Hybridoma

technology

A general representation of the hybridoma method

used to produce monoclonal antibodies.
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 antibody
producing B-cells are then harvested from
the mouse and, 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
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.[1]

Method
 

(1) Immunisation of a mouse

(2) Isolation of B cells from the spleen
(3) Cultivation of myeloma cells
(4) Fusion of myeloma and B cells
(5) Separation of cell lines
(6) Screening of suitable cell lines
(7) in vitro (a) or in vivo (b) multiplication
(8) Harvesting
 

Agile Pulse In Vivo Electroporation System for

enhanced vaccine delivery manufactured by BTX
Harvard Apparatus, Holliston MA USA

BTX ECM 2001 Electrofusion generator for hybridoma

production
 

Hybridoma cells grown in tissue culture. The image

shows a single clone of cells each of which is

producing large amounts of a specific monoclonal
antibody which the cells secrete and which can be
readily purified from the culture media.

Laboratory animals (mammals, e.g. mice)

are first exposed to the antigen against
which an antibody is to be generated.
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[2], western blot, and
immunoprecipitation-mass spectrometry
screening can also be used. Unlike
western blot assays, immunoprecipitation-
mass spectrometry facilitates screening
and ranking of clones which bind to the
native (non-denaturated) forms of antigen
proteins.[3]

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.[2]

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.[2]

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.[2]
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.[2]

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.[2]

One study[4] 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.[5] 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.[6]

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.[7] 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[8] 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[9] 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.[10]

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.[11]

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.
References
1. Milstein, C (1999). "The hybridoma
revolution: an offshoot of basic
research". BioEssays. 21 (11): 966–73.
doi:10.1002/(SICI)1521-
1878(199911)21:11<966::AID-
BIES9>3.0.CO;2-Z . PMID 10517870 .
2. Nelson, PN; Reynolds, GM; Waldron,
EE; Ward, E; Giannopoulos, K; Murray,
PG (2000). "Demystified ...:
Monoclonal antibodies" . Molecular
Pathology. 53 (3): 111–7.
doi:10.1136/mp.53.3.111 .
PMC 1186915 . PMID 10897328 .
3. Korbakis, D; Brinc, D; Schiza, C;
Soosaipillai, A; Jarvi, K; Drabovich, AP;
Diamandis, E (2015). "Immunocapture-
selected reaction monitoring
screening facilitates the development
of ELISA for the measurement of
native TEX101 in biological fluids" .
Molecular & Cellular Proteomics. 14
(6): 1517–1526.
doi:10.1074/mcp.M114.047571 .
PMC 4458717 . PMID 25813379 .
4. Bretton, PR; Melamed, MR; Fair, WR;
Cote, RJ (1994). "Detection of occult
micrometastases in the bone marrow
of patients with prostate carcinoma".
Prostate. 25 (2): 108–14.
 doi:10.1002/pros.2990250208 .
PMID 7518596 .
5. Kvalheim, G (1996). "Detection of
occult tumor cells in bone marrow and
blood in breast cancer patients—
methods and clinical significance" .
Acta Oncol. 35: 13–18.
doi:10.3109/02841869609098516 .
PMID 9073044 .
. Kvalheim, G (1998). "Diagnosis of
minimal residual disease in bone
marrow and blood in cancer patients--
methods and clinical implications".
Acta Oncologica. 37 (5): 455–62.
 doi:10.1080/028418698430403 .
PMID 9831374 .
7. Franklin, WA; Shpall, EJ; Archer, P;
Johnston, CS; Garza-Williams, S; Hami,
L; Bitter, MA; Bast, RC; Jones, RB
(1996). "Immunocytochemical
detection of breast cancer cells in
marrow and peripheral blood of
patients undergoing high dose
chemotherapy with autologous stem
cell support". Breast Cancer Res Treat.
41 (1): 1–13.
doi:10.1007/BF01807031 .
PMID 8932871 .
 . Riesenberg, R; Oberneder, R; Kriegmair,
M; Epp, M; Bitzer, U; Hofstetter, A;
Braun, S; Riethmüller, G; Pantel, K
(1993). "Immunocytochemical double
staining of cytokeratin and prostate
specific antigen in individual prostatic
tumour cells". Histochemistry. 99 (1):
61–6. doi:10.1007/BF00268022 .
PMID 7682210 .
9. Ghosh, AK; Mason, D Y; Spriggs, A I
(1983). "Immunocytochemical staining
with monoclonal antibodies in
cytologically "negative" serous
effusions from patients with malignant
disease" . J Clin Pathol. 36 (10):
1150–53.
 doi:10.1136/jcp.36.10.1150 .
PMC 498493 . PMID 6194182 .
10. Ghosh, AK; Spriggs, AI; Taylor-
Papadimitriou, J; Mason, DY (1983).
"Immunocytochemical staining of cells
in pleural and peritoneal effusions with
a panel of monoclonal antibodies" . J
Clin Pathol. 36 (10): 1154–64.
doi:10.1136/jcp.36.10.1154 .
PMC 498494 . PMID 6194183 .
11. Vissers, CJ; Flohil, CC; De Jong, AA;
Dinjens, WN; Bosman, FT (1996). "A
new monoclonal antibody for specific
immunocytochemical staining of
nucleoli". Acta histochemica. 98 (2):
 113–21. doi:10.1016/S0065-
1281(96)80028-6 . PMID 8739296 .

External links
Hybridomas at the US National Library
of Medicine Medical Subject Headings
(MeSH)
"Hybridoma Technology" .
Understanding Cancer Series: The
Immune System. National Cancer
Institute. Archived from the original on
5 October 2014.
"Hybridoma Cell Culture" .
Retrieved from
"https://en.wikipedia.org/w/index.php?
title=Hybridoma_technology&oldid=951620958"

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