The production and characterization of monoclonal anti-bodies directed against the GABA sub a /benzodiazepine receptor

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5671405

Gallombardo, P A

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
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AMINOBUTYRIC ACID
ANIMAL CELLS
ANIMALS
ANTIBODIES
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOASSAY
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MARKERS
BODY
BRAIN
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHEMICAL COMPOSITION
DIAGNOSTIC TECHNIQUES
DRUGS
ELECTROPHORESIS
HYBRIDOMAS
IMMUNOASSAY
IMMUNOLOGY
ISOTOPE APPLICATIONS
KINETICS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MONOCLONAL ANTIBODIES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNODETECTION
RADIOIMMUNOLOGY
RATS
REACTION KINETICS
RECEPTORS
RODENTS
TRACER TECHNIQUES
TWO-DIMENSIONAL ELECTROPHORESIS
VERTEBRATES