Mouse monoclonal antibodies

Human autoantibodies" and mice monoclonal antibodieS " that bind to GPIa and block platelet adhesion to collagen rpes I and III and collagen-induced platelet aggregation have been described. 

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... 

The first mouse monoclonal antibody specific for human CD3 was produced in 1979 and named orthoclone OKT3. Aside from its use in the laboratory, OKT3 became the first anti-CD3 antibody to be utilized in transplantation medicine, but its wider application was hampered by its immunogenic and mitogenic properties (reviewed in [6]). Consequently, humanized and engineered anti-CD3 antibodies were developed to circumvent these limitations (Table 1). Since T cells and the TCR are involved in many immunological diseases, it is not surprising that the application of CD3 antibodies is not restricted to the field of transplantation. For example, CD3 antibodies are tested in clinical studies of diseases such as autoimmune diabetes (type 1 diabetes), immune-mediated inflammatory arthritis and inflammatory bowel disease [7]. 

Hirayama N, Hirano T Kohler G, Kurata A, Okumura K, Ovary Z Biological activities of anti-trinitrophenyl and antidinitrophenyl mouse monoclonal antibodies. Proc Natl Acad Sci USA 1982 79 613-615. 

Trastuzumab (Herceptin) -humanized mouse monoclonal antibody directed against HER-2/new receptor -fevers, chills, nausea, vomiting, headache during administration -cardiotoxicity (the FDA has not approved concurrent use with doxorubicin)... 

M.-G. Baek and R. Roy, Simultaneous binding of mouse monoclonal antibody and streptavidin to heterobifunctional dendritic L-lysine core bearing T-antigen tumor marker and biotin, Bioorg. Med. Chem., 9 (2001) 3005-3011. 

When primary mouse monoclonal antibodies are of different IgG isotypes/ subclasses, they can be selectively detected with secondary antibodies directed against the corresponding IgG isotype (Fig. 8.2). 

The ratio of primary antibodies to Fab fragments required for the formation of complexes that produce optimal immunolabeling of specific antigen does not appear to vary dramatically with primary antibody specificity or species. Primary antibody to Fab fragment ratios of 1 2 1 4 (weight for weight, based on concentration data supplied by manufacturers) typically produces optimal results with primary mouse monoclonal antibodies. 

Incubate sections with primary mouse monoclonal antibody at its optimal dilution for 30 min at room temperature. 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Manil, L., Motte, P., Pernas, P., Troalen, F. Bohuon, C., and Belief D. (1986) Evaluation of protocols for purification of mouse monoclonal antibodies. J. Immunol. Methods 90, 25-37. 

Burchiel S. W Billman, J. R and Alber, T. R. (1984) Rapid and efficient purification of mouse monoclonal antibodies from ascites fluid using high performance liquid chromatography. J. Immunol. Methods 69, 33-42. 

The primary antibody in NGG-sap-PBS (e.g., 10 pg/mL mouse monoclonal antibody) is then added to the fixed cells in NGG-sap-PBS and incubated for 30 min at room temperature. Do not pipet directly onto the cells, but add antibody solutions at the edge of the dish, and add wash solutions from a wide-mouth bottle or beaker to minimize cell disattachment. The minimum volume to cover a 35-mm dish surface completely is 1 mL. 

If the antigen of interest is abundant and additional sensitivity is not required, then a two-step direct method, rather than a three-step indirect method, can be anployed. With the two-step method, the primary antibody is a biotin-labeled mouse monoclonal antibody, which is followed directly by the ABC incubation step. Because of the abundance of human immunoglobulins in tissue sections, methods for staining immunoglobulin often employ biotin-labeled mouse antihuman immunoglobulin reagents. 

Primary antibody (e.g., mouse monoclonal antibody to a surface or intracellular antigen) (see Note 2). 

Sheep anti-FITC-POD (Roche). Primary antibodies in this study we have used a mouse monoclonal antibody against smooth muscle a-actin (sm-a-actin, Dako) and rabbit polyclonal antibody against programmed cell death 4 (PDCD4, Rockland Immunochemicals). 

There was a problem with using mouse monoclonal antibody proteins to treat some human diseases, however. The human immune system recognizes the mouse antibody as the foreign protein it is. 

Figure 4.4 The process of developing a mouse monoclonal antibody is illustrated here. Antibody-producing cells from a mouse immunized with material containing the intended antibody target are fused with tumor cells to produce hybridomas. The hybridomas are cloned and the culture fluids are tested to find the clone producing the desired antibody. Large amounts of the monoclonal antibody can then be isolated from the culture medium.

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