Monoclonal antibodies

antibody
An antibody is a protein used by the immune system to identify
and neutralize foreign objects like bacteria and viruses. Each
antibody recognizes a specific antigen unique to its target.

Monoclonal antibodies (mAb) are antibodies that are identical

because they were produced by one type of immune cell, all
clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from

different cell lines.

Isotypes
According to differences in their heavy chain constant
domains, immunoglobulins are grouped into five classes, or
isotypes: IgG, IgA, IgM, IgD, and IgE.
 Monoclonal Antibodies
 Monoclonal antibodies (mAb) are antibodies that are
identical because they were produced by one type of
immune cell, all clones of a single parent cell
 Given (almost) any substance, it is possible to create
monoclonal antibodies that specifically bind to that
substance; they can then serve to detect or purify that
substance
 This has become an important tool in biochemistry,
molecular biology and medicine
 The structure of antibodies

 http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
 Hybridoma technology
 It is technique of producing hybrid cell lines called
“hybridomas” by the fusion of fusing a specific antibody-
producing lymphocyte B cell with a myeloma cell that has
an ability to grow in tissue culture
 Hydridoma produce antibodies produced that have single
specificity and are called monoclonal antibodies.
 This technique was discovered Georges Kohler of West
Germany, Cesal Milstein of Argentina and Niels Jerne of
Denmark in 1975.
 They were awarded Nobel Prize for Physiology and Medicine
the 1984
 Principal
 This is based on fusion between myeoma cell (malignant plasma
cell) and spleen cell from suitable immunised animal.
 Spleen can produce antibody but die in short period under culture
condition
 Myeloma cell are adapted to grow permanently in cell culture
 Myeloma cells cannot synthesize antibodies as they lack HGPRT
gene required for the synthesize the enzyme, hypoxanthine
guanine phosphoribosyl transferase (azaquinine resistant) or
thymidine kinase (bromodeoxy uridine resistant)
 Mutant myeloma cell can not grow in HAT (Hypoxanthine
Aminopterin Thymidine) medium
 Only hybrid cell can grow in HAT medium and produce
monoclonal antibody
 Method
v Immunization of a mouse
v Isolation of B cells from the
spleen
v Cultivation of myeloma cells
v Fusion of myeloma and B
cells
v Separation of cell lines
v Screening of suitable cell
lines
v in vitro (a) or in vivo (b)
multiplication
v Harvesting
Immunization of a mouse
 Immunize a rabbit through repeated injection of a specific
antigen for the production of specific antibody, facilitated due
to proliferation of the desired B cells
Produce tumors in a mouse or a rabbit
 Culture separately the spleen cells that produce specific
antibodies and the myeloma cells that produce tumors
 Fusion
 Fusion of spleen cells to myeloma cells is induced
using polyethylene glycol (PEG), to produce
hybridoma
 Hybridomas are grown in selective hypoxanthine
aminopterin thymidine (HAT) medium.
 HAT medium contains a drug, aminopterin that
blocks one pathway for nucleotide synthesis,
making the cells dependent on another pathway
that needs HGPRT enzyme, which is absent in
myeloma cells.
 Myeloma cells that do not fuse with B cells will die.
B cells that do not fuse will also die because they
lack tumorigenic property of immortal growth.
Selection and Storage

 Select desired hybridoma for cloning and antibody

production
 Prepare single cell colonies that can grow and use
them to screen of antibody producing hybridomas
 Only one in several hundred cell hybrids will
produce antibodies of the desired specificity
 Culture selected hybridoma cells for the
production of monoclonal antibodies in large
quantities
 Hybridoma cells can be frozen for future use or
can be injected in the body of an animal, so that
antibodies will be produced in the body and
recovered later from the body fluid.