Hybridoma technology
Hybridoma technology is a method for producing large numbers of 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
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 (or vulnerable) to the HAT medium (see below).
Fused cells are incubated in HAT medium (
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. 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] 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.[4] 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. [4]
The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing
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
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.
Monoclonal antibodies are especially useful in distinguishing morphologically similar lesions, like
One study[5] 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.[6] 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.[7]
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.
One of the main reasons for
One study[9] reports on having developed an immuno-cytochemical procedure for simultaneous labeling of cytokeratin component no. 18 (CK18) and
In most cases of malignant disease complicated by effusion,
Ghosh, Mason and Spriggs[10] 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
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.[11]
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.[12]
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
- Hybridomas at the U.S. 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".