Hybridoma Technology

Hybridoma are cells that have been engineered to produce a desired antibody in large amounts. To produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies. They have to be selected and subsequently cloned by limiting dilution. Supplemental media containing Interleukin-6 (such as briclone) are essential for this step.The production of monoclonal anti-bodies was first invented by Cesar Milstein, Georges J. F. Köhler and Niels Kaj Jerne in 1975.

Selection occurs via culturing the newly fused primary hybridoma cells in selective-media, specifically media containing 1x concentration HAT for roughly 10-14 days. After using HAT it is often desirable to use HT containing media. Cloning occurs after identification of positive primary hybridoma cells. Clone by limited dilution. While some may believe that IL-6 is essential for this step, it is not necessary to add that expensive supplement, rather use 50% heat-inactivated FBS for the first week. Add 10% FBS DMEM to the clone culture plate after screening for single colony wells.

Method
A hybridoma, which can be considered as a hybrid cell, is produced by the injection of a specific antigen into a mouse, procuring the antibody-producing cell from the mouse's spleen and the subsequent fusion of this cell with a cancerous immune cell called a myeloma cell. The hybrid cell, which is thus produced, can be cloned to produce many identical daughter clones. These daughter clones then secrete the immune cell product. Since these antibodies come from only one type of cell (the hybridoma cell) they are called monoclonal antibodies. The advantage of this process is that it can combine the qualities of the two different types of cells; the ability to grow continually, and to produce large amounts of pure antibody.

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 leukaemia's and (Hypoxanthine, Aminopterin, and Thymidine (HAT). Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells, especially weakly established hybridomas.

HAT medium is used for preparation of monoclonal antibodies. Laboratory animals (eg. mice) are first exposed to an antigen to which we are interested in isolating an antibody against. Once splenocytes are isolated from the mammal, the B cells are fused with HGPRT negative, immortalized myeloma cells using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT medium. Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies.

Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and foetal bovine serum) and produce antibody (Nelson et al., 2000.)

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 labelled conjugate, and chromogenic substrate, are then incubated, and the formation of a coloured product indicates a positive hybridoma. Alternatively, immunocytochemical screening can also be used (Nelson et al., 2000.)

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 1to 60 Î¼g/ml of monoclonal antibody, which is maintained at âˆ’20Â°C or lower until required (Nelson et al., 2000.)

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 (Nelson et al., 2000.)

In Diagnostic Histopathology
With the help of monoclonal antibodies, tissues and tumours 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, Î± fetoprotein and others are organ-associated antigens and the production of monoclonal antibodies against these antigens helps in determining the nature of a primary tumour (Nelson et al., 2000.)

Monoclonal antibodies are especially useful in distinguishing morphologically similar lesions, like mesothelioma and 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. (Nelson et al., 2000.)

One study (Bretton et al., 1994) 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 tumour-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 (Kvalheim, 1996.) One major drawback of immuno-cytochemistry is that only tumour-associated and not tumour-specific monoclonal antibodies are used, and as a result, some cross-reaction with normal cells can occur (Kvalheim, 1998.)

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%. (Nelson et al., 2000.)

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 (1996) performed a sensitive immuno-cytochemical assay by using a combination of four monoclonal antibodies (260F9, 520C9, 317G5 and BrE-3) against tumour 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 tumour cells in the bone marrow.

One study (Riesenberg et al., 1993) reports on having developed an immuno-cytochemical procedure for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). This would help in the further characterization of disseminated individual epithelial tumour 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 (1983) 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 -20Â°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 (Ghosh et al., 1983.)

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 (Vissers et al., 1996.)

A hybridoma produces antibodies called monoclonal antibodies, which are more pure than the polyclonal antibodies produced by conventional techniques. The uses of monoclonal antibodies are varied. They are used to identify different types of leukaemia's and lymphomas, to track cancer antigens, to attack cancer metastases, and to prevent graft-versus-host disease in bone marrow transplants.

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.