# US 6,297,023 B1 · Assay for plasminogen activator inhibitor-1 and tissue plasminogen activator activity

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- Number: US 6,297,023 B1
- Document ID: US6297023B1
- Status: Granted
- Kind: B1
- Application number: 08/300,447
- Filed: 1994-09-02
- Granted: 2001-10-02
- Earliest claimed: 1994-09-02
- Assignees: Beth Israel Deaconess Medical Center, Inc.
- Inventors: Jan McDonagh; Myoung H. Lee; Marcin J. Mankowski
- Web page: https://patentgrep.com/patent/US6297023B1

## CPC

- C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
- G01N 2333/8132: Plasminogen activator inhibitors
- G01N 2333/968: Plasmin, i.e. fibrinolysin
- G01N 2333/9726: Tissue plasminogen activator
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention is based upon the discovery that PAI-1 activity of a sample can be measured with sensitivity and correlation to in vivo activity without the use of standard curves. The assay determines the PAI-1 activity of a sample upon utilizing the second order rate equation for the reaction of PAI-1 and t-PA, as measured by their activities, in that sample.

## Claims

### Claim 1

A method for obtaining an initial activity of plasminogen activator inhibitor-1, an initial activity of tissue plasminogen activator, or a half-life of tissue plasminogen activity in a sample comprising the steps of:
(a) measuring activity of tissue plasminogen activator in the sample as a function of time; and
(b) obtaining a value of (PAI)0, (t-PA)0 or ½ (t-PA)0; from formula:
kt=−1/((PAI)0−(tPA)0)ln{(tPA)0[(PAI)0−(tPA)0+(tPA)]}/(PAI)0(tPA)
wherein (PAI)0 is the initial activity of plasminogen activator inhibitor-1 in the sample;
(tPA)0 is the initial activity of tissue plasminogen activator in the sample;
(tPA) is an activity of tissue plasminogen activator at time t; and
the half-life of tissue plasminogen activator in the sample is a value for t wherein (tPA) is ½ (t-PA)0;
k is a second order rate constant for a reaction:

### Claim 2 (depends on 1)

The method of claim 1 wherein the sample is a body fluid.

### Claim 3 (depends on 2)

The method of claim 2 wherein the sample is blood, plasma or serum.

### Claim 4 (depends on 2)

The method of claim 2 further comprising adding tissue plasminogen activator to the sample.

### Claim 5 (depends on 2)

The method of claim 2 further comprising acidifying the sample, thereby obtaining an acidified sample.

### Claim 6 (depends on 5)

The method of claim 5 wherein the sample is acidified with a sodium acetate buffer.

### Claim 7 (depends on 5)

The method of claim 5 wherein the acidified sample has a pH between about 3.5 to about 4.5.

### Claim 8 (depends on 7)

The method of claim 7 wherein the pH of the acidified sample is about 4.

### Claim 9 (depends on 5)

The method of claim 5 wherein the sample is maintained at about room temperature.

### Claim 10 (depends on 5)

The method of claim 5 wherein the activity of tissue plasminogen activator in step (a) is measured in a coupled assay.

### Claim 11 (depends on 5)

The method of claim 5 wherein the activity of tissue plasminogen activator in step (a) is measured by the steps:
(a) contacting a preselected amount of the sample with a preselected amount of plasminogen, thereby obtaining a mixture:
(b) maintaining the mixture under conditions sufficient to permit tissue plasminogen activator-catalyzed conversion of plasminogen to plasmin; and
(c) measuring an amount of plasmin formed.

### Claim 12 (depends on 11)

The method of claim 11 wherein the amount of plasmin formed is measured by contacting the plasmin with a chromogenic substrate and measuring an increase in absorbance of the sample.

### Claim 13 (depends on 12)

The method of claim 12 wherein the plasminogen is glu plasminogen.

### Claim 14 (depends on 5)

The method of claim 5 wherein the activity of tissue plasminogen activator in step (a) is measured at three or more distinct intervals.

### Claim 15 (depends on 14)

The method of claim 14 wherein the activity of tissue plasminogen activator in step (a) is measured at five or more distinct intervals.

### Claim 16

A method for obtaining an initial activity of plasminogen activator inhibitor-1, an initial activity of tissue plasminogen activator, or a half-life of tissue plasminogen activity in a sample, wherein the sample is a body fluid, comprising the steps of:
(a) adding tissue plasminogen activator to the sample;
(b) acidifying the sample;
(c) contacting a first preselected amount of the sample with plasminogen, thereby obtaining a mixture:
(d) maintaining the mixture under conditions sufficient to permit tissue plasminogen activator-catalyzed conversion of plasminogen to plasmin;
(e) measuring the plasmin formed in step (d), thereby measuring activity of tissue plasminogen activator at a first time, t;
(f) repeating steps (c) through (d) at least at two different times, t;
(g) obtaining a value of (PAI)0, (tPA)0 or ½ (t-PA)0 from a formula:
kt=−1/((PAI)0−(tPA)0)ln{(tPA)0[(PAI)0−(tPA)0+(tPA)]}/(PAI)0(tPA)
wherein (PAI)0 is the initial activity of plasminogen activator inhibitor-1 in the sample;
(tPA)0 is the initial activity of tissue plasminogen activator in the sample;
(tPA) is an activity of tissue plasminogen activator at time t; and
the half-life of tissue plasminogen activator in the sample is the value for t wherein (tPA) is ½ (t-PA)0;
k is a second order rate constant for a reaction: