The production and characterization of monoclonal anti-bodies directed against the GABA sub a /benzodiazepine receptor
Genetic techniques have indicated that several subunits exist which may combine to form a family a GABA{sub a} receptor subtypes. Further investigations of the localization, structure and function of these receptor subtypes will require the use of subunit specific probes. In order to develop immunochemical markers for the GABA{sub a} subunits mice were immunized with purified receptor and antibody secreting hybridomas were formed. From these hybridomas six monoclonal antibodies were derived. All six monoclonal antibodies recognized the purified receptor in a solid-phase radioimmunoassay and immunoblotted to a 50kD protein in the purified preparation. The mAbs A2, B2, E9, and H10 specifically recognized a 50kD protein band from rat brain membranes which was shown by two-dimensional electrophoresis to be the receptor subunit identified by photolabeling. The mAbs D5 and F7 preferentially recognized unique proteins in addition to the 50kD subunit. A procedure was developed for using mAbs B2 and F7 to immunoprecipitate the benzodiazepine binding site from solubilized brain membranes. A competitive binding assay and an analysis of crossreactivity were combined to divide the six monoclonal antibodies into groups recognizing at least four district epitopes. The monoclonal antibodies were used to demonstrate that the 50kD subunit can be phosphorylated and they were used to follow the development of this subunit in the neonatal rat. The antibodies were able to label immunoreactive proteins in rat astrocytes and in three nematode species. These proteins may be structurally related to subunits of the GABA{sub a} or acetylcholine receptor.
- Research Organization:
- Yale Univ., New Haven, CT (United States)
- OSTI ID:
- 5671405
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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