Hybridoma
Encyclopedia
Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific 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. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies
(in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by Cesar 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.
s, e.g. mice) are first exposed to an antigen to which we are interested in isolating an antibody against. Usually this is done by a series of injections of the antigen in question, over the course of several weeks. Once splenocyte
s are isolated from the mammal's spleen
, the B cells are fused with immortalized myeloma cells. 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). The fusion is accomplished using polyethylene glycol
or the Sendai virus
. It is performed by making the cell membrane
s more permeable.
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 pathway
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 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.
, 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 by immuno-cytological analysis of bone marrow, other tissue aspirates, as well as lymph nodes and other tissues.
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.
The detection of small quantities of invasive or metastatic cells by normal histopathological staining with haematoxylin and eosin is not always sensitive. The use of monoclonal antibodies increases the sensitivity to a large extent. For example, the use of monoclonal antibodies to cytokeratin in the investigation of the sentinel axillary lymph node for metastatic breast cancer increases nodal positivity by up to 10%.
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 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.
The use of immuno-cytochemical techniques can help to avoid performing procedures, which are painful, uncomfortable and expensive to the patient. It can also help to speed up the start of appropriate treatment.
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. Immuno-cytochemical staining can also detect the presence of two antigens in the same smear.
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
-producing B cell
B cell
B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...
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. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
(in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by Cesar Milstein
César Milstein
César Milstein FRS was an Argentine biochemist in the field of antibody research. Milstein shared the Nobel Prize in Physiology or Medicine in 1984 with Niels K. Jerne and Georges Köhler.-Biography:...
and Georges J. F. Köhler
Georges J. F. Köhler
-External links:* http://www.nobel.se/medicine/laureates/1984/...
in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with Niels Kaj Jerne
Niels Kaj Jerne
Niels Kaj Jerne, FRS was a Danish immunologist. He was awarded the Nobel Prize in Physiology or Medicine in 1984. The citation read "For theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies"....
, who made other contributions to immunology.
Method
Laboratory animals (mammalMammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
s, e.g. mice) are first exposed to an antigen to which we are interested in isolating an antibody against. Usually this is done by a series of injections of the antigen in question, over the course of several weeks. Once splenocyte
Splenocyte
A splenocyte can be any one of the different white blood cell types as long as it is situated in the spleen or purified from splenic tissue.It sometimes explicitly refers to monocytes or macrophages....
s are isolated from the mammal's spleen
Spleen
The spleen is an organ found in virtually all vertebrate animals with important roles in regard to red blood cells and the immune system. In humans, it is located in the left upper quadrant of the abdomen. It removes old red blood cells and holds a reserve of blood in case of hemorrhagic shock...
, the B cells are fused with immortalized myeloma cells. 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
Hypoxanthine-guanine phosphoribosyltransferase 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 reaction transfers the 5-phosphoribosyl group from...
(HGPRT) gene, making them sensitive to the HAT medium
HAT medium
HAT 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 reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis...
(see below). The fusion is accomplished using polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
or the Sendai virus
Sendai virus
Sendai virus , also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan , is a negative sense, single-stranded RNA virus of the Paramyxoviridae family, a group of viruses featuring, notably, the Morbillivirus and Rubulavirus genera...
. It is performed by making the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
s more permeable.
Fused cells are incubated in HAT medium (hypoxanthine
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. Hypoxanthine is a necessary additive in certain cell,...
-aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
-thymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...
medium) for roughly 10 to 14 days. Aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the de novo
De novo
In general usage, de novo is a Latin expression meaning "from the beginning," "afresh," "anew," "beginning again." It is used in:* De novo transcriptome assembly, the method of creating a transcriptome without a reference genome...
or salvage pathway
Salvage pathway
A salvage pathway is a pathway in which nucleotides are synthesized from intermediates in the degradative pathway for nucleotides....
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 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 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 antigenProstate specific antigen
Prostate-specific antigen also known as gamma-seminoprotein or kallikrein-3 is a glycoprotein that in humans is encoded by the KLK3 gene. KLK3 is a member of the kallikrein-related peptidase family that are secreted by the epithelial cells of the prostate gland...
, 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
Peritoneal mesothelioma
Peritoneal mesothelioma is the name given to the cancer that attacks the lining of the abdomen. This type of cancer affects the lining that protects the contents of the abdomen and which also provides a lubricating fluid to enable the organs to move and work properly.The peritoneum is made of two...
, adenocarcinoma
Adenocarcinoma
Adenocarcinoma is a cancer of an epithelium that originates in glandular tissue. Epithelial tissue includes, but is not limited to, the surface layer of skin, glands and a variety of other tissue that lines the cavities and organs of the body. Epithelium can be derived embryologically from...
, and in the determination of the organ or tissue origin of undifferentiated metastases. Selected monoclonal antibodies help in the detection of occult metastases by immuno-cytological analysis of bone marrow, other tissue aspirates, as well as lymph nodes and other tissues.
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.
The detection of small quantities of invasive or metastatic cells by normal histopathological staining with haematoxylin and eosin is not always sensitive. The use of monoclonal antibodies increases the sensitivity to a large extent. For example, the use of monoclonal antibodies to cytokeratin in the investigation of the sentinel axillary lymph node for metastatic breast cancer increases nodal positivity by up to 10%.
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 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.
The use of immuno-cytochemical techniques can help to avoid performing procedures, which are painful, uncomfortable and expensive to the patient. It can also help to speed up the start of appropriate treatment.
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. Immuno-cytochemical staining can also detect the presence of two antigens in the same smear.
See also
- Trifunctional antibodyTrifunctional antibodyA trifunctional antibody is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen, making it a type of bispecific monoclonal antibody. In addition, its intact Fc-part can bind to an Fc receptor on accessory cells like conventional monospecific...
, describing the application of hybridoma technology to produce so-called hybrid-hybridomas or quadroma cell lines