2. Hybridoma
technology
Hybridoma technology is a
technology of forming hybrid
cell lines called hybridomas by
fusing an antibody-producing B
cell with a myeloma (B cell
cancer) cell that is selected for its
ability to grow in tissue culture
and for an absence of antibody
chain synthesis
3. The production of monoclonal antibodies was
invented by Cesar Miltein and Georges J.F. Kohler
in 1975.
They shared the Nobel Prize of 1984 for Medicine
and Physiology with Niels kai Jerne, who made
other contributions to immunology.
The term hybridoma was coined by Leonard
Harzenberg during his sabbatical in cesar milteins
's laboratory in 1976–1977
4. PRINCIPLE
Antibodies are produced by B lymphocyte,each B
lymphocyte being pre programmed to respond to a
single antigenic determinant.
.
When an antigen reacts to the cell surface reactor of B
lymphocyte,it proliferates rapidly to yield a population
of B cells ,all of which produces antibodies of same
specificity,this is called as “clonal selection”.
6. Thus a B lymphocyte produces antibodies of only one
specificity. In addition , an antibody producing B
lymphocyte cells called plasma cell is fully differentiated
and does not divide when cultured in vitro,these features
are critical to hybridoma technology.
B lymphocyte are isolated from spleen of an animal eg
mouse wich had been immunized with antigen against
which monoclonal antibodies are to be raised.
Immunization is achived by injecting the antigen
along with the suitable adjuvant either subcutaneously or
in peritoneal cavity followed by the booster doses of
antigen.
7. Steps in production of
monoclonal
antibodies……
1. Immunisation
2. Fusion
3. Culture in hat
10. Fusion…….
Lymphocytes are fused with myeloma cells
Myeloma cells are malignant lymphocytes…….
Cells are fused using PEG(polyethyleneglycol)
to promote membrane fusion…………..
11.
12.
13. Culture in HAT………….
Hence the myeloma cells would not survive in culture…unless this enzyme is added to the media………..
Myeloma cells are engineered to be deficient in an enzyme…hypoxanthine guanine phosphoribosyl transferase(HGPRT)…..
Only hybrid cells survive…….
Spleen are short lived i.e, their life span is 7-10 days,so they die gradually…..
HAT kills myeloma cells…..
The medium in which the culture is done contains….hypoxanthine,aminopterin,thymidine….
15. Non fused, - HGPRT plasma cells and all non fused B cells die
soon…….
Hybridoma cells consisting of immortalized plasma cells fused
with B cells will survive in the absence of supplemented
HGPRT in culture medium……
Fused cells survive ………
The powerful toxins of the media block the metabolic pathway
“Denovo pathway”..essential for purine metabolism…in DNA
synthesis……
The cells are cultured in HAT medium which is lacking the
enzyme HGPRT…
