Mice, monoclonal antibody production

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

A further advance in antibody technology is the development of transgenic mouse human strains. XenoMouse animals have been engineered in such a way that they now produce exclusively human antibodies rather than murine antibodies when immunized. The use of XenoMouse animals to produce MAbs avoids the need for any engineering of the antibody genes, since the products are already 100% human protein. XenoMouse animals are fully compatible with standard hybridoma technology and can be readily adopted by laboratories experienced in monoclonal antibody production [56]. 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

All of the processes and considerations described for polyclonal antibody production are also relevant to monoclonal antibody production. The only variation between the two, with respect to the the immunization process, is species selection. Monoclonal antibodies are most commonly produced in mice because of the commercial availability of mouse myeloma cells that are particularly suited as partner cells in hybridoma preparation (discussed below). 

EBV), or by fusing it with another immortalized cell. The latter approach is called hybridoma technology because it involves fusion of two cells to produce a hybrid cell. This is the approach used most widely for the production of mouse monoclonal antibodies. 

Wilson PO, Barber PC, Hamid QA, et al. The immunolocal-ization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol. 1988 69 91-104. 

Using 1° antibodies made in the different species is the easiest way to localize multiple proteins (Chapter 11). However, it is not possible to get all 1° antibodies needed from a different species. This is especially true because there are so many mouse monoclonal antibodies available. Eventually, an experiment will need two mouse 1° antibodies or two rabbit 1° antibodies. This chapter presents the concept of combining multiple 1° antibodies made in the same species of animals. Two different approaches include block-between method and labeled Fab procedure (Lewis et al., 1993) that is available as the commercial product, Zenon (Molecular Probes/Invitrogen). 

Yoshimura T, Takeya M, Takahashi K, Kuratsu JI, Leonard EJ. Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1. J Immunol 1991 147 2229-2233. 

Monoclonal Antibody Production. Six-month old BALB/cBkl mice (Bantin and Kingman Laboratories, Fremont, CA) were injected intraperitoneally (IP) with 100 ig of the heptachlor-KLH conjugate mixed 1 1 with complete Freund s adjuvant. Mice received a single IP injection every other week. A total of three injections were administered. Four days prior to fusion, each mouse was given an intrasplenic injection of 100 ig of the heptachlor-BSA conjugate in sterile saline. The spleen was removed and the splenocytes fused with SP2/0 myeloma cells and grown under conditions described by Stanker ct al. (131. 

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