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The amount of mAb needed and the importance of such factors as cost, turnaround time, and regulatory compliance depends on the purpose. The very competitive diagnostic industry is concerned with cost, turnaround time, and regulatory requirements.

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Step 2: Screening of Mice for Antibody Production
After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies. Several humane techniques have been developed for collection of small volumes of blood from mice (Loeb and Quimby 1999). Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry. If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production

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Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells
Mice are immunized with an antigen that is prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes used as immunogens. In almost all laboratories, mice are used to produce the desired antibodies. In general, mice are immunized every 2-3 weeks but the immunization protocols vary among investigators. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.

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Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies

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Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies

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Akerstrom, B., T. Brodin, K. Reis, L. Bjorck. 1985. Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 135:2589-2592.

Capiaumont, J., C. Legrand, D. Carbonell, B. Dousset, F. Belleville, P. Nabet. 1995. Methods for reducing the ammonia in hybridoma cell cultures. J Biotech 39:49-58.

Chandler, J. 1987. Factors influencing monoclonal antibody production in mouse ascites fluid. Pp. 75-92 in Commercial Production of Monoclonal Antibodies. S. Seaver, ed. New York:Marcel Dekker, Inc.

Chandler, J. 1998. In Testimony Before the National Research Council's Committee on Monoclonal Antibody Production. November 10, 1998: Washington, DC.

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GENERATION OF HYBRIDOMAS: PERMANENT CELL LINES SECRETING MONOCLONAL ANTIBODIES

IN VITRO PRODUCTION OF MONOCLONAL ANTIBODY

SCIENTIFIC NEEDS FOR MOUSE ASCITES PRODUCTION OF MAB

DVANTAGES AND DISADVANTAGES OF IN VITRO AND IN VIVO METHODS

LARGE-SCALE PRODUCTION OF MONOCLONAL ANTIBODIES

ANIMAL-WELFARE ISSUES RELATED TO THE ASCITES METHOD FOR PRODUCING MONOCLONAL ANTIBODIES .

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https://grants.nih.gov/grants/policy/antibodies.pdf

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monoclonal antibodies in biomedical research

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monoclonal antibodies in biomedical research

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Monoclonal antibodies (mAb) are used extensively in basic biomedical research, in diagnosis of disease, and in treatment of illnesses, such as infections and cancer. Antibodies are important tools used by many investigators in their research and have led to many medical advances.

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Producing mAb requires immunizing an animal, usually a mouse; obtaining immune cells from its spleen; and fusing the cells with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. A tumor of the fused cells is called a hybridoma, and these cells secrete mAb. The development of the immortal hybridoma requires the use of animals; no commonly accepted nonanimal alternatives are available.

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The Antibody Discovery Programme’s team led by Dr Sophia Karagiannis have a world leadership position in the development of Immunoglobulin E (IgE) antibody therapeutics for cancer.

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Step 3: Preparation of Myeloma Cells
Step 4:  Fusion of Myeloma Cells with Immune Spleen Cells
Step 5 : Clones by Ascites Production