Hybridoma technology is a method for producing
large numbers of identical antibodies (also
called monoclonal antibodies). This process
starts by injecting a mouse (or other mammal)
with an antigen that provokes an immune response.
A type of white blood cell, the B cell, produces
antibodies that bind to the injected antigen.
These newly produced antibodies are then harvested
from the mouse. These isolated B cells are
in turn fused with immortal B cell cancer
cells, a myeloma, to produce a hybrid cell
line called a hybridoma, which has both the
antibody-producing ability of the B-cell and
the exaggerated longevity and reproductivity
of the myeloma. The hybridomas can be grown
in culture, each culture starting with one
viable hybridoma cell, producing cultures
each of which consists of genetically identical
hybridomas which produce one antibody per
culture (monoclonal) rather than mixtures
of different antibodies (polyclonal). The
myeloma cell line that is used in this process
is selected for its ability to grow in tissue
culture and for an absence of antibody synthesis.
In contrast to polyclonal antibodies, which
are mixtures of many different antibody molecules,
the monoclonal antibodies produced by each
hybridoma line are all chemically identical.
The production of monoclonal antibodies was
invented by César Milstein and Georges J.
F. Köhler in 1975. They shared the Nobel
Prize of 1984 for Medicine and Physiology
with Niels Kaj Jerne, who made other contributions
to immunology. The term hybridoma was coined
by Leonard Herzenberg during his sabbatical
in César Milstein's laboratory in 1976–1977.
== Method ==
Laboratory animals (mammals, e.g. mice) are
first exposed to the antigen that an antibody
is to be generated against. Usually this is
done by a series of injections of the antigen
in question, over the course of several weeks.
These injections are typically followed by
the use of in vivo electroporation, which
significantly enhances the immune response.
Once splenocytes are isolated from the mammal's
spleen, the B cells are fused with immortalised
myeloma cells. The fusion of the B cells with
myeloma cells can be done using electrofusion.
Electrofusion causes the B cells and myeloma
cells to align and fuse with the application
of an electric field. Alternatively, the B-cells
and myelomas can be made to fuse by chemical
protocols, most often using polyethylene glycol.
The myeloma cells are selected beforehand
to ensure they are not secreting antibody
themselves and that they lack the hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) gene, making
them sensitive to the HAT medium (see below).
Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine
medium) for roughly 10 to 14 days. Aminopterin
blocks the pathway that allows for nucleotide
synthesis. Hence, unfused myeloma cells die,
as they cannot produce nucleotides by the
de novo or salvage pathways because they lack
HGPRT. Removal of the unfused myeloma cells
is necessary because they have the potential
to outgrow other cells, especially weakly
established hybridomas. Unfused B cells die
as they have a short life span. In this way,
only the B cell-myeloma hybrids survive, since
the HGPRT gene coming from the B cells is
functional. These cells produce antibodies
(a property of B cells) and are immortal (a
property of myeloma cells). The incubated
medium is then diluted into multi-well plates
to such an extent that each well contains
only one cell. Since the antibodies in a well
are produced by the same B cell, they will
be directed towards the same epitope, and
are thus monoclonal antibodies.
The next stage is a rapid primary screening
process, which identifies and selects only
those hybridomas that produce antibodies of
appropriate specificity. The first screening
technique used is called ELISA. The hybridoma
culture supernatant, secondary enzyme labeled
conjugate, and chromogenic substrate, are
then incubated, and the formation of a colored
product indicates a positive hybridoma. Alternatively,
immunocytochemical screening can also be used.The
B cell that produces the desired antibodies
can be cloned to produce many identical daughter
clones. Supplemental media containing interleukin-6
(such as briclone) are essential for this
step. Once a hybridoma colony is established,
it will continually grow in culture medium
like RPMI-1640 (with antibiotics and fetal
bovine serum) and produce antibodies.Multiwell
plates are used initially to grow the hybridomas,
and after selection, are changed to larger
tissue culture flasks. This maintains the
well-being of the hybridomas and provides
enough cells for cryopreservation and supernatant
for subsequent investigations. The culture
supernatant can yield 1 to 60 µg/ml of monoclonal
antibody, which is maintained at -20 °C or
lower until required.By using culture supernatant
or a purified immunoglobulin preparation,
further analysis of a potential monoclonal
antibody producing hybridoma can be made in
terms of reactivity, specificity, and cross-reactivity.
== Applications ==
The use of monoclonal antibodies is numerous
and includes the prevention, diagnosis, and
treatment of disease. For example, monoclonal
antibodies can distinguish subsets of B cells
and T cells, which is helpful in identifying
different types of leukaemias. In addition,
specific monoclonal antibodies have been used
to define cell surface markers on white blood
cells and other cell types. This led to the
cluster of differentiation series of markers.
These are often referred to as CD markers
and define several hundred different cell
surface components of cells, each specified
by binding of a particular monoclonal antibody.
Such antibodies are extremely useful for fluorescence-activated
cell sorting, the specific isolation of particular
types of cells.
=== In diagnostic histopathology ===
With the help of monoclonal antibodies, tissues
and organs can be classified based on their
expression of certain defined markers, which
reflect tissue or cellular genesis. Prostate
specific antigen, placental alkaline phosphatase,
human chorionic gonadotrophin, α-fetoprotein
and others are organ-associated antigens and
the production of monoclonal antibodies against
these antigens helps in determining the nature
of a primary tumor.Monoclonal antibodies are
especially useful in distinguishing morphologically
similar lesions, like pleural and peritoneal
mesothelioma, adenocarcinoma, and in the determination
of the organ or tissue origin of undifferentiated
metastases. Selected monoclonal antibodies
help in the detection of occult metastases
(cancer of unknown primary origin) by immuno-cytological
analysis of bone marrow, other tissue aspirates,
as well as lymph nodes and other tissues and
can have increased sensitivity over normal
histopathological staining.One study performed
a sensitive immuno-histochemical assay on
bone marrow aspirates of 20 patients with
localized prostate cancer. Three monoclonal
antibodies (T16, C26, and AE-1), capable of
recognizing membrane and cytoskeletal antigens
expressed by epithelial cells to detect tumour
cells, were used in the assay. Bone marrow
aspirates of 22% of patients with localized
prostate cancer (stage B, 0/5; Stage C, 2/4),
and 36% patients with metastatic prostate
cancer (Stage D1, 0/7 patients; Stage D2,
4/4 patients) had antigen-positive cells in
their bone marrow. It was concluded that immuno-histochemical
staining of bone marrow aspirates are very
useful to detect occult bone marrow metastases
in patients with apparently localized prostate
cancer.
Although immuno-cytochemistry using tumor-associated
monoclonal antibodies has led to an improved
ability to detect occult breast cancer cells
in bone marrow aspirates and peripheral blood,
further development of this method is necessary
before it can be used routinely. One major
drawback of immuno-cytochemistry is that only
tumor-associated and not tumor-specific monoclonal
antibodies are used, and as a result, some
cross-reaction with normal cells can occur.In
order to effectively stage breast cancer and
assess the efficacy of purging regimens prior
to autologous stem cell infusion, it is important
to detect even small quantities of breast
cancer cells. Immuno-histochemical methods
are ideal for this purpose because they are
simple, sensitive, and quite specific. Franklin
et al. performed a sensitive immuno-cytochemical
assay by using a combination of four monoclonal
antibodies (260F9, 520C9, 317G5 and BrE-3)
against tumor cell surface glycoproteins to
identify breast tumour cells in bone marrow
and peripheral blood. They concluded from
the results that immuno-cytochemical staining
of bone marrow and peripheral blood is a sensitive
and simple way to detect and quantify breast
cancer cells.
One of the main reasons for metastatic relapse
in patients with solid tumours is the early
dissemination of malignant cells. The use
of monoclonal antibodies (mAbs) specific for
cytokeratins can identify disseminated individual
epithelial tumor cells in the bone marrow.
One study
reports on having developed an immuno-cytochemical
procedure for simultaneous labeling of cytokeratin
component no. 18 (CK18) and prostate specific
antigen (PSA). This would help in the further
characterization of disseminated individual
epithelial tumor cells in patients with prostate
cancer. The twelve control aspirates from
patients with benign prostatic hypertrophy
showed negative staining, which further supports
the specificity of CK18 in detecting epithelial
tumour cells in bone marrow.
In most cases of malignant disease complicated
by effusion, neoplastic cells can be easily
recognized. However, in some cases, malignant
cells are not so easily seen or their presence
is too doubtful to call it a positive report.
The use of immuno-cytochemical techniques
increases diagnostic accuracy in these cases.
Ghosh, Mason and Spriggs
analysed 53 samples of pleural or peritoneal
fluid from 41 patients with malignant disease.
Conventional cytological examination had not
revealed any neoplastic cells. Three monoclonal
antibodies (anti-CEA, Ca 1 and HMFG-2) were
used to search for malignant cells. Immunocytochemical
labelling was performed on unstained smears,
which had been stored at -20 °C up to 18
months. Twelve of the forty-one cases in which
immuno-cytochemical staining was performed,
revealed malignant cells. The result represented
an increase in diagnostic accuracy of approximately
20%. The study concluded that in patients
with suspected malignant disease, immuno-cytochemical
labeling should be used routinely in the examination
of cytologically negative samples and has
important implications with respect to patient
management.
Another application of immuno-cytochemical
staining is for the detection of two antigens
in the same smear. Double staining with light
chain antibodies and with T and B cell markers
can indicate the neoplastic origin of a lymphoma.One
study has reported the isolation of a hybridoma
cell line (clone 1E10), which produces a monoclonal
antibody (IgM, k isotype). This monoclonal
antibody shows specific immuno-cytochemical
staining of nucleoli.Tissues and tumours can
be classified based on their expression of
certain markers, with the help of monoclonal
antibodies. They help in distinguishing morphologically
similar lesions and in determining the organ
or tissue origin of undifferentiated metastases.
Immuno-cytological analysis of bone marrow,
tissue aspirates, lymph nodes etc. with selected
monoclonal antibodies help in the detection
of occult metastases. Monoclonal antibodies
increase the sensitivity in detecting even
small quantities of invasive or metastatic
cells. Monoclonal antibodies (mAbs) specific
for cytokeratins can detect disseminated individual
epithelial tumour cells in the bone marrow
