Monoclonal antibodies isolation from tissue culture

Monoclonal antibodies are produced in a series of steps beginning with the immunization of a mouse and removal of its spleen after an appropriate period of time. Antibody-producing cells are isolated from the spleen and fused with "immortal" myeloma cells from tissue culture through the use of polyethylene glycol. Cells resulting from the fusion of a B-cell and a myeloma cell are called hybridomas. Hybridomas derive the ability to produce a single type of antibody from the B-cell partner and the ability to survive and proliferate outside the body of the animal for an extended period of time from the myeloma cell partner. Through a series of manipulations in tissue culture, individual hybridomas are isolated and allowed to divide and produce antibody. Antibody in the cell culture medium is then tested for... 

Isolation and purificcition of monoclonal antibodies from tissue culture supernatant... 

An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. 

Note that so far the expanded bed mainly has been used with rather uncomplicated systems such as for purification of monoclonal antibodies from culture broth, isolation of extracellular substances from microbial cultivations, harvesting of fusion proteins from Escherichia coli cell homogenates, affinity isolation of certain enzymes from microbial homogenates, and separation of serum proteins from serum. To our knowledge there are very few reports on the isolation from homogenates of mammalian tissues or plant material. 

Ultimately, the cost of immunosorbent isolation will depend on the entire process and must be evaluated against alternative processes. Consider, as an example, the costs and decisions involved in the purification of urokinase. One course of drug therapy consists of 33 mg of urokinase (4,000,000 CTA units). At the hospital pharmacy the drug costs for one course of treatment are currently 3,000 (9), or 91,000/gram. There are approximately 76,000 patients in the U.S. that could be treated with urokinase therapy each year requiring an annual production of approximately 2,500 g. We have selected a monoclonal antibody that has allowed the purification of urokinase from urine, tissue culture media, and bacterial culture media in a single step with 85% retention of urokinase activity (6). This monoclonal antibody was immobilized at 2 gL l with an immobilization yield of 0.8 and a cycle half number of 300 cycles. The urokinase capacity for the first cycle would be 1.2 gL l of immunosorbent. 

Antibodies can come in a variety of forms and purities. Polyclonal antibodies can come as whole serum or as purified antibodies with an IgG concentration of 1 mg/ml. Monoclonal antibodies come as isolated tissue culture media from hybridoma cells called supernatant. The antibody from supernatants is between 50 and 100 tig/ml, which means that the working antibody dilution for immunocytochemistry will be lower than whole serum. In addition, monoclonal antibodies can be ascites fluid giving antibodies that are highly concentrated of 1 mg/ml. Today, generation of ascites may be restricted by federal regulations for care of research animals. 

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