Immunoblotting studies of coagulation factor XII, plasma prekallikrein, and high molecular weight kininogen
Immunoblotting techniques for the qualitative and quantitative analysis of FXII, PK, and HMWK in whole plasma are presented. Sensitive, specific, and quantitative immunodetection of FXII and PK can be achieved by developing the blots with polyclonal antiserum followed by radiolabeled FXII or PK, respectively. This approach is based on the assumption that bivalent antibodies bind monovalently to the NC-bound antigen and have available binding sites to bind radiolabeled antigen derived from the fluid phase. This radiolabeled antigen overlay principle may be generally useful for immunodetection of any trace protein in complex mixtures, provided that the radiolabeled purified antigen is available. Immunoblotting may also be helpful for the partial characterization of the structural or functional abnormalities of CRM-positive variant molecules. For example, earlier studies of a FXII-variant molecule that had been purified and characterized were supported by immunoblotting studies of the CRM-positive deficient plasma. Quantitative measurement of HMWK is possible using a monoclonal antibody directed against the light chain of HMWK followed by radiolabeled secondary antibody. Quantitation of cleaved and single-chain HMWK is possible using dilutions of dextran sulfate-activated NHP on unreduced SDS-PAGE and dilutions of unactivated NHP with reduced SDS-PAGE as standards. These assays allow assessment of the degree of in vivo activation of the contact system in various disease states.
- Research Organization:
- Scripps Clinic and Research Foundation, La Jolla, CA
- OSTI ID:
- 6792895
- Journal Information:
- Semin. Thromb. Hemostasis; (United States), Vol. 1
- Country of Publication:
- United States
- Language:
- English
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BLOOD COAGULATION
ANTIGEN-ANTIBODY REACTIONS
BLOOD COAGULATION FACTORS
QUALITATIVE CHEMICAL ANALYSIS
QUANTITATIVE CHEMICAL ANALYSIS
ANTIBODIES
BIOCHEMICAL REACTION KINETICS
BLOOD PLASMA
ELECTROPHORESIS
KININOGENIN
MOLECULAR WEIGHT
MONOCLONAL ANTIBODIES
PAPER
PROTEINS
RADIOISOTOPES
TRACER TECHNIQUES
VALENCE
BIOLOGICAL MATERIALS
BLOOD
BODY FLUIDS
CHEMICAL ANALYSIS
COAGULANTS
DRUGS
ENZYMES
HEMATOLOGIC AGENTS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MATERIALS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
SERINE PROTEINASES
550201* - Biochemistry- Tracer Techniques