.g. from plants and mammals to bacteria.
-The future of genetic engineering is considered almost unlimited in its commercial applications.
-The main impact of classic genetics on biotechnology was the improvement of the industrial strains which resulted in increased yield and reduction in costs.
-In genetic engineer the genes could move from one cell type to another e.g. from plants and mammals to bacteria.
-The future of genetic engineering is considered almost unlimited in its commercial applications.