Methods of determination of activation or inactivation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) hormonal systems

1. An in vitro method of determining activation or inactivation of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) hormonal systems in a subject, the method comprising detecting in a single reading, in a single assay the combined presence of atrial and brain natriuretic peptide prohormones (proANP and proBNP) or fragments thereof in a sample from the subject, wherein, compared to a reference level, detection of an increase in the combined presence of proANP and proBNP, or fragments thereof, in the sample indicates activation of the ANP and BNP hormonal systems, and wherein, compared to a reference level, detection of a decrease in the combined presence of proANP and proBNP, or fragments thereof, in the sample indicates inactivation of these systems, wherein said method does not comprise detection of the presence of proANP and proBNP or fragments thereof individually; wherein detection of activation of the ANP and BNP hormonal systems is diagnostic of cardiac impairment or detection of inactivation of ANP and BNP hormonal systems indicates successful treatment of cardiac impairment.

2. The method according to claim 1 , which comprises contacting the sample with a bi- or oligo-specific first binding substance that is able to bind to:

(a) proANP (SEQ ID NO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3); and

(b) proBNP (SEQ ID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6).

3. The method according to claim 1 which comprises contacting the sample with

a fusion polypeptide agent comprising both:

(a) proANP (SEQ ID NO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3);

and

(b) proBNP (SEQ ID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6);

wherein said fusion polypeptide agent can be bound by a first binding substance, and said fusion polypeptide agent is used as a calibration agent or a competitive inhibitor;

and

said first binding substance, which is able to bind to:

(c) proANP (SEQ ID NO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3);

(d) proBNP (SEQ ID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6); and

(e) said fusion polypeptide agent.

4. The method according to claim 3 wherein the first binding substance comprises:

(a) a bi- or oligo-specific binding substance; or

(b) a mixture of mono-specific binding substances.

5. The method according to claim 2 wherein the first binding substance comprises an antibody or a fragment or derivative thereof, wherein said fragment or derivative thereof retains the binding characteristics of said antibody.

6. The method according to claim 5 wherein the antibody comprises a polyclonal antibody, monoclonal antibody, oligoclonal antibody, bifunctional antibody or crossreacting polyclonal antibody.

7. The method according to claim 3 wherein in the fusion polypeptide agent, (a) is SEQ ID NO:3 and (b) is SEQ ID NO: 6, or (a) is SEQ ID NO:2 and (b) is SEQ ID NO:5.

8. The method according to claim 1 which comprises contacting the sample with

a fusion polypeptide agent comprising:

(a) proBNP 15-24 and proANP 82-96 ;

(b) proBNP 1-37 and proANP 29-98 ;

(c) proBNP 10-29 and proANP 20-80 ;

(d) proBNP 1-76 and proANP 1-98 ;

(e) proBNP 10-29 and proANP 60-80 ;

(f) proBNP 1-108 and proANP 1-126 ; or

(g) proBNP 77-92 and proANP 112-126 ;

wherein said fusion polypeptide agent can be bound by a first binding substance, and said fusion polypeptide agent is used as a calibration agent or a competitive inhibitor; and

said first binding substance, which is able to bind to:

(h) proANP (SEQ ID NO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3); and

(i) proBNP (SEQ ID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6).

9. The method according to claim 2 wherein the first binding substance and/or the fusion polypeptide agent is:

(a) labelled with a detectable label; and/or

(b) immobilised.

10. The method according to claim 2 which additionally comprises contacting the sample with a second binding substance which is able to bind to the first binding substance.

11. The method according to claim 10 wherein the second binding substance is:

(a) labelled with a detectable label; and/or

(b) immobilised.

12. The method according to claim 10 wherein the second binding substance causes precipitation of the first binding substance and any peptide which is bound to it.

13. The method according to claim 1 which comprises an immunoassay.

14. The method of claim 1 , wherein said reference level is determined from a previous measurement from said subject.

15. The method of claim 1 , wherein said reference level is based on the normal level of a population of subjects.

16. The method of claim 15 , wherein said population of subjects is the general population.

17. The method of claim 1 , wherein said assay is calibrated so that a particular reading in the assay is known to represent the normal peptide level.

18. The method of claim 1 , wherein said assay is calibrated so that a normal level will produce a negligible or insignificant result.

19. The method of claim 3 , wherein said assay is calibrated by use of said fusion polypeptide agent.

20. The method of claim 1 , wherein said subject is a human.