Method of determining equine NT-proBNP

• US 9,103,839 B2
• Filed: 03/07/2008
• Issued: 08/11/2015
• Est. Priority Date: 03/07/2007
• Status: Expired due to Fees

• Current Assignee: The Antibody Lab GmbH
• Original Assignee: Biomedica Medizinprodukte GmbH & Co., KG
• Inventors: Woloszczuk, Wolfgang, Hawa, Gerhard
• Primary Examiner(s): Gabel, Gail R
• Assistant Examiner(s): GRUN, JAMES LESLIE
• Application Number:
  US12/530,122
• Publication Number:
  US 20100136590A1
• Time in Patent Office: 2,713 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/7.92, 435/7.93, 435/7.94, 435/28, 435/40.5, 435/40.52, 435/69.4, 435/69.6, 435/70.21, 435/331, 435/335, 435/287.2, 435/287.9, 435/975, 436/503, 436/504, 436/514, 436/516, 436/518, 436/536, 436/547, 436/548, 436/8, 436/169, 436/172, 436/807, 436/811, 530/300, 530/317, 530/328, 530/350, 530/387.9, 530/388.24, 530/388.25, 530/389.1, 530/389.2, 530/389.3, 530/391.1, 530/391.3, 530/806, 422/400, 422/420, 422/425, 422/430, 536/23.51
• US Class Current: 1/1
• CPC Class Codes: C07K 14/58 Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• 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 2800/324 Coronary artery diseases, e.g. angina pectoris, myocardial infarction
• CPC Class Codes: G01N 2800/325 Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
• 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

Abstract

The invention relates to a method of determining equine NT-proBNP, or fragments thereof, comprising the steps of: providing an equine sample, contacting the sample with at least one antibody which specifically binds to equine NT-proBNP, and determining the presence and/or concentration of the equine NT-proBNP or fragments thereof existing in the sample.

Claims

1. A method of determining a presence of equine NT-proBNP (N-terminal form of Brain Natriuretic Peptide), or fragments thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO:

1 in an equine sample, comprising;

providing an equine sample;

contacting the sample with at least one isolated antibody that specifically binds to equine NT-proBNP and/or to at least one of said fragments thereof; and

detecting binding of the at least one antibody to the equine NT-proBNP or fragments thereof, thereby determining that the equine NT-proBNP or fragments thereof are present in the sample.

2. The method of claim 1, wherein the at least one antibody specifically binds to at least one specific NT-proBNP-epitope within the sequence of amino acid residues 8 to 81 of SEQ ID NO:

1.

3. The method of claim 2, wherein the NT-proBNP-epitope from SEQ ID NO:

1 is an epitope comprising the sequence PLGGLGPASEQS (SEQ ID NO;

     3), PASEQSGIQELL (SEQ ID NO;

     4), LLDRLGDSVLEP (SEQ ID NO;

     5), SVLEPQAERMTL (SEQ ID NO;

     6), PQAERMTLEPLQ (SEQ ID NO;

     7), EPLQQDRGPAEA (SEQ ID NO;

     8), LQQDRGPAEASE (SEQ ID NO;

     9), DRGPAEASETRG (SEQ ID NO;

     10), PAEASETRGAAP (SEQ ID NO;

     11), RGAAPTGVLGPR (SEQ ID NO;

     12), LGPRTKVLQALR (SEQ ID NO;

     13), or PRTKVLQALRGL (SEQ ID NO;

     14).

4. The method of claim 1, wherein the at least one antibody is polyclonal or monoclonal.

5. The method of claim 1, wherein the at least one antibody is labelled.

6. The method of claim 5, wherein the at least one antibody is labelled with peroxidase, biotin, fluorescent dye, gold colloid or radionuclides.

7. The method of claim 6, wherein the at least one antibody is labelled with horseradish peroxidase.

8. The method of claim 1, wherein the at least one antibody is bound to a solid phase.

9. The method of claim 1, further comprising contacting the sample with at least one isolated anti-NT-proBNP-antibody that binds to equine NT-proBNP but is not specific for equine NT-proBNP.

10. The method of claim 1, further comprising contacting the sample with at least one isolated antibody that is specific to equine pre-proBNP.

11. The method of claim 10, wherein the antibody that is specific to equine pre-proBNP is an antibody that specifically binds to an epitope comprising the sequence SPKMMRNSGCFG (SEQ ID NO:

16) and/or DRIGSFSGLGCN (SEQ ID NO;

     17).

12. The method of claim 1, wherein detecting the binding of the at least one antibody to the equine NT-proBNP or fragments thereof comprises radioimmunoassay, immunobinding assay, Western blot, immunohistochemistry, enzyme-immunoassay, lateral flow device (LFD), or a combination thereof.

13. The method of claim 1, further comprising contacting the at least one antibody that specifically binds to equine NT-proBNP and/or to at least one of said fragments thereof with a polypeptide comprising the amino acid sequence of SEQ ID NO:

1 or the at least one fragment thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO;

1 as a positive control or a standard for a concentration determination.

14. The method of claim 1 further comprising determining a concentration of equine NT-proBNP or fragments thereof in the sample.

15. An isolated antibody or an isolated antibody mixture that specifically binds to equine NT-proBNP (N-terminal form of Brain Natriuretic Peptide) or fragments thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO:

1.

16. The isolated antibody or isolated antibody mixture of claim 15, wherein the isolated antibody or antibody mixture is further defined as binding to at least one epitope with the sequence comprising PLGGLGPASEQS (SEQ ID NO:

3), PASEQSGIQELL (SEQ ID NO;

     4), LLDRLGDSVLEP (SEQ ID NO;

     5), SVLEPQAERMTL (SEQ ID NO;

     6), PQAERMTLEPLQ (SEQ ID NO;

     7), EPLQQDRGPAEA (SEQ ID NO;

     8), LQQDRGPAEASE (SEQ ID NO;

     9), DRGPAEASETRG (SEQ ID NO;

     10), PAEASETRGAAP (SEQ ID NO;

     11), RGAAPTGVLGPR (SEQ ID NO;

     12), LGPRTKVLQALR (SEQ ID NO;

     13), or PRTKVLQALRGL (SEQ ID NO;

     14).

17. A kit adapted for determining equine NT-proBNP (N-terminal form of Brain Natriuretic Peptide) or fragments thereof, comprising the at least one antibody or at least one antibody mixture of claim 15 and a polypeptide comprising the amino acid sequence of claim SEQ ID NO:

1 or specific fragment thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO;

1.

18. The kit of claim 17, further comprising at least one further antibody that specifically binds to the at least one antibody or that specifically binds to equine NT-proBNP or fragments thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO:

1.

19. The kit of claim 17, wherein the at least one antibody is a labelled antibody.

20. The kit of claim 19, wherein the at least one antibody is labelled with peroxidase, biotin, fluorescent dye, gold colloid or radionuclides.

21. An isolated antibody mixture comprising at least one isolated anti-NT-proBNP-antibody that binds to equine NT-proBNP but is not specific for equine NT-proBNP and further comprising an isolated antibody or an isolated antibody mixture that specifically binds to equine NT-proBNP (N-terminal form of Brain Natriuretic Peptide) or fragments thereof comprising at least 8 contiguous amino acids from the sequence of amino acid residues 8 to 81 of SEQ ID NO:

1.

22. The isolated antibody mixture of claim 21, wherein the isolated antibody or antibody mixture that specifically binds to equine NT-proBNP or said fragments thereof is further defined as binding to at least one epitope from SEQ ID NO:

1, further defined as the sequence comprising PLGGLGPASEQS (SEQ ID NO;

     3), PASEQSGIQELL (SEQ ID NO;

     4), LLDRLGDSVLEP (SEQ ID NO;

     5), SVLEPQAERMTL (SEQ ID NO;

     6), PQAERMTLEPLQ (SEQ ID NO;

     7), EPLQQDRGPAEA (SEQ ID NO;

     8), LQQDRGPAEASE (SEQ ID NO;

     9), DRGPAEASETRG (SEQ ID NO;

     10), PAEASETRGAAP (SEQ ID NO;

     11), RGAAPTGVLGPR (SEQ ID NO;

     12), LGPRTKVLQALR (SEQ ID NO;

     13), or PRTKVLQALRGL (SEQ ID NO;

     14).