METHOD, COMPOSITION AND DEVICE FOR SAMPLING NATRIURETIC PEPTIDES IN A BIOLOGICAL FLUID

• US 20090215182A1
• Filed: 02/27/2008
• Published: 08/27/2009
• Est. Priority Date: 02/27/2008
• Status: Active Grant

• Current Assignee: Crimson U.S. Assets LLC (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Crimson U.S. Assets LLC (The Carlyle Group LP (Corporate Private Equity))
• Inventors: O'Rear, Jessica, Schellenberger, Ute, Kiernan, Urban A., Nelson, Randall W., Niederkofler, Eric E.
• Granted Patent: US 9,482,677 B2
• Time in Patent Office: Days
• US Class Current: 436/17
• CPC Class Codes: G01N 1/28 Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• CPC Class Codes: G01N 2333/58 Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
• CPC Class Codes: G01N 2560/00 Chemical aspects of mass spectrometric analysis of biological material
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

Disclosed is a composition that synergistically prevents proteolysis or modification of peptides in sampled biological fluids using sulfonyl fluoride family protease inhibitors at high concentrations combined with at least one additional protease inhibitor of a different type, preferably a broad spectrum protease inhibitor, and a chelator. A preferred embodiment uses AEBSF at 10 mM, Benzamidine at 20 mM and EDTA as the chelator. The disclosed composition may be combined with other protease inhibitors to further modulate its specificity, for instance to additionally target acidic proteases. Additional protease inhibitors, reducing agents, stabilizers and buffering agents may be combined with the disclosed compositions in devices for sampling or testing biological fluids for levels of peptides of interest, or methods therefore. The disclosed devices, compositions and methods are of particular use in sampling and testing for the level of natriuretic peptides.

Claims

1. A composition of matter for sampling a protein profile in a biological fluid of interest, comprising:

an effective amount of a first protease inhibitor selected from the group consisting of leupeptin and benzamidine;

an effective amount of chelator; and

an effective amount of a second protease inhibitor, which includes a sulfonyl fluoride functional group, wherein the effective amount of the second protease inhibitor results in the formation of protein adducts in the absence of the first protease inhibitor and the chelator; and

whereby essentially no adducts are formed in the sampled protein profile by the composition of matter.

2. A composition according to claim 1 wherein the protein profile is of a natriuretic peptide selected from the set consisting of ANP, BNP and CNP.

3. A composition according to claim 1 wherein the natriuretic peptide is a synthetic natriuretic peptide.

4. A composition according to claim 1 suitable for preserving a sampled protein profile for at least one of (i) two hours at room temperature, and (ii) at least six months at seventy degrees centigrade below zero.

5. A composition according to claim 1 further comprising the biological fluid,

6. A composition according to claim 5, wherein the biological fluid is derived from blood, sweat, blister fluid, lung fluid, saliva, lacrimal gland secretion, cerebrospinal fluid, seminal fluid, lymph or urine.

7. A composition according to claim 1, wherein the chelator is ethylenediamine tetraacetic acid, the first protease inhibitor is benzamidine and the second protease inhibitor is AEBSF.

8. A composition according to claim 7, wherein AEBSF is present at about 10 mM, benzamidine is present at about 20 mM and ethylenediamine tetraacetic acid is present at least at about 1 mM.

9. A composition according to claim 1, wherein the first protease inhibitor is leupeptin at about 2.5 mM.

10. A device useful for sampling a protein profile in a biological fluid of interest, comprising:

a component for receiving a fluid fraction of the biological fluid, the component containing an effective amount of a first protease inhibitor selected from the group consisting of leupeptin and benzamidine; and

an effective amount of a second protease inhibitor, based on sulfonyl fluoride, suitable for inactivating serine proteases, wherein the effective amount of the second protease inhibitor results in the formation of protein adducts in the absence of the first protease inhibitor; and

wherein the sampled protein profile is not substantially modified by the formation of adducts by the first protease inhibitor and the second protease inhibitor in combination.

11. The device of claim 10, wherein the sampled protein profile is stable for at least one of (i) two hours at room temperature, and (ii) at least six months at seventy degrees centigrade below zero.

12. The device of claim 10 is configured to collect samples in the form of a biological fluid from the group consisting of blood, sweat, blister fluid, lung fluid, saliva, lacrimal gland secretion, cerebrospinal fluid, seminal fluid, lymph or urine.

13. A device according to claim 10, wherein the second protease inhibitor is AEBSF present at a concentration of about 10 mM and benzamidine is at a concentration of about 20 mM in the sampled biological fluid, or AEBSF is at a concentration of about 2.5 mM and leupeptin as the second protease inhibitor is at a concentration of about 2.5 mM.

14. A device according to claim 10, wherein the fluid fraction of the biological fluid includes a chelator and the biological fluid is blood or urine.

15. A method of sampling a protein profile in a biological fluid of interest, comprising:

adding an effective amount of chelator to a sample of the biological fluid;

adding an effective amount of a first protease inhibitor selected from the group consisting of leupeptin and benzamidine; and

adding an effective amount of a second protease inhibitor, based on sulfonyl fluoride, suitable for inactivating serine proteases, wherein the effective amount of the second protease inhibitor results in the formation of protein adducts in the absence of the first protease inhibitor; and

wherein the sampled protein profile is essentially unmodified by the formation of adducts by a combination of the effective amount of the first protease inhibitor and the effective amount of the second protease inhibitor in combination.

16. A method according to claim 15, wherein a fluid fraction of the sampled biological fluid is stored at one or more of seventy degrees centigrade below zero, below zero degrees centigrade, at two to four degrees centigrade, and room temperature.

17. A method according to claim 15, wherein the biological fluid is sampled by adding the chelator followed by one or more of the first protease inhibitor and the second protease inhibitor.

18. A method according to claim 15, wherein the biological fluid is blood or urine.

19. A method according to claim 15, wherein the second protease inhibitor is AEBSF.

20. A method according to claim 19, wherein AEBSF is at a concentration of about 10 mM and benzamidine is at a concentration of about 20 mM in a sampled biological fluid;

or AEBSF is at a concentration of about 2.5 mM and leupeptin is at a concentration of about 2.5 mM.