[Production of various antibodies and single chain Fv molecule against signal-transducing proteins].
In the present review, we described the procedures of production of polyclonal and monoclonal antibodies and single chain Fv molecule (scFv). Among several animal species, the rabbit is the best animal for polyclonal antibody production and the mouse is the best animal for monoclonal antibody production. In this review, we discussed problems that might be encountered when trying to produce antibodies. Polyclonal antibodies are easily produced in rabbits by immunizing them with glutathione-S-transferase fusion proteins. However, it is difficult to eliminate nonspecifically reacting antibodies, even after the antibodies were purified from sera by an antigen column. Monoclonal antibody production is a time-consuming process, but it successful, will produce a very useful reagent due to no limitation of supply and constant quality. We described monoclonal antibody production by means of glutathione-S-transferase fusion protein. scFv is a portion of the antibody and is constructed by PCR of VH and VL regions of the antibody. We recommend that scFv should be constructed from a hybridoma that secretes monoclonal antibody, although some researchers have claimed that scFv can be constructed from the spleen of immunized mice. The expression of scFv is a promising approach to analyze the function of one of the subtypes, when the original monoclonal antibody can block the function of the protein.
Kurose
H
H
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
Takikawa
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D005260:Female; D005982:Glutathione Transferase / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D051379:Mice; D021382:Protein Sorting Signals / Q000276:immunology*; D011817:Rabbits; D015398:Signal Transduction / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D021382:Protein Sorting Signals; D005982:Glutathione Transferase