Production of monoclonal antibody (MoAB) CS-1 against platelet glycoprotein IIb-IIIa (GP IIb-IIIa)
Platelet aggregation is mediated by the binding of fibrinogen (FGN) to the platelet GP IIb-IIIa complex. Receptor complex was purified from a solubilized platelet membrane fraction followed by lentil-lectin affinity chromatography and gel filtration. SDS-PAGE showed the product to be the complex, GP IIb-IIIa; light scattering intensity measurements indicated a Ca/sup 2 +/-dependent complex of the appropriate MW. Purified complex was then used to raise antibodies in Balb-c mice by standard hybridoma technology; positive cells were found by ELISA enabling the detection of antibody concentrations as low as 3 ng/ml. The MoABs were purified from both culture supernatant and ascites fluid by ion exchange and protein-A affinity chromatography. Western blotting determined that CS-1 is specific for nonreduced GP IIIa but not reduced GP IIIa or any form of GP IIb. CS-1 inhibited binding of radiolabeled FGN to ADP-stimulated platelets by 40-50% over a concentration range of 5-70 ..mu..g/ml. Flow cytometric analysis confirmed that CS-1 recognized a protein on the surface of both resting and stimulated platelets. The determined specificity of CS-1 and its limited ability to inhibit FGN binding to platelets make this MoAB useful for analysis of the structural and functional relationships between fibrin(ogen) and GP IIb-IIIa.
- Research Organization:
- Bowman Gray School of Medicine, Winston-Salem, NC
- OSTI ID:
- 6127204
- Report Number(s):
- CONF-870644-; TRN: 87-033967
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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BLOOD PLATELETS
BLOOD COAGULATION
FIBRINOGEN
BIOCHEMICAL REACTION KINETICS
MONOCLONAL ANTIBODIES
BIOSYNTHESIS
CELL FLOW SYSTEMS
CELL MEMBRANES
CHROMATOGRAPHY
ELECTROPHORESIS
ENZYME IMMUNOASSAY
GLUCOPROTEINS
HYBRIDOMAS
LABELLED COMPOUNDS
MICE
TRACER TECHNIQUES
ANIMAL CELLS
ANIMALS
ANTIBODIES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD COAGULATION FACTORS
BODY FLUIDS
CARBOHYDRATES
CELL CONSTITUENTS
COAGULANTS
DRUGS
GLOBULINS
HEMATOLOGIC AGENTS
HEMOSTATICS
IMMUNOASSAY
ISOTOPE APPLICATIONS
KINETICS
MAMMALS
MATERIALS
MEMBRANES
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
RODENTS
SACCHARIDES
SEPARATION PROCESSES
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques