Direct renin assay system and method
AssigneeUniversity of Utah
InventorHans J. Keutel
Life of this patent
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Abstract
An assay system and method for the direct determination of the enzymatic activity of the enzyme renin in a biological sample. An enzyme specific octopeptide is incorporated into substrate which is immobilized at one end on an insoluble carrier and is tagged with a marker at the other. Through enzymatic activity, renin reacts with the substrate to cleave the octopeptide and thereby permit solubilization of the marker segment of the substrate. The solubilized marker can then be separated and quantified to indicate the amount of active renin in the sample.
Claims12
A substrate having a specificity for the enzymatic action of renin enzyme comprising:
- an octopeptide having the general formulation:
- PRO-PHE-HIS-LEU-LEU-VAL-TYR-SER;
- a marker attached at one end of the substrate;
- an insoluble carrier attached at the other end of the substrate so as to immobilize the substrate and attached marker.
An immobilized substrate having specificity for enzymatic action by active renin enzyme comprising:
- an insoluble carrier;
- a first extender attached to the carrier;
- an octopeptide comprising an amino acid chain in the sequence of PRO-PHE-HIS-LEU-LEU-VAL-TYR-SER, the octopeptide being attached at a first end to the first extender;
- a second extender attached to a second end of the octopeptide;
- a marker attached to the second extender.
A method of providing a system for the assay of enzymatic activity of renin comprising the steps of:
- obtaining an octopeptide having a formulation of amino acids in a sequence of PRO-PHE-HIS-LEU-LEU-VAL-TYR-SER;
- incorporating the octopeptide into a polypeptide so as to form a substrate;
- immobilizing the substrate by attaching one end to an insoluble carrier;
- reacting the other end of the substrate with a material which enhances the coupling of an amino acid with the substrate, thereby preparing an activated end of the substrate;
- and
- coupling a radioactive isotope labelled amino acid to the activated end of the substrate.
A method for assaying enzymatic activity of active renin enzyme in a biological sample comprising the steps of:
- obtaining an octopeptide having a formulation of amino acids in a sequence of PRO-PHE-HIS-LEU-LEU-VAL-TYR-SER;
- incorporating the octopeptide in a soluble polypeptide substrate;
- tagging the substrate by attaching a marker at one end;
- immobilizing the tagged substrate by attaching the other end of the substrate to an insoluble carrier;
- contacting the immobilized, tagged substrate with a biological sample so as to permit enzymatic activity of active renin enzyme in the sample to sever the LEU-LEU bond of the octopeptide thereby liberating the marker tagged segment of the substrate;
- solubilizing the marker tagged segment of the substrate;
- separating the solubilized marker tagged segment of the substrate from the remaining immobilized substrate;
- and
- measuring the marker in the separated portion.
Dependencies are inferred from the claim text.