Specific antibodies for diagnosing heart failure
View Patent ↗The invention relates to an in vitro diagnosis of heart failure. More specifically, the invention relates to specific antibodies of a peptide domain which is situated on either side of hinge region R76S77 of proBNP(1-108). In particular, the invention relates to a method of obtaining the aforementioned antibodies and to the use thereof in detecting blood proBNP(1-108) with the exception of BNP(1-76) and BNP(77-108). The invention also relates to a method of detecting blood proBNP(1-108), reagents and a kit for same.
1. An isolated anti-proBNP(1-108) antibody, characterized in that, firstly, it specifically recognizes the sequence RAPR 76 S 77 P (SEQ ID NO: 5) of proBNP(1-108) and does not substantially recognize the peptides BNP(1-76) or BNP(77-108) and, secondly, it has the ability to specifically recognize circulating proBNP(1-108) in human serum or plasma samples.
2. The anti-proBNP(1-108) antibody as claimed in claim 1 , which specifically recognizes the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 (SEQ ID NO: 4) of proBNP(1-108).
3. The anti-proBNP(1-108) antibody as claimed in claim 1 , which specifically recognizes the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108) of proBNP(1-108).
4. A method for obtaining an anti-proBNP(1-108) antibody as defined in claim 1 , in which an animal is immunized with the whole proBNP(1-108) molecule, and then the antiserum obtained is depleted using the BNP(77-108) peptide and/or the BNP(1-76) peptide.
5. A method for obtaining an anti-proBNP(1-108) antibody as defined in claim 1 , in which an animal is immunized with a peptide chosen from
a peptide of formula
a 1 -X 1 -RAPRSP-X 2 -a 2 (SEQ ID NO: 5) (I)
where
a1 may be H or may represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary amine or secondary amine function, an aminocarboxyl group, a biotinyl group and an acetyl group,
X 1 represents a peptide sequence of 0 to 3 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
X 2 represents a peptide sequence of 0 to 7 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
a2 may represent an OH function, an NH2 function or an alkoxyl group;
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID NO: 108) (III)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide comprising a sequence derived from the sequence
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (II) or from the sequence
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (III) by substitution of one or more among the amino acids Y 70 , T 71 , L 72 , K 79 , M 80 , V 81 , Q 82 , G 83 , S 84 and G 85 , with it being possible for X to be H or to represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where
Z may be an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G (C13P30: SEQ ID NO: 16);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S (CN32: SEQ ID No. 109);
and, optionally, the antiserum obtained is depleted using the BNP(77-108) peptide and/or the BNP(1-76) peptide.
6. A method for obtaining a hybridoma that secretes an anti-proBNP(1-108) antibody as defined in claim 1 , in which an animal is immunized with a peptide chosen from
a peptide of formula
a 1 -X 1 -RAPRSP-X 2 -a 2 (SEQ ID NO: 5) (I)
where
a 1 may be H or may represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary amine or secondary amine function, an aminocarboxyl group, a biotinyl group and an acetyl group,
X 1 represents a peptide sequence of 0 to 3 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
X 2 represents a peptide sequence of 0 to 7 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
a 2 may represent an OH function, an NH 2 function or an alkoxyl group;
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID NO: 108) (III)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide comprising a sequence derived from the sequence
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (II) or from the sequence
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (III) by substitution of one or more among the amino acids Y 70 , T 71 , L 72 , K 79 , M 80 , V 81 , Q 82 , G 83 , S 84 and G 85 , with it being possible for X to be H or to represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where
Z may be an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G (C13P30: SEQ ID NO: 16);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S (CN32: SEQ ID NO: 109);
immunoglobulin-secreting lymphocytes are taken from this animal,
and the lymphocytes are fused with myeloma cells so as to obtain at least one immunoglobulin-secreting hybridoma.
7. The method as claimed in claim 5 , in which the peptide of formula (II) has the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 (SEQ ID NO: 4).
8. The method as claimed in claim 5 , in which the peptide of formula (III) has the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108).
9. A hybridoma which can be produced by the method as claimed in claim 6 , wherein the antibody secreted by the hybridoma specifically recognizes the sequence RAPR 76 S 77 P (SEQ ID NO: 5) of proBNP(1-108) and does not substantially recognize the peptides BNP(1-76) or BNP(77-108).
10. An anti-proBNP(1-108) monoclonal antibody secreted by a hybridoma as claimed in claim 9 .
11. A method of in vitro diagnosis of heart failure in a human subject, comprising bringing a biological sample of the human subject into contact with an anti-proBNP(1-108) antibody as defined in claim 1 and detecting the proBNP(1-108) in the sample, whereby, if the proBNP(1-108) concentration in the biological sample is higher than that of normal individuals, then the human subject is diagnosed as having heart failure.
12. A method of in vitro diagnosis of heart failure in a human subject, comprising:
a) bringing a biological sample of the human subject into contact with an anti-proBNP(1-108) antibody as defined in claim 1 ,
b) incubating the mixture under conditions that allow the formation of antigen-antibody complexes, and
c) revealing the antigen-antibody complexes formed, optionally using a labeled detection antibody capable of binding specifically to the proBNP(1-108) present in the primary complex, or using a labeled detection antigen capable of binding to the antibody directed against said proBNP(1-108) present in the primary complex, and
d) correlating the amount of antigen-antibody complexes revealed with the clinical condition of the human subject, wherein, if the amount of antigen-antibody complexes is higher than that of normal individuals, then the human subject is diagnosed as having heart failure.
13. A kit for detecting proBNP(1-108) in a biological sample, containing at least one antibody as defined in claim 1 .
14. The kit for detecting proBNP(1-108) in a biological sample, as claimed in claim 13 , containing:
(i) in a container, the at least one antibody;
(ii) in another container, at least one peptide chosen from
a peptide of formula
a 1 -X 1 -RAPRSP-X 2 -a 2 (SEQ ID NO: 5) (I)
where
a 1 may be H or may represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary amine or secondary amine function, an aminocarboxyl group, a biotinyl group and an acetyl group,
X 1 represents a peptide sequence of 0 to 3 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
X 2 represents a peptide sequence of 0 to 7 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
a 2 may represent an OH function, an NH 2 function or an alkoxyl group;
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID NO: 108) (III)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
a peptide comprising a sequence derived from the sequence X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II) or from the sequence X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID NO: 108) (III) by substitution of one or more among the amino acids Y 70 , T 71 , L 72 , K 79 , M 80 , V 81 , Q 82 , G 83 , S 84 and G 85 , with it being possible for X to be H or to represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may be an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108);
the peptide having the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 (SEQ ID NO: 4);
the peptide having the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G (C13P30: SEQ ID NO: 16);
the peptide having the sequence C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S (CN32: SEQ ID NO: 109).
15. A peptide of formula:
a 1 -X 1 -RAPRSP-X 2 -a 2 (SEQ ID NO: 5) (I)
where
a 1 may be H or may represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary amine or secondary amine function, an aminocarboxyl group, a biotinyl group or an acetyl group,
X 1 represents a peptide sequence of 0 to 3 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
X 2 represents a peptide sequence of 0 to 7 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
a 2 may represent an OH function, an NH 2 function, or an alkoxyl group.
16. A peptide of formula
X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108).
17. The peptide as claimed in claim 16 , having the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 (SEQ ID NO: 4).
18. A peptide of formula:
X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID NO: 108) (III)
where X may be H or may represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108).
19. The peptide as claimed in claim 18 , having the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108).
20. A peptide comprising a sequence derived from the sequence X-Y 70 TLRAPR 76 S 77 PKMVQGSG 85 -Z (SEQ ID NO: 4) (II) or from the sequence X-Y 70 TLRAPR 76 S 77 PKMVQGS 84 -Z (SEQ ID No. 108) (III) by substitution of one or more among the amino acids Y 70 , T 71 , L 72 , K 79 , M 80 , V 81 , Q 82 , G 83 , S 84 and G 85 , with it being possible for X to be H or to represent either an acetyl group, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108), and where Z may be an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP(1-108).
21. The peptide as claimed in claim 15 , having a sequence chosen from the group consisting of the following sequences
SEQ ID NO: 16:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G
(peptide C13P30)
SEQ ID NO: 109:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S
(peptide CN32)
SEQ ID NO: 6:
C-G-R-A-P-R-S-P
SEQ ID NO: 7:
Acetyl-C-G-R-A-P-R-S-P
SEQ ID NO: 8:
C-G-R-A-P-R-S-P-K
SEQ ID NO: 9:
Acetyl-C-G-R-A-P-R-S-P-K
SEQ ID NO: 10:
C-G-R-A-P-R-S-P-K-M-V
SEQ ID NO: 11:
C-G-R-A-P-R-S-P-K-M-V-Q-G-S-G
SEQ ID NO: 12:
R-A-P-R-S-P-G-C
SEQ ID NO: 13:
Acetyl-R-A-P-R-S-P-G-C
SEQ ID NO: 110:
C-Y-T-L-R-A-P-R-S-P-K
SEQ ID NO: 111:
C-Y-T-L-R-A-P-R-S-P-K-M-V
SEQ ID NO: 112:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q
SEQ ID NO: 113:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G
SEQ ID NO: 19:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-bA
SEQ ID NO: 20:
C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-A-T-bA
SEQ ID NO: 114:
Acetyl-C-T-L-R-A-P-R-S-P-K-M-V-Q
SEQ ID NO: 115:
C-T-L-R-A-P-R-S-P-K-M-V-Q-G
SEQ ID NO: 116:
C-T-L-R-A-P-R-S-P-K-M-V-Q-G-S
SEQ ID NO: 117:
C-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G
SEQ ID NO: 118:
C-L-R-A-P-R-S-P-K-M-V
SEQ ID NO: 119:
C-L-R-A-P-R-S-P-K-M-V-Q
SEQ ID NO: 120:
L-R-A-P-R-S-P-K-M-V-Q-C
SEQ ID NO: 121:
C-L-R-A-P-R-S-P-K-M-V-Q-G-S
SEQ ID NO: 122:
C-L-R-A-P-R-S-P-K-M-V-Q-G-S-G.
22. A method for obtaining anti-proBNP(1-108) antibodies that specifically recognize the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 , (SEQ ID NO: 4) the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108) and/or the sequence RAPR 76 S 77 P (SEQ ID NO: 5) of proBNP(1-108) with the substantial exclusion of the BNP(1-76) and BNP(77-108) peptides, and that have the ability to specifically recognize circulating proBNP(1-108) in human serum or plasma samples, in which method an animal is immunized with a peptide as defined in claim 15 , and, optionally, the antiserum obtained is depleted using the BNP(77-108) peptide and/or the BNP(1-76) peptide.
23. A method for obtaining a hybridoma that secretes an anti-proBNP(1-108) antibody that specifically recognizes the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG85 (SEQ ID NO: 4), the sequence Y 70 TLRAPR 76 S 77 PKMVQGS84 (SEQ ID NO: 108) and/or the sequence RAPR 76 S 77 P (SEQ ID NO: 5) of proBNP(1-108) with the substantial exclusion of the BNP(1-76) and BNP(77-108) peptides, and that has the ability to specifically recognize circulating proBNP(1-108) in human serum or plasma samples, in which method an animal is immunized with a peptide as defined in claim 15 , followed by removal of immunoglobulin-secreting lymphocytes from this animal, and said lymphocytes are fused with myeloma cells so as to obtain at least one immunoglobulin-secreting hybridoma.
24. An anti-proBNP(1-108) antibody, characterized in that it is obtained by a method as claimed in claim 22 .
25. The anti-proBNP(1-108) antibody as claimed in claim 24 , which specifically recognizes the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG 85 (SEQ ID NO: 4) or the sequence Y 70 TLRAPR 76 S 77 PKMVQGS 84 (SEQ ID NO: 108) of proBNP(1-108).
26. A hybridoma which can be produced by the method as claimed in claim 23 , wherein the antibody secreted by the hybridoma specifically recognizes the sequence Y 70 TLRAPR 76 S 77 PKMVQGSG85 (SEQ ID NO: 4), the sequence Y 70 TLRAPR 76 S 77 PKMVQGS84 (SEQ ID NO: 108) and/or the sequence RAPR 76 S 77 P (SEQ ID NO. 5) of proBNP(1-108) with the substantial exclusion of the BNP(1-76) and BNP(77-108) peptides.
27. An anti-proBNP(1-108) monoclonal antibody secreted by a hybridoma as claimed in claim 26 .
28. The anti-proBNP(1-108) monoclonal antibody as claimed in claim 27 , secreted by the hybridoma 3D4 deposited with the CNCM under the No. CNCM I-3073.
29. A method of in vitro diagnosis of heart failure in a human subject, comprising bringing a biological sample of the human subject into contact with an anti-proBNP(1-108) antibody as defined in claim 24 and detecting the proBNP(1-108) in the sample, whereby, if the proBNP(1-108) concentration in the biological sample is higher than that of normal individuals, then the human subject is diagnosed as having heart failure.
30. A method of in vitro diagnosis of heart failure in a human subject, comprising:
a) bringing a biological sample of the human subject into contact with an anti-proBNP(1-108) antibody as defined in claim 24 ,
b) incubating the mixture under conditions that allow the formation of antigen-antibody complexes, and
c) revealing the antigen-antibody complexes formed, optionally using a labeled detection antibody capable of binding specifically to the proBNP(1-108) present in the primary complex, or using a labeled detection antigen capable of binding to the antibody directed against said proBNP(1-108) present in the primary complex, and
d) correlating the amount of antigen-antibody complexes revealed with the clinical condition of the human subject, wherein if the amount of antigen-antibody complexes is higher than that of normal individuals then the human subject is diagnosed as having heart failure.
31. A kit for detecting proBNP(1-108) in a biological sample, containing at least one antibody as defined in claim 24 .
32. A kit for detecting proBNP(1-108) in a biological sample, containing, as standard and/or control, at least one peptide as defined in claim 15 .
33. A kit for detecting proBNP(1-108) in a biological sample, containing:
in a container, at least one antibody as defined in claim 24 ;
in another container, at least one peptide of formula:
a 1 -X 1 -RAPRSP-X 2 -a 2 (SEQ ID NO: 5) (I)
where
a 1 may be H or may represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary amine or secondary amine function, an aminocarboxyl group, a biotinyl group or an acetyl group,
X 1 represents a peptide sequence of 0 to 3 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
X 2 represents a peptide sequence of 0 to 7 amino acids, which may or may not be derived from the natural sequence of proBNP(1-108),
a 2 may represent an OH function, an NH 2 function, or an alkoxyl group.