PURIFICATION OF A FIBRINOLYSIS ENHANCING PROTEIN

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: N/A
Agency Tracking Number: 19299
Amount: $50,000.00
Phase: Phase I
Program: SBIR
Awards Year: 1992
Solicitation Year: N/A
Solicitation Topic Code: N/A
Solicitation Number: N/A
Small Business Information
Elcatech Inc
#64-1001 South Marshall Street, Winston-salem, NC, 27101
DUNS: N/A
HUBZone Owned: N
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 Mark X Triscott
 (919) 777-3624
Business Contact
Phone: () -
Research Institution
N/A
Abstract
DURING THE DEVELOPMENT OF OUR FIBRINOLYTIC ASSAY SYSTEM WE NOTED AN ENHANCEMENT OF FIBRINOLYTIC ACTIVITY IN PLASMA WHICH COULD NOT BE ACCOUNTED FOR BY ANY KNOWN FIBRINOLYTIC FACTOR. USING HEPARING AFFINITY CHROMATOGRAPHY, WITH A 0.5M NAC1 ELUTION AND AN IMMUNO AFFINITY COLUMN WE HAVE BEEN ABLE TO PARTIALLY PURIFY THIS FACTOR. THE IMMUNO AFFINITY COLUMN USES IMMOBILIZED MCAB-3, A MONOCLONAL ANTIBODY AGAINST A PLASMA ELUATE FROM A HEPARIN AFFINITY COLUMN. WE HAVE DEMONSTRATED THE ENHANCING ACTIVITY OF THE PROTEIN IN THE ENZYME LINKED FIBRINOLYTIC ASSAY (ELFA), A CLOT LYSIS ASSAY WITH ADDED T-PA, AND A BIO IMMUNOASSAY USING A TRANSFERABLE SOLID PHASE ENZYME LABELLED FIBRIN. USING THE BIO IMMUNOASSAY WE HAVE ESTIMATED PLASMA LEVELS TO BE APPROXIMATELY 4.5 UNITS. PLASMA PROTEIN LEVELS FOR THE COFACTOR APPEAR TO BE LESS THAN 50UG/ML. HPLC GEL PERMEATION CHROMATOGRAPHY REVEALS A MCAB-3 BINDING PROTEIN OF 78KD. THE PROTEIN COULD NOT BE ACTIVATED IN THE PRESENCE OF HIGH CONCENTRATIONS OF EXTRINSIC FIBRINOLYTIC ACTIVATORS, PRECLUDING THE POSSIBILITY OF PLASMINOGEN BEING RESPONSIBLE FOR THE ACTIVITY. THE PROTEIN DID NOT ACTIVATE PLASMINOGEN AND HAD NO MEASURABLE FIBRINOLYTIC ACTIVITY. IMMUNIDIFFUSION ANALYSIS OF THE INTACT PROTEIN REVEALED NON IDENTITY WITH VITRONECTIN OR HISTIDEINE RICH GLYCOPROTEIN, OTHER HEPARIN BINDING PLASMA PROTEINS IMPLICATED IN FIBRINOLYSIS MODULATION. WE WILL PURIFY THE PROTEIN TO HOMOGENEITY AND POSITIVELY IDENTIFY IT BY N-TERMINAL SEQUENCE ANALYSIS. WE WILL RAISE A POLYCLONAL ANTIBODY AGAINST THE PROTEIN TO USE IN THE DEVELOPMENT OF IMMUNOASSAYS FOR THE MEASUREMENT OF THE PROTEIN IN PLASMA.

* information listed above is at the time of submission.

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