Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1992-02-07
2001-06-05
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S013000, C435S028000, C436S501000, C436S536000, C436S063000, C436S086000, C436S124000, C530S300000
Reexamination Certificate
active
06242173
ABSTRACT:
BACKGROUND
Thrombosis and thrombolysis are maintained in delicate balance by a series of high regulated enzymatic reactions. The component enzymes are are largely serine proteases which are produced as needed from zymogens, inactive forms of the enzymes which circulate in the blood at relatively high concentrations. It has become increasingly clear that the active forms of the pro- and anti-coagulant enzymes may be useful for evaluating patients afflicted with or at risk for thrombosis-related disorders.
For example, epidemiologic studies have demonstrated that elevated levels of fibrinogen and factor VII are associated with an increased risk of coronary artery disease and stroke. Some investigators have differentiated between activated and unactivated factor VII and find that the level of activated factor VII is the better risk indicator. In addition, there is evidence which suggests that the subset of the population that develops atherosclerosis may have a form of thrombotic diathesis which might manifest itself in the form of a chronic increase in activation of the coagulation/fibrinolytic system. Thrombotic manifestations of atherosclerotic disease such as stroke and myocardial infarction represent the major causes of mortality in the western industrialized world.
Identifying and evaluating patients at risk for thrombotic disorders will require accurate measurement of plasma levels of active pro- and anti-coagulant factors of the coagulation/fibrinolytic system. The need for this capability will grow in years to come. Heart disease, stroke and the many other thrombosis-related disorders occur most frequently in the older segment of the population. With the rapid growth of the older cohort of our population, the capability to monitor the activation state of coagulation/fibrinolytic system will be of increasing clinical utility.
SUMMARY OF THE INVENTION
This invention pertains to assays for detecting and/or quantifying catalytically-active, serine proteases in a biological fluid. The methods are useful for measuring the active enzymes of the coagulation/fibrinolytic system and evaluating the system or components of the system as indicative of thrombosis-related disorders. The methods involve the combined use of halomethyl ketone probes having broad specificity for catalytically-active serine proteases and immunological reagents specific for serine proteases of particular types. The halomethyl ketone probes are active site specific; they are only incorporated into catalytically-active serine proteases. An antibody is used to provide specificity for the particular type of serine protease. By the combined active-site-specificity of the halomethyl ketone probes and the type-specificity of the antibody, the catalytically-active fraction of a particular serine protease is determined.
In the assays of this invention, the halomethyl ketone probe is incubated with a sample of biological fluid so that it is incorporated generally into catalytically-active serine proteases in the sample. Depending on the format of the assay, the halomethyl ketone probe is coupled to a detectable label, to a group which can be labeled or to a group that provides a secondary recognition site for capture onto a solid phase. Thus, incorporation of the halomethyl ketone labels the active serine proteases either for detection or for separation. A mono-specific antibody is incubated with the biological fluid to selectively bind serine proteases of the type to be determined. In formats of the assay where the halomethyl ketone provides a label for detection, the anti-body can be linked to a solid phase to effect type-specific separation of the serine proteases to be determined. In formats of the assay where the halomethyl ketone provides a secondary recognition site for separation, the antibody is used to type-specifically label the serine protease for detection.
The invention also pertains to a method of separating catalytically-active serine proteases from a biological fluid. The method involves the incorporation of a biotinylated halomethyl ketone into the active site of serine proteases and the adsorption of the serine proteases onto an avidin-coated solid phase to effect separation.
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Mann Kenneth G.
Tracy Russell P.
Williams Brady
DeConti, Jr. Giulio A.
Lahive & Cockfield LLP
Marschel Ardin H.
University of Vermont and State Agriculatural College
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