[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.

[Functional analysis of G protein-coupled receptor kinase (beta ARK1) by intracellular immunization].

G protein-coupled receptor kinases (GRKs) are believed to involve in desensitization of the G protein-coupled receptors. So far, cDNAs of six GRKs were cloned from several species including human and rat. However, it is unknown whether single GRK phosphorylates various receptors and desensitizes them in the cells. To determine whether GRK2 (also called beta ARK1) involves desensitization of the beta 1-adrenergic receptor-mediated response in heart, we tried to apply monoclonal antibody which could recognize only beta ARK1 and inhibit its phosphorylating activity to the heart cells. Monoclonal antibody was obtained by immunization of carboxyl terminus of beta ARK1 as fusion protein of glutathione-S-transferase (GST). The resulting monoclonal antibody specifically reacted with beta ARK1, and inhibited the binding of purified beta gamma subunit to the carboxyl terminus. Monoclonal antibody completely inhibited phosphorylation of the m2 muscarinic acetylcholine receptor as well as phosphorylation of GST-intracellular third loop fusion protein of the m2 receptor. When monoclonal antibody was applied to myocyte prepared from guinea pig heart, the desensitization of the beta 1-adrenergic receptor was partially inhibited as measured by Ca2+ channel activation. Thus intracellular application of monoclonal antibody is promising approach to analyze function of GRKs.