6 PROLIFERATION(^4) FUSION (with PEG)
5 SELECTION ON HAT MEDIUM
Hybridoma Technology
BIO-GRAM
Kohler and Milstein in 1975, devised a technique for preparing monoclonal antibody, since direct biochemical
purification of monoclonal antibodies from polyclonal antibodies is not feasible. This technique is called
Hybridoma Technology. The process involves the fusion of a normal activated antibody producing B cell
with a myeloma i.e., cancerous plasma cell to produce a hybrid cell called Hybridoma. Hence, named so.
3 CLONAL SELECTION
When an antigen reacts to
the cell surface receptor of a B
lymphocyte, it proliferates rapidly
to yield a population or clone of
B cells producing antibodies of
the same specificity. Antibodies
of only desired specificity are
selected. They are specific to only
one antigenic determinant. One
such plasma or B cell for each is
selected.
Normal plasma cells
(Capable of producing specific
antibodies and HGPRT+)
Antibodies
Myeloma cells
Abnormal plasma cells, capable of continous
growth but cannot produce antibodies and
are HGPRT– (i.e., lacks hypoxanthine-guanine
phosphoribosyl transferase enzyme).
The plasma cells and
myeloma cells are mixed
together. Some of the plasma
cells and myeloma cells fuse
to form hybrid cells which
are now capable of growing
continuously in culture while
producing antibodies.
Fused hybrid
cells
Unfused
myeloma cells
Unfused
plasma cells
Suspension of cells
The mixture of cells is transferred and incubated in a selective
HAT medium that allows only hybrid cells to grow.
It contains hypoxanthine, aminopterin and thymidine.
Hybrid cells
(HGPRT+) survives
Produce antibodies
and grows
successfully
(immortally) on
HAT medium.
Myeloma cells (HGPRT–)
Cannot survive in HAT
medium since the drug
aminopterin blocks the
cellular biosynthesis of
purines and pyrimidines
by de novo pathway.
Unfused
plasma (B) cells
(HGPRT+)
Cannot survive
for long in tissue
culture and
eventually dies.
Hybrid cells proliferate into
clones called hybridomas.
They are screened for
production of the desired
antibody.
Possess the immortal growth properties of myeloma
cell as well as the antibody producing properties of
B cell. Thus, the resulting hybrid cell secrete large
quantities of monoclonal antibodies that can be
cultured indefinitely.
Hybridomas
Monoclonal antibodies
(Are produced by a single clone of
cells, therefore MAb is a single pure
type of antibody. They can be made in
large quantities in the laboratory and
are increasingly coming into use as
therapeutic agents.)
Ab-4
3 4
Ab-3
2
Ab-1 Ab-2
Clones
1
Hybridomas
7 SCREENING AND PRODUCTION
The selected
hybridomas are
then cultured
to produce
large amounts
of monoclonal
antibodies.
Ab-1
Ab-2
Ab-3
Ab-4
Polyclonal
antiserum
Ab-4
Ab-3
Ab-1
Ab-2
Polyclonal
antiserum,
containing
a mixture of
monoclonal
antibodies
each specific
for different
epitope of
an antigen is
isolated.
2 ISOLATION OF SERUM AND SPLEEN CELLS
Spleen cells
are isolated
which
comprises of
B cells, each
producing
specific
antibodies.
Plasma
cells
1
3
2
4
A mouse is
injected with
a specific
antigen
against which
antibodies
are to be
generated.
1 IMMUNISATION
Epitopes
4
2
3
1
Antigen
(A) (B)