Hybridoma technology is a method for producing large numbers of identical
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
(also called
monoclonal antibodies
A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies ca ...
). This process starts by injecting a mouse (or other mammal) with an
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
that provokes an immune response. A type of white blood cell, the
B cell
B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or ...
, 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
Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, anemi ...
, to produce a hybrid
cell line
An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cell ...
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
Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism. This technique is also called micropropagation. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, su ...
and for an absence of antibody synthesis. In contrast to
polyclonal antibodies
Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a ...
, 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
César Milstein, CH, FRS (8 October 1927 – 24 March 2002) was an Argentine biochemist in the field of antibody research. Milstein shared the Nobel Prize in Physiology or Medicine in 1984 with Niels Kaj Jerne and Georges J. F. Köhler for d ...
and
Georges J. F. Köhler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with
Niels Kaj Jerne
Niels Kaj Jerne, FRS (23 December 1911 – 7 October 1994) was a Danish immunologist. He shared the Nobel Prize in Physiology or Medicine in 1984 with Georges J. F. Köhler and César Milstein "for theories concerning the specificity in dev ...
, who made other contributions to immunology. The term hybridoma was coined by
Leonard Herzenberg
Leonard Arthur "Len" Herzenberg (November 5, 1931 – October 27, 2013) was an immunologist, geneticist and professor at Stanford University. His contributions to the development of cell biology made it possible to sort viable cells by their spe ...
during his sabbatical in
César Milstein
César Milstein, CH, FRS (8 October 1927 – 24 March 2002) was an Argentine biochemist in the field of antibody research. Milstein shared the Nobel Prize in Physiology or Medicine in 1984 with Niels Kaj Jerne and Georges J. F. Köhler for d ...
's laboratory in 1976–1977.
Method
![Hybridoma technology](https://upload.wikimedia.org/wikipedia/commons/3/3f/Hybridoma_technology.png)
Laboratory animals (
mammal
Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...
s, 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
Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...
, which significantly enhances the immune response. Once
splenocyte A splenocyte can be any one of the different white blood cell types as long as it is situated in the spleen
The spleen is an organ found in almost all vertebrates. Similar in structure to a large lymph node, it acts primarily as a blood filte ...
s are isolated from the mammal's
spleen
The spleen is an organ found in almost all vertebrates. Similar in structure to a large lymph node, it acts primarily as a blood filter. The word spleen comes . , the B cells are fused with immortalised myeloma cells. The fusion of the B cells with myeloma cells can be done using electrofusion.
Electrofusion
The word electrofusion is also used when fusing cells with electricity.
Electrofusion is a method of joining MDPE, HDPE and other plastic pipes using special fittings that have built-in electric heating elements which are used to weld the joi ...
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
Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
. The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves and that they lack the
hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is an enzyme encoded in humans by the ''HPRT1'' gene.
HGPRT is a transferase that catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. This r ...
(HGPRT) gene, making them sensitive to the
HAT medium
HAT Medium (hypoxanthine-aminopterin-thymidine medium) is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reducta ...
(see below).
Fused cells are incubated in HAT medium (
hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-hydroxypurine. Hyp ...
-
aminopterin
Aminopterin (or 4-aminopteroic acid), the 4-amino derivative of folic acid, is an antineoplastic drug with immunosuppressive properties often used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inh ...
-
thymidine
Thymidine (symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. I ...
medium) for roughly 10 to 14 days.
Aminopterin
Aminopterin (or 4-aminopteroic acid), the 4-amino derivative of folic acid, is an antineoplastic drug with immunosuppressive properties often used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inh ...
blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the ''
de novo'' or
salvage pathway A salvage pathway is a pathway in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to nucleotide salvage in particular, in which nucleotides (purine and py ...
s 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 enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
. 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,
western blot, and immunoprecipitation-mass spectrometry. Unlike western blot assays, immunoprecipitation-mass spectrometry facilitates screening and ranking of clones which bind to the native (non-denaturated) forms of antigen proteins.
Flow cytometry screening has been used for primary screening of a large number (~1000) of hybridoma clones recognizing the native form of the antigen on the cell surface.
In the flow cytometry-based screening, a mixture of antigen-negative cells and antigen-positive cells is used as the antigen to be tested for each hybridoma supernatant sample.
The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing
interleukin-6
Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. In humans, it is encoded by the ''IL6'' gene.
In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth ...
(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
A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell re ...
, which is helpful in identifying different types of
leukaemias. In addition, specific monoclonal antibodies have been used to define
cell surface markers
The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophen ...
on
white blood cells
White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
and other cell types. This led to the
cluster of differentiation
The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophen ...
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
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...
, 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
Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), P-30 antigen, is a glycoprotein enzyme encoded in humans by the ''KLK3'' gene. PSA is a member of the kallikrein-related peptidase family and is secreted b ...
,
placental alkaline phosphatase
Alkaline phosphatase, placental type also known as placental alkaline phosphatase (PLAP) is an allosteric enzyme that in humans is encoded by the ''ALPP'' gene.
Gene
There are at least four distinct but related alkaline phosphatases: intestina ...
,
human chorionic gonadotrophin,
α-fetoprotein
Alpha-fetoprotein (AFP, α-fetoprotein; also sometimes called alpha-1-fetoprotein, alpha-fetoglobulin, or alpha fetal protein) is a protein that in humans is encoded by the ''AFP'' gene. The ''AFP'' gene is located on the ''q'' arm of chromosome 4 ...
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
The pleural cavity, pleural space, or interpleural space is the potential space between the pleurae of the pleural sac that surrounds each lung. A small amount of serous pleural fluid is maintained in the pleural cavity to enable lubrication bet ...
and
peritoneal mesothelioma,
adenocarcinoma
Adenocarcinoma (; plural adenocarcinomas or adenocarcinomata ) (AC) is a type of cancerous tumor that can occur in several parts of the body. It is defined as neoplasia of epithelial tissue that has glandular origin, glandular characteristics, or ...
, and in the determination of the organ or tissue origin of undifferentiated
metastases
Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then, ...
. Selected monoclonal antibodies help in the detection of
occult metastases (
cancer of unknown primary origin
Cancer of unknown primary origin (CUP) is a cancer that is determined to be at the metastatic stage at the time of diagnosis, but a primary tumor cannot be identified. A diagnosis of CUP requires a clinical picture consistent with metastatic dise ...
) 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
Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...
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
Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...
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
Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...
is a sensitive and simple way to detect and quantify breast cancer cells.
One of the main reasons for
metastatic relapse
Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then, ...
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
Prostate-specific antigen (PSA), also known as gamma-seminoprotein or kallikrein-3 (KLK3), P-30 antigen, is a glycoprotein enzyme encoded in humans by the ''KLK3'' gene. PSA is a member of the kallikrein-related peptidase family and is secreted b ...
(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
A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
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.
References
External links
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{{Biotechnology
Cell culture techniques
Immunology