Hybridoma Technology - Coursework Example

Introduction 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 mouses 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 lymphomas. Monoclonal antibodies are being used to track cancer antigens, to attack cancer metastases, and preventing graft-versus-host disease in bone marrow transplants. The other applications of monoclonal antibodies include the use in immuno-histochemistry for the localization of specific antigens in situ, immunogold labelling, Western-type blotting, radioimmune assays, ELISA, and functional assays. Hybridoma Technology After the injection of a specific antigen into a mouse and procuring the B cells from the mouses spleen, the splenic B cells are mixed with HGPRT (hypoxanthine guanine phosphoribosyltransferase) negative myeloma cells and polyethylene glycol (a fusing agent) before being fused with histocompatible myeloma cells like Sp2/0. The mixing and centrifugation process generates myeloma–splenic B cell hybridomas, which are plated into tissue culture wells. After this, the unfused myeloma cells are removed by using a selective medium containing 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. 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. 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. 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 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 (Nelson et al., 2000.) Application of monoclonal antibodies 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, 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 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 -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 (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.) Conclusion 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. *************************************************************************************************** References Bretton, PR, Melamed, MR, Fair, WR, Cote, RJ (1994). Detection of occult micrometastases in the bone marrow of patients with prostate carcinoma. Prostate. 1994 Aug; 25(2): 108-14. 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. 1996; 41(1): 1-13. Ghosh, AK, Mason, DY, Spriggs, Al (1983). Immunocytochemical staining with monoclonal antibodies in cytologically "negative" serous effusions from patients with malignant disease. J Clin Pathol 1983;36:1150-1153. Ghosh, AK, Spriggs, Al, Taylor-papadimitriou, J and Mason, DY(1983). Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies. J Clin Pathol 1983;36:1154-1164. Kvalheim, G (1996). Detection of occult tumour cells in bone marrow and blood in breast cancer patients--methods and clinical significance. Acta Oncol. 1996; 35 Suppl 8:13-8. Kvalheim, G (1998). Diagnosis of minimal residual disease in bone marrow and blood in cancer patients--methods and clinical implications. Acta Oncol. 1998; 37(5): 455- 62. Nelson, PN, Reynolds, GM, Waldron, EE, Ward, E, Giannopoulos, K and Murray, PG (2000). Demystified …Monoclonal antibodies. Mol Pathol. 2000 June; 53(3): 111– 117. Riesenberg, R, Oberneder, R, Kriegmair, M, Epp, M, Bitzer, U, Hofstetter, A, Braun, S, Riethmuller, G, Pantel, K (1993). Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells. Histochemistry. 1993 Jan;99(1):61-6. Vissers, CJ, Flohil, CC, De Jong, AA, Dinjens, WN, Bosman, FT (1996). A new monoclonal antibody for specific immunocytochemical staining of nucleoli. Acta Histochem. 1996 Apr; 98(2): 113-21. Read More