# US 2017/0285048 A1 · Method for measurement of peptidic degradation products of a proteolytic cascade in blood samples

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- Number: US 2017/0285048 A1
- Document ID: US20170285048A1
- Status: Published application
- Kind: A1
- Application number: 15/588,272
- Filed: 2017-05-05
- Published: 2017-10-05
- Earliest claimed: 2014-10-22
- Assignees: Attoquant Diagnostics GmbH
- Applicants: Attoquant Diagnostics GmbH
- Inventors: Marko Poglitsch; Cornelia Schwager; Hans Loibner; Manfred Schuster
- Web page: https://patentgrep.com/patent/US20170285048A1

## CPC

- G01N 33/86: involving blood coagulating time {or factors, or their receptors}
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G01N 2333/515: Angiogenesic factors; Angiogenin
- G01N 2333/81: Protease inhibitors
- G01N 2333/96472: Aspartic endopeptidases (3.4.23)
- G01N 2410/02: Angiotensins; Related peptides
- G01N 2410/06: Kallidins; Bradykinins; Related peptides
- 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 discloses a method for measurement of peptidic degradation products of a proteolytic cascade in biological samples, especially blood samples, wherein the sample is incubated until a steady state equilibrium is reached for at least one peptidic degradation product involved in said proteolytic cascade and wherein said at least one peptidic degradation product in steady state equilibrium of the proteolytic cascade is quantified in the sample.

## Claims

### Claim 1

A method for measurement of peptidic degradation products of a proteolytic cascade in a biological sample comprising:
incubating a biological sample ex vivo until a steady state equilibrium is reached for at least one peptidic degradation product involved in a proteolytic cascade; and
quantifying at least one peptidic degradation product in a steady state equilibrium concentration in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample is a blood sample.

### Claim 3 (depends on 2)

The method of claim 2, where the sample comprises full blood, plasma, serum, fresh or frozen anti-coagulated full blood, or fresh or frozen anti-coagulated plasma.

### Claim 4 (depends on 1)

The method of claim 1, wherein at least two degradation products of the proteolytic cascade are quantified in the sample in steady state equilibrium concentration.

### Claim 5 (depends on 4)

The method of claim 4, wherein a ratio is calculated of the steady state equilibrium quantifications of the at least two peptidic degradation products.

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample is incubated for up to 300 minutes.

### Claim 7 (depends on 1)

The method of claim 1, wherein the sample is incubated at a temperature of 30 to 50° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the proteolytic cascade is a renin-angiotensin system (RAS) or a bradykinin system, or both.

### Claim 9 (depends on 1)

The method of claim 1, wherein the peptidic degradation product is angiotensinogen, angiotensin I, angiotensin 2-10, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-9, angiotensin 1-7, angiotensin 2-7, angiotensin 3-7, angiotensin 1-5, kallidin, bradykinin 1-9, bradykinin 2-9, bradykinin 1-8, bradykinin 1-7, or bradykinin 1-5.

### Claim 10 (depends on 1)

The method of claim 1, further defined as comprising quantifying angiotensin I, angiotensin II, angiotensin 1-7, and angiotensin 1-5.

### Claim 11 (depends on 1)

The method of claim 1, further defined as comprising quantifying angiotensin II, angiotensin 1-7, and angiotensin 1-5.

### Claim 12 (depends on 1)

The method of claim 1, further defined as comprising quantifying bradykinin 1-9, bradykinin 1-8, bradykinin 1-7, and bradykinin 1-5.

### Claim 13 (depends on 1)

The method of claim 1, further defined as comprising quantifying bradykinin 1-8, bradykinin 1-7, and bradykinin 1-5.

### Claim 14 (depends on 1)

The method of claim 1, further comprising adding one or more protease inhibitor and/or one or more chaotropic agent after the incubation until a steady state equilibrium is reached for at least one peptidic degradation product.

### Claim 15 (depends on 1)

The method of claim 1, wherein substrates or substrate analogues of any enzyme(s) involved in the proteolytic cascade are not added before and/or during the incubation until a steady state equilibrium is reached.

### Claim 16 (depends on 1)

The method of claim 1, wherein the matrix of the biological sample and/or the concentrations of the components of the proteolytic cascade to be analysed are not modified.

### Claim 17 (depends on 1)

The method of claim 1, wherein the at least steady state equilibrium peptide level does not vary more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% over a time period of at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes or 300 minutes.

### Claim 18 (depends on 1)

The method of claim 1, wherein the proteolytic cascade comprises at least two consecutive proteolytic reactions.

### Claim 19 (depends on 17)

The method of claim 17, wherein at least one of the at least two proteolytic reactions is performed by a feeding enzyme, said feeding enzyme having a turnover rate, and wherein the turnover rate of the feeding enzyme does not vary more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% over a time period of at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes or 300 minutes.

### Claim 20 (depends on 1)

A kit for practicing the method of claim 1, comprising one or more chemical, biochemical and/or biotechnological reagent, further defined as a peptide, enzyme, enzyme inhibitor, buffer, solvent, chaotropic agent, or detergent.

### Claim 21 (depends on 20)

The kit of claim 20, further comprising one or more chemical, biochemical and/or biotechnological laboratory item, further defined as a solid phase extraction material, purification materials, or container.

### Claim 22 (depends on 20)

The kit of claim 20, further comprising one or more biological sample further defined as comprising a blood sample, serum sample, plasma sample, or tissue sample.

### Claim 23 (depends on 20)

The kit of claim 20, further comprising one or more protease inhibitor or inhibitor cocktail, feeding substrate, or blood collection tube.

### Claim 24 (depends on 20)

The kit of claim 20, further comprising one or more anti-coagulant, one or more standard, one or more quality control, one or more solid phase extraction material, or one or more solvents.

### Claim 25 (depends on 1)

A physical or electronic representation of the result of quantification of the method of claim 1, comprising at least two quantified amounts of the at least two degradation products provided in sequence of the proteolytic cascade.

### Claim 26 (depends on 25)

The physical or electronic representation of claim 25, comprising quantified amounts of at least two of angiotensin I, angiotensin 2-10, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-9, angiotensin 1-7, angiotensin 2-7, angiotensin 3-7, angiotensin 1-5, kallidin, bradykinin 1-9, bradykinin 2-9, bradykinin 1-8, bradykinin 1-7, and bradykinin 1-5 in dependence of their relative quantities.

### Claim 27 (depends on 25)

The physical or electronic representation of claim 25, comprising quantified amounts of angiotensin I, angiotensin 2-10, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-9, angiotensin 1-7, angiotensin 2-7, angiotensin 3-7, and angiotensin 1-5 in dependence of their relative quantities.

### Claim 28 (depends on 25)

The physical or electronic representation of claim 25, comprising quantified amounts of kallidin, bradykinin 1-9, bradykinin 2-9, bradykinin 1-8, bradykinin 1-7, and bradykinin 1-5 in dependence of their relative quantities.