Conjunctive analysis of biological marker expression for predicting cardiac mortality

• US RE39,816 E1
• Filed: 04/29/2005
• Issued: 09/04/2007
• Est. Priority Date: 09/04/2001
• Status: Active Grant

• Current Assignee: Nexus Dx, Inc. (Sphingotec GMBH)
• Original Assignee: Nanogen, Inc.
• Inventors: Jackowski, George, Stanton, Eric B.
• Primary Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US11/117,772
• Time in Patent Office: 858 Days
• Field of Search: 435/7.92, 435/7.93, 435/7.94, 435/969, 435/970, 435/973, 435/975, 435/528, 435/530, 435/807, 435/808, 435/810, 436/514, 436/518
• US Class Current: 435/7.92
• CPC Class Codes: G01N 2800/325 Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: Y10S 435/969 Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A diagnostic tool is disclosed for accurately and rapidly diagnosing the condition of an ailing organ. Although applicable to numerous organ and organ systems, this application particularly illustrates the concept of conjunctive marker utilization as it relates to diagnosing and distinguishing congestive heart failure. The invention particularly relates to the conjunctive utilization of cardiac Troponin I (cTn-I) and natriuretic peptide, e.g. ANP, pro-ANP, BNP, pro-BNP and CNP as a retrospective tool for diagnosing the underlying mechanism of heart failure and as a prospective analytical device for monitoring disease progression and efficacy of therapeutic agents.

Claims

1. A method for predicting cardiac mortality rate in a patient, said method comprising the steps of:

drawing a sample of serum or plasma from a patient,

depositing the sample in a sample window of a diagnostic test kit, said test kit comprising

a front panel comprising a sample window and a display window;

a back panel; and

a dry chemistry membrane affixed between the front and back panels positioned for display in at least the display window, wherein said membrane comprises;

a sample region, and a control region, said sample region positioned to receive the sample from the sample window; and

at least two antibody pairs located at discrete locations along said membrane between the sample region and the control region, each of said antibody pairs comprising an antibody reagent member and an immobilized capture antibody member, each capture antibody member being located on said membrane closer to the control region than the corresponding antibody reagent member, each antibody pair having a measurable or observable moiety labeled or chemically bonded to the antibody reagent member of each said antibody pairs,

the antibody pairs being monoclonal or polyclonal and comprising;

a first antibody pair that specifically binds to a marker of cell injury selected from the group consisting of Troponin-T, cardiac Troponin-I, MLC-1, MLC-2, Glycogen Phosphorylase BB, Ca ATPase, Phospholamban, Myosin Heavy Chain, Actin, Tropomyosin, Calmodulin, Caldosmon Phospholamban phosphatase Calsequestrin, Ca⁺⁺ pumping adenosine triphosphatase, Ca⁺⁺ transport ATPase, Adenylate cyclase, Protein kinase, Histidine rich calcium binding protein, Protein phosphatase, Protein phosphatase 2C, High affinity calcium binding protein, Low density lipoprotein-binding sarcoplasmic reticulum protein, Ca⁺⁺-requiring protease (m-calpain), and Pyruvate dehydrogenase, and

a second antibody pair that specifically bind to a marker of organ adaptation selected from the group consisting of ANP, pro-ANP, BNP, pro-BNP and CNP,

such that upon adding sample to the sample window, analytes present in the sample and complementary to the antibody pairs will migrate toward the control region, binding to the antibody pair each of said analytes, producing a color change proportional to each concentration of analyte present, and

visualizing or measuring the moiety and determining cardiac mortality rate.

2. The method in accordance with claim 1 wherein:

the marker of cell injury is cardiac Troponin-I; and

the marker of organ adaptation is atrial natriuretic peptide or pro-atrial natriuretic peptide.

3. The method of claim 1 wherein:

said body fluid is selected from the group consisting of blood, a blood product, plasma, serum, or urine.

4. A method for predicting cardiac mortality rate in a patient, said method comprising the steps of:

drawing a sample of a body fluid from a patient,

contacting said sample with a first antibody that specifically binds to a marker of cell injury selected from the group consisting of Troponin-T, cardiac Troponin-I, MLC-1, MLC-2, Glycogen Phosphorylase BB, Ca ATPase, Phospholamban, Myosin Heavy Chain, Actin, Tropomyosin, Calmodulin, Caldesmon Phospholaiban phosphatase Calsequestrin, Ca⁺⁺ pumping adenosine triphosphatase, Ca⁺⁺ transport ATPase, Adenylate cyclase, Protein kinase, Histidine rich calcium binding protein, Protein phosphatase, Protein phosphatase 2C, High affinity calcium binding protein, Low density lipoprotein-binding sarcoplasmic reticulum protein, Ca⁺⁺-requiring protease (m-calpain), and Pyruvate dehydrogenase,

contacting said sample with a second antibody that specifically binds to a marker of organ adaptation selected from the group consisting of ANP, pro-ANP, BNP, pro-BNP and CNP, and

providing means for determining binding between each of said respective markers and each of said respective antibodies,

whereby said binding provides a means for determining cardiac mortality rate.

5. The method of claim 4 wherein:

said body fluid is selected from the group consisting of blood, a blood product, plasma, serum, or urine.

6. A method for determining a relative risk of cardiac mortality in a chronic congestive heart failure patient, said method comprising the steps of:

(A) drawing a sample of a body fluid from said patient,

(B) assaying for the presence of a cardiac marker of cell injury in said sample using;

a first antibody that specifically binds to a cardiac marker of cell injury, wherein said cardiac marker of cell injury is selected from the group consisting of cardiac Troponin-T, cardiac Troponin-I, MLC-1, and MLC-2; and

(C) assaying for the presence of a marker of organ adaptation in a body fluid sample drawn from said patient using;

a second antibody that specifically binds to said marker of organ adaptation, wherein said marker of organ adaptation is selected from the group consisting of ANP, N-terminal ANP, BNP, N-terminal BNP and CNP,

wherein when both said marker of cell injury and said marker of organ adaptation are present in said sample at increased levels as compared to control samples, said patient has a greater risk of cardiac mortality.

7. The method of claim 6, wherein said body fluid is selected from the group consisting of blood, a blood product, plasma, serum, or urine.

8. The method of claim 7, wherein said body fluid is serum or plasma.

9. The method of claim 7 or 8, wherein:

said first antibody specifically binds to a cardiac marker of cell injury selected from the group consisting of cardiac Troponin-T and cardiac Troponin-I; and

said second antibody specifically binds to a marker of organ adaptation selected from the group consisting of BNP and N-terminal BNP.

10. The method of claim 9, wherein:

said first antibody specifically binds to cardiac Troponin-I and said second antibody specifically binds to N-terminal BNP.

11. The method of claim 6, 7 or 8, wherein both said assaying for the presence of a cardiac marker and said assaying for the presence of a marker of organ adaptation, comprise the steps of:

(a) depositing said sample in a sample window of a diagnostic test kit,

wherein said test kit comprises a front panel comprising the sample window and a display window;

a back panel; and

a dry chemistry membrane affixed between the front and back panels positioned for display in at least the display window,

wherein said membrane comprises;

a sample region positioned to receive the sample from the sample window,

a control region, and

at least a first antibody pair and a second antibody pair located at discrete locations along said membrane between the sample region and the control region,

each of said antibody pairs comprising an antibody reagent member and an immobilized capture antibody member, each capture antibody member being located on said membrane closer to the control region than the corresponding antibody reagent member, each antibody reagent member having a measurable or observable moiety labeled or chemically bonded thereto, wherein the antibody reagent member of said first antibody pair is said first antibody that specifically binds a cardiac marker of cell injury, and the antibody reagent member of said second antibody pair is said second antibody that specifically binds a marker of organ adaptation,

such that the cardiac marker of cell injury and the marker of organ adaptation present in the sample will migrate toward the control region, bind to a cognate antibody pair to form an immobilized immunocomplex, and produce a color change proportional to the concentration of each marker in the sample, and

(b) visualizing or measuring the moiety labeled or chemically bonded to said first antibody and said second antibody so as to detect the presence of each marker in said sample.

12. The method of claim 9, wherein both said assaying for the presence of a cardiac marker and said assaying for the presence of a marker of organ adaptation, comprise the steps of:

(a) depositing said sample in a sample window of a diagnostic test kit,

wherein said test kit comprises a front panel comprising the sample window and a display window;

a back panel; and

a dry chemistry membrane affixed between the front and back panels positioned for display in at least the display window,

wherein said membrane comprises;

a sample region positioned to receive the sample from the sample window,

a control region, and

at least a first antibody pair and a second antibody pair located at discrete locations along said membrane between the sample region and the control region,

each of said antibody pairs comprising an antibody reagent member and an immobilized capture antibody member, each capture antibody member being located on said membrane closer to the control region than the corresponding antibody reagent member, each antibody reagent member having a measurable or observable moiety labeled or chemically bonded thereto, wherein the antibody reagent member of said first antibody pair is said first antibody that specifically binds cardiac Troponin-T or cardiac Troponin-I and the antibody reagent member of said second antibody pair is said second antibody that specifically binds BNP or N-terminal BNP,

such that the cardiac marker of cell injury and the marker of organ adaptation present in the sample will migrate toward the control region, bind to a cognate antibody pair to form an immobilized immunocomplex, and produce a color change proportional to the concentration of each marker in the sample, and

(b) visualizing or measuring the moiety labeled or chemically bonded to said first antibody and said second antibody so as to detect the presence of each marker in said sample.

13. The method of claim 10, wherein both said assaying for the presence of a cardiac marker and said assaying for the presence of a marker of organ adaptation, comprise the steps of:

(a) depositing said sample in a sample window of a diagnostic test kit,

wherein said test kit comprises a front panel comprising the sample window and a display window;

a back panel; and

a dry chemistry membrane affixed between the front and back panels positioned for display in at least the display window,

wherein said membrane comprises;

a sample region positioned to receive the sample from the sample window,

a control region, and

at least a first antibody pair and a second antibody pair located at discrete locations along said membrane between the sample region and the control region,

each of said antibody pairs comprising an antibody reagent member and an immobilized capture antibody member, each capture antibody member being located on said membrane closer to the control region than the corresponding antibody reagent member, each antibody reagent member having a measurable or observable moiety labeled or chemically bonded thereto, wherein the antibody reagent member of said first antibody pair is said first antibody that specifically binds cardiac Troponin-I and the antibody reagent member of said second antibody pair is said second antibody that specifically binds N-terminal BNP,

such that the cardiac marker of cell injury and the marker of organ adaptation present in the sample will migrate toward the control region, bind to a cognate antibody pair to form an immobilized immunocomplex, and produce a color change proportional to the concentration of each marker in the sample, and

(b) visualizing or measuring the moiety labeled or chemically bonded to said first antibody and said second antibody so as to detect the presence of each marker in said sample.

14. The method of claim 10, wherein said first antibody is a monoclonal antibody and said second antibody is a monoclonal antibody.

15. The method of claim 10, wherein said first antibody is a polyclonal antibody and said second antibody is a polyclonal antibody.

16. The method of claim 6, wherein said increased levels are two-fold greater than in the control samples.

17. The method of claim 6, wherein steps (B) and (C) are carried out simultaneously.

18. The method of claim 10, wherein said increased levels are two-fold greater than in the control samples.

19. The method of claim 10, wherein steps (B) and (C) are carried out simultaneously.

20. The method of claim 10, wherein said first antibody is a monoclonal antibody and said second antibody is a polyclonal antibody.

21. The method of claim 10, wherein said first antibody is a polyclonal antibody and said second antibody is a monoclonal antibody.

22. The method of claim 6, wherein said first antibody specifically binds to cardiac Troponin-I;

and said second antibody specifically binds to a N-terminal ANP.