FR2863269A1 - New peptides containing an epitope from the hinge region of pro-brain natriuretic peptide, useful as standard or control in assay of pro-BNP for diagnosis of cardiac insufficiency - Google Patents

Abstract

Peptides (I) that contain a pro-BNP (brain natriuretic peptide) epitope are new. Peptides of formula (I) that contain a pro-BNP (brain natriuretic peptide) epitope are new. a1-X1-ArgAlaProArgSerPro-X2-a2 (I) a1 : hydrogen, thiol, hydroxy, aminoxy, primary or secondary amino, aminocarbonyl, biotin residue or acetyl; X1 : sequence of 0-3 amino acids (aa) optionally derived from the natural sequence of proBNP(1-108); X2 : sequence of 0-8 aa optionally derived from the natural sequence of proBNP(1-108); and a2 : hydroxy, primary amino or alkoxy. Independent claims are also included for the following: (1) peptides (II) or their derivatives (IIa); and (2) kit, for detecting proBNP(1-108) in a biological sample, that contains at least one (I) or (II) as standard and/or control. X-Tyr(70)ThrLeuArgAlaProArg(76)Ser(77)ProLysMetValGlnGlySerGly(85)-Z (II) X : hydrogen, acetyl, 1-3 aa that do not belong to the sequence of proBNP(1-108); and Z : hydroxy or 1-3 aa that do not belong to the sequence of proBNP(1-108).

Description

The invention relates to the field of in vitro diagnosis of insufficiency

ventricular heart.

  Congestive heart failure is a common clinical syndrome, especially in the elderly. It usually presents as an insidious onset of nonspecific symptoms such as exertional coughing, fatigue, and the appearance of peripheral edema. The diagnosis is classically based on the study of various parameters such as the clinical signs (classified in four stages: NYHA stages I to IV) (ie, the New York Heart Association). echocardiography, scintigraphy, stress test, etc.

  Due to the severity of the heart disease and also the high costs of its management, an early diagnosis of this syndrome is, of course, very highly desirable: it would help to prevent the rapid progression of the syndrome to heart failure strict. Identifying people at risk of heart failure is therefore a necessity. This would also make it possible to adapt therapeutic monitoring faster, easier and less expensive. Unfortunately, there is no fully satisfactory and fully informative method of diagnosing heart failure.

  Presymptomatic markers predictive of heart failure have long been sought. In this regard, it has been demonstrated that cardiomyocytes manufacture and secrete peptides with natriuretic activity: a peptide of atrial origin, Atrial Natriuretic Peptide (ANP) discovered in the rat by Bold et al. Life Science 1981, vol. 28 (1): 89-94, and an atrioventricular peptide of atrioventricular origin called BNP (Brain Natriuretic Peptide) discovered by Sudoh et al., Nature 1988, vol. 332: 78-81 in pigs, and then in humans.

  The precursor of BNP is preproBNP (1-134), a form of storage of the molecule inside the cardiomyocytes. This is cleaved to release a signal peptide and proBNP (1-108). ProBNP (1-108) consists of a polypeptide of 108 amino acids, of the following sequence: ## STR1 ## (SEQ ID NO: 1) [SEQ. ID. It is cleaved, before and / or during its secretion, between the amino acids Arg76 and Ser77, on the one hand, the BNP, also designated BNP (77-108) or BNP-32, or even BNP (1-32), and the N-terminal portion of the prohormone, BNP (1-76), also referred to as the N-terminal fragment of proBNP or NT-proBNP.

  BNP or BNP (77-108), a vasoactive form of the molecule, consists of a 32 amino acid peptide of sequence: S77PKMVQGSGCFGRKMDRISSSSGLGCKVLRRH108 (SEQ ID No. 2). NT-proBNP or BNP (1-76) consists of the 76 N-terminal amino acids of proBNP (1-108) constituting the following sequence: ## STR1 ##.

  The plasma level of BNP hormonal BNP (77-108) is elevated in patients with ventricular dystrophy. Plasma assays for BNP (77-108) have been used as a predictive marker for ventricular heart failure. However, it is well known that the hormone BNP (77-108) is unstable. As a result, its dosage requires special precautions (Davidson, N.C. et al., Circulation 1995: 91: 1276) (Gobinet-Georges et al., Clin Chem Chem Med 2000: 38: 519-23). In addition, the half-life of BNP very short and its plasma concentration is low. As a result, a number of false negative responses are observed in subjects at risk of heart failure. Thus, the BNP (77-108) assay does not correctly discriminate the NYHA stage I patients from healthy subjects (Clerico A. et al., J. Endocrinol Invest 1998; 21: 170-9 ) (Del Ry S. et al Scand J. Clin Lab.Invest 2000, 60: 81-90).

  To circumvent this difficulty, the patent application WO 93/24531 describes a method for the in vitro diagnosis of heart failure based on the detection of BNP (1-76) (N-terminal fragment of proBNP), an abundant compound and which has a long half-life compared to that of the hormone BNP (77-108). However, the method described in the application WO 93/24531 does not seem easy to implement on the BNP (1-76) in blood samples. Indeed, the only examples shown are carried out, not on actual sera, but on standard ranges obtained with the aid of a synthetic peptide, the peptide BNP (47-64), subsequence of BNP (1-76). ). To overcome this disadvantage, a highly sophisticated automated system has since proved necessary.

  The article Hunt et al. Biochemical and Biophysical Research Communications, Vol. 214 (3), 1995, pp. 1175-1183 discloses a competitively-type RIA assay of BNP (1-76) on plasmas of patients with heart failure, involving antiserum directed against the N-terminal fragment of proBNP (1-13). The article specifically shows that, in patients with heart failure, the level of BNP (1-76), which correlates very well with that of BNP (77-108), is considerably high compared to the level observed in control subjects. However, the protocol described for specifically extracting the only plasmatic BNP (1-76) is complex, since it requires extraction of plasma from Sep-pak C18TM cartridge (Millipore-Waters), followed by HPLC chromatography. Moreover, this article emphasizes that, in the RIA assay thus implemented, proBNP (1-108) does not appear to be recognized. Instead, it suggests that proBNP (1-108) could be secreted into the circulation from cardiac tissue but would then be rapidly degraded to a smaller peptide by cleavage of N-terminal acids. Or, according to the article, proBNP (1-108) would be presented in such a way that anti-proBNP antiserum (1-108) would be unable to bind to it. Finally, the authors suggest that BNP (1-76) (N-terminal fragment of proBNP) could even be a more specific marker of cardiac dysfunction than BNP (77-108) or the Nterminal fragment of proANF '.

  Karl et al. (Scand J. Clin, Lab.Invest 1999; 59 (suppl 230): 177,181) describes a method of detection of BNP (1-76) similar to that of patent application WO93 / 24531, but it does not provide no results obtained on patient samples.

  The article Schulz et al. Scand. J. Clin. Lab. lnvest., 2001, vol. 61, pp. 33-42, also discloses a radioimmunoassay specific for BNP (176) (N-terminal fragment of proBNP), without extraction, with the aid of an antiserum directed against the amino acids 1-21 of this fragment. The authors confirm the interest of the assay of BNP (1-76) in the diagnosis of ventricular heart failure as well as its good correlation with the BNP assay (77-108). In a study of the different circulating forms of proBNP (1-108), they hypothesized that proBNP (1108) would circulate in the blood, both in the form of intact prohormone and cleavage products, BNP (1). -76) (N-terminal fragment of proBNP) and BNP (77-108). However nothing is said or suggested in the article concerning a possible physiological activity of proBNP (1-108) or any diagnostic value of proBNP (1-108) as a predictive or diagnostic marker of ventricular heart failure.

  The article Shimizu et al. Clinica Chimica Acta, 2002, vol. 316, pp. 129135, presents a study on the degradation of human BNP in blood and circulating molecular forms of immunoreactive BNP in the plasma of heart failure. He observed in the plasma of the latter the presence of two forms of immunoreactive BNP: a high molecular weight BNP (36 KD, which could correspond to a proBNP trimer (1108)) and a low molecular weight BNP. The latter corresponds to the simultaneous presence of a form of degradation product of BNP-32 which has lost the N-terminal serine and proline (it is the des-SerPro-BNP (BNP332)) of the hormonal form of BNP-32. 32 (here called BNP (1-32)). ProBNP (1108) and hormonal BNP (BNP-32, BNP (1-32) or BNP (77-108)) are therefore secreted by the heart into the blood. Nevertheless, the authors seem to suggest that proBNP (1-108) in its oligomerized form (trimer) is present in a concentration close to that of circulating BNP (77-108), but they do not measure it. As a result, the correlation of the concentration of proBNP (1-108) with the clinical status of patients is not studied. It follows that the diagnostic or prognostic value of serum proBNP (1-108) is not demonstrated. It is also not suggested that it is possible to measure the latter routinely.

  There is therefore still a need, in the context of the early diagnosis of heart failure, to have a method which avoids the disadvantages of the prior art. In particular, there is a need for a simple method, routinely and reliably usable, which avoids the disadvantages of detecting BNP (77-108), a scarcely abundant and unstable molecule, while avoiding complex extractions, caused by the assay of other molecular forms of BNP, and which may eventually go as far as requiring sophisticated automation.

  The authors of the present invention have therefore endeavored to develop an alternative method for solving the problem. At the heart of the present invention lies the unexpected discovery, made by the inventors, of an epitope with unique properties located in the region of the hinge peptide Y70TLRAPR76S77PKMVQGSG85 (SEQ ID No. 4) of proBNP (1-108) and comprising at least the sequence RAPR76S77P (SEQ ID No. 5).

  Indeed, during an immunization performed in rabbits with a peptide of the hinge region of proBNP (1-108), of sequence Y70TLRAPR76S77PKMVQGSGS5 (SEQ ID No. 4), they surprisingly found that the antiserum obtained not only contained antibodies that specifically recognized said peptide in the hinge region without substantially recognizing the forms of BNP (1-76) and BNP (77-108), but in addition had the ability to recognize circulating proBNP (1108).

  The present inventors have furthermore shown for the first time that the circulating proBNP (1-108) was indeed a predictive marker of heart failure and that it was present at a significantly higher concentration in heart failure patients. only in healthy control subjects.

  The authors have also discovered that another way to obtain this type of antibody is to immunize animals with the complete proBNP molecule (1-108). Indeed, the authors found that immunization with the complete proBNP (1-108) molecule made it possible to induce the appearance of antibodies specifically recognizing a peptide in the hinge region.

  In addition, the authors of the present invention have demonstrated that the minimum epitope recognized by the antibodies according to the invention has the following sequence: RAPR76S77P. They have furthermore shown that a good way to obtain the antibodies which are the subject of the present invention is to immunize animals with a peptide of general formula: ## STR1 ## where to be H or to represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary or secondary amine function, an amino-carboxyl group, a biotinyl group and an acetyl group, X, represents a peptide sequence of 0 to 3 acids amino, resulting from the natural sequence of proBNP (1-108) or not, X2 represents a peptide sequence of 0 to 8 amino acids, resulting from the natural sequence of proBNP (1-108) or not, a2 can represent a function OH, NH2 or an alkoxyl group.

  Similarly, the authors of the present invention have shown that it is possible to obtain the same specific antibodies by immunizing an animal with a peptide comprising the sequence RAPR76S77P or with a peptide of formula: X-Y70TLRAPR76S77PKMVQGSG85-Z ( II) where X can be H or represent either an acetyl group or 1-3 amino acids not belonging to the proBNP (1-108) sequence, and where Z can represent an OH function, or 1 to 3 amino acids not belonging to the proBNP sequence (1-108).

  The authors of the present invention have further developed a simple and reliable method of early diagnosis of heart failure based on the detection of proBNP (1-108) circulating in the blood and a kit allowing the implementation of this detection of circulating proBNP (1-108).

  The subject of the present invention is therefore an anti-proBNP (1-108) antibody, characterized in that, on the one hand, it specifically recognizes the proBNP (1-108) peptide Y7OTLRAPR76S77PKMVQGSG85 and does not substantially recognize the BNP (1 -76) and BNP (77-108), and, on the other hand, has the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples.

  The present invention more particularly relates to an anti-proBNP antibody (1-108), characterized in that, on the one hand, it specifically recognizes the RAPR76S77P peptide of proBNP (1-108) and does not substantially recognize BNP ( 1-76) and BNP (77-108), and, on the other hand, has the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples.

  The present invention furthermore relates to a process for obtaining anti-proBNP (1-108) antibodies specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1108) with the substantial exclusion of BNP (1). -76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples characterized in that an animal is immunized with the entire proBNP molecule (1-108), and then, using the BNP peptide (77-108) and / or BNP peptide (1-76), the antiserum obtained is depleted.

  By exhaustion of an antiserum obtained against a given specific antigen (the immunizing antigen) is meant the removal of non-specific antibodies, potentially present in this antiserum, by contacting and incubating said antiserum with antigens. non-specific, ie different antigens of the immunizing antigen, then immunological separation and elimination of the antibodies reacted with said non-specific antigens and recovery of the antiserum thus exhausted (that is to say depleted of antibodies non-specific). Exhaustion is typically used to make specific antiserum directed against a given antigen.

  In the present case, an antiserum recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 or the RAPR76S77P peptide of proBNP (1-108) can be exhausted, ie made specific, by contact with BNP (77-108) and / or BNP (1-76) supraommés, the latter may, for example, be immobilized in solid phase and serve as a support for immunoadsorption chromatography according to conventional techniques, known to those skilled in the art. The antibody finally present in the exhausted antiserum is here a monospecific polyclonal antibody.

  The subject of the present invention is also a peptide of formula: a1-X1 RAPRSP-X2-a2 (I) in which a1 may be H or represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary or secondary amine function , an amino-carboxyl group, a biotinyl group and an acetyl group, X1 represents a peptide sequence of 0 to 3 amino acids, derived from the natural sequence of proBNP (1-108) or not, X2 represents a peptide sequence of 0 At 8 amino acids, resulting from the natural sequence of proBNP (1-108) or not, a2 can represent an OH, NH2 or an alkoxyl group. The present invention also relates to a peptide of formula: XY70TLRAPR76S77PKMVQGSG85-Z (II) where X can be H, or represent either an acetyl group, or 1 to 3 amino acids not belonging to the proBNP (1-108) sequence, and where Z can represent an OH function, or 1 to 3 amino acids not belonging to the sequence of proBNP (1-108).

  Note that in the formula (II) above, the amino acid number (Y70, R76S77, G85) has been maintained simply to assist in the understanding of the invention and the identification of this sequence vis-à-vis the proBNP sequence (1-108). The same is true of the other sequences presented with numbers in this application.

  The invention also relates to any peptide containing the XY70TLRAPR76S77PKMVQGSG85-Z (II) sequence, in substituted form, conservatively or non-conservatively, in any amino acid from position 70 to position 85 provided that it remains intact. (in particular not substituted) RAPR76S77P portion.

  It is therefore a peptide comprising a sequence derived from the sequence X-Y70TLRAPR76S77PKMVQGSG85-Z (II) by substitution of one or more of the amino acids Y70, T71, L72, K79, M80, V81, Q82, G83 , S84 and G85, with X being absent or representing either an NH2 function or 1-3 amino acids not belonging to the proBNP sequence (1-108), and where Z can be absent or represent either an OH function or 1 to 3 amino acids not belonging to the proBNP (1-108) sequence.

  The invention also relates to the peptide of sequence Y70TLRAPR76S77PKMVQGSG85.

  The subject of the invention is also a process for obtaining anti-proBNP (1-108) antibodies specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) with the substantial exclusion of BNP ( 1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples characterized in that an animal is immunized with a peptide of formula where X1, X2, and a2 have the same meaning as above, and, optionally, that one exhausts, using the peptide BNP (77-). 108) and / or BNP peptide (1-76), the antiserum obtained. The antibody thus obtained is a monospecific antibody.

  The subject of the invention is also a process for obtaining anti-proBNP (1-108) antibodies specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) with the substantial exclusion of BNP ( 1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples characterized in that an animal is immunized with a peptide of formula : XY70TLRAPR76S77PKMVQGSG85-Z (II) wherein X and Z are as defined above and, optionally, in that one exhausts, using peptide BNP (77-108) and / or peptide BNP (1-76 ), the antiserum obtained.

  The subject of the invention is also a process for obtaining an anti-proBNP antibody secreting hybridoma (1-108) specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) excluding substantial amount of BNP (1-76) and BNP (77-108), and Io having the ability to specifically recognize proBNP (1-108) circulating in serum or human plasma samples characterized in that an animal is immunized with a peptide of formula: X-Y70TLRAPR76S77PKMVQGSG85-Z (II) in substituted form, conservatively or otherwise, provided that it retains intact (in particular unsubstituted) portion RAPR76S77P, where X and Z are as defined herein above and, optionally, in that the antiserum obtained is depleted with the aid of the peptide BNP (77-108) and / or the peptide BNP (1-76).

  The subject of the invention is also a process for obtaining anti-proBNP (1-108) antibodies specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) with the substantial exclusion of peptides. BNP (1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples, characterized in that an animal is immunized with the peptide sequence Y70TLRAPR76S77PKMVQGSG85, and, optionally, that one exhausts, with the BNP peptide (77-108) and / or dru peptide BNP (1-76), the antiserum obtained.

  The subject of the invention is also a process for obtaining an anti-proBNP antibody secreting hybridoma (1-108) specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) excluding substantial amount of BNP (1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples characterized in that: - an animal is immunized with a peptide chosen from the peptides of the following formulas: ## STR2 ## where al, XI, X2, and a2 have the same meaning as above, X-Y70TLRAPR76S77PKIMVQGSG85-Z (II) where X and Z have the same meaning as above, X-Y70TLRAPR76S77PKMVQGSG85- Z (II) II in substituted form, conservatively or not, provided that it retains intact (in particular unsubstituted) portion RAPR76S77P, where X and Z have the same meaning as above, Y70TLRAPR76S77PKMVQGSG85, - we are In this animal, immunoglobulin-secreting lymphocytes are derived, and lymphocytes are fused with myeloma cells to obtain at least one immunoglobulin-secreting hybridoma.

  This corresponds to the conventional technique for obtaining hybridomas whose principle is described in Kohler and Milstein, (1975) Nature (London), 256: 495-497.

  The subject of the invention is also such a hybridoma and the monoclonal anti-proBNP (1-108) antibody secreted by said hybridoma.

  The present invention further relates to a method for in vitro diagnosis of heart failure in a human comprising contacting a biological sample, preferably blood, plasma or serum, with an anti-proBNP antibody (1- 108) specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or RAPR76S77P peptide of proBNP (1-108) to the substantial exclusion of BNP (1-76) and BNP (77-108), and having the ability to recognize specifically proBNP (1-108) circulating in human serum or plasma samples, and detection of proBNP (1-108) in the sample.

  The invention generally provides a method for in vitro diagnosis of heart failure in a human comprising: a) contacting a biological sample, preferably blood, plasma or serum, with an anti-proBNP antibody ( 1-108) specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) to the substantial exclusion of BNP (1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples; b) incubation of the mixture under conditions allowing the formation of antigen-antibody complexes; and c) revealing the antigen-antibody complexes formed, optionally with the aid of a labeled detection antibody, capable of binding specifically to the proBNP (1-108) present in the first complex, or with the aid of a labeled detection antigen capable of binding to the antibody directed against said proBNP (1-108) present in the first complex.

  In particular, the invention provides a method of diagnosing heart failure which comprises, in addition to steps a, b and c above, a step d) of correlating the amount of antigen-antibody complexes revealed in the clinical state. from subject.

  The present invention furthermore relates to a kit for detecting proBNP (1-108) in a biological sample, in particular in a sample of blood, plasma or serum, containing at least one anti-proBNP antibody (1-108) recognizing in a manner Y7OTLRAPR76S77PKMVQGSGS5 peptide and / or proBNP (1-108) RAPR76S77P peptide except for the substantial exclusion of BNP (1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1- 108) circulating in human serum or plasma samples.

  The invention finally relates to a kit for detecting proBNP (1-108) in a biological sample, in particular in a sample of blood, plasma or serum, containing, as a standard and / or control, a compound containing at least one peptide chosen from the group of peptides of the following formulas: ## STR2 ## where a, XI, X2, and a2 have the same meaning as above, X-Y70TLRAPR76S77PKMVQGSG85-Z (II) where X and Z have the same meaning as above, X-Y70TLRAPR76S77PKMVQGSG85- Z (II) in substituted form, conservatively or not, provided that it retains intact (in particular unsubstituted) portion RAPR76S77P, where X and Z have the same meaning as above, Y7OTLRAPR76S77PKMVQGSG85.

  Definitions In the context of the invention, a biological sample or a sample of biological fluid is preferably constituted by a biological fluid, such as blood, plasma, serum, urine, cerebrospinal fluid, saliva, etc ...

  lo Heart failure means the condition in which an abnormal cardiac function is responsible for the heart's inability to pump enough blood to meet the metabolic needs of the organism and / or in which the heart provides but with abnormally high filling pressures. It can especially be a right and / or left ventricular failure.

  The term antibody refers to any whole antibody or functional fragment of an antibody (genetically engineered or otherwise), comprising, or consisting of, at least one antigenic combining site, allowing said antibody to bind to at least one determinant antigen of an antigenic compound. By way of example of antibody fragments, mention may be made of the Fab, Fab ', F (ab') 2 fragments as well as the single-chain variable fragments (scFv chains).

  The anti-proBNP (1-108) antibodies according to the invention may be of polyclonal or monoclonal type. An anti-proBNP (1-108) polyclonal antibody according to the invention may, inter alia, be obtained by immunization of an animal such as a rabbit, a mouse, etc. with the aid of proBNP (1-108). whole, sampling and exhaustion of the antiserum obtained on, for example, an immunoadsorbent containing BNP (77-108) and / or BNP (1-76) according to methods known per se to those skilled in the art. An anti-proBNP monoclonal antibody (1-108) according to the invention may, inter alia, be obtained by the conventional method of Kbhier and Milstein (Nature (London), 256 495-497 (1975)).

  The production of monoclonal antibodies or monospecific polyclonal sera, or antibodies obtained by screening genomic libraries, useful in the context of the invention is based on conventional techniques, which are detailed below.

  By capture antibody is meant an antibody or part of an antibody, preferably fixed on a solid phase, which is capable of retaining the proBNP (1-108) antigen present in a biological sample by affine binding.

  The presence of the antigen in the biological sample is revealed by detection means. As regards the detection of the antigen, the invention provides in particular detection using at least one detection antibody. Such a labeled detection antibody is capable of binding to the captured antigen by affine binding, recognizing an epitopic site different from that recognized by the capture antibody.

  The term labeled refers both to direct labeling (via enzymes, radioisotopes, fluorochromes, luminescent compounds, etc.) as to indirect labeling (for example, by means of antibodies themselves labeled directly or indirectly). using reagents of a labeled affinity pair, such as, but not limited to, the labeled avidin-biotin pair, etc.).

  By antigenic fragment is meant any portion of proBNP (1-108) capable of inducing antibody synthesis substantially specific to only proBNP (1-108) in an immunized animal.

  In accordance with the present invention, an antigenic fragment contains at least the epitope or epitope site RAPR76S77P. An epitope site or epitope is an amino acid sequence that is recognized by at least one antibody and allows specific binding thereof.

  The term monospecific polyclonal antibody applies to any polyclonal antibody with specificity for a single epitope. This means that the antibody is capable of binding an amino acid sequence of the proBNP (1-108) sequence containing the amino acids comprising the epitope, but is incapable of binding an amino acid sequence of the sequence of the amino acid sequence. proBNP (1-108) which does not contain the amino acids comprising the epitope.

  The term specifically, when it refers to a specific recognition or binding of an antibody to an antigen, means that the antibody interacts with the antigen without substantial interaction with other antigens, or if one speaks of recognition specific with an epitope, by almost exclusive recognition of this epitope. Association constants greater than 108 L.mol-1 are preferable.

  By conservative substitution is meant in particular the substitution of an amino acid of a class by an amino acid of the same class, which substitution does not significantly modify the immunoreactivity of the peptide obtained relative to that of the original peptide. Among the various amino acid classes, there are generally polar side chain amino acids (such as asparagine, glutamine, serine, threonine and tyrosine), nonpolar side chain amino acids (such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan and cysteine), amino acids with a basic side chain (such as lysine, arginine and histidine ), and acid side chain amino acids (such as aspartic acid and glutamic acid).

  By non-conservative substitution is meant any other type of substitution, which does not significantly modify the immunoreactivity of the peptide obtained relative to that of the original peptide.

  The substantial non-recognition of the BNP (1-76) and BNP (77108) peptides refers to an absence or near-absence of reaction of the antibodies of the invention with these peptides.

  Production of specific antibodies The antibodies used in the present invention are antibodies specific for the antigen, and for this reason are monoclonal antibodies or monospecific polyclonal antibodies, ie they specifically recognize only that an epitope.

  The monoclonal antibodies can be obtained according to the conventional method of lymphocyte fusion and hybridoma culture described by Kbhler and Milstein, Nature, 1975, 256: 495-497. Other methods of preparing monoclonal antibodies are also known (Harlow et al., 1988 Antibodies: a laboratory manual). The monoclonal antibodies can be prepared by immunizing a mammal (for example a mouse, a rat, a rabbit or even a human, etc.) and using the lymphocyte fusion technique leading to hybridomas (Kêhler and Milstein, 1975). ).

  Alternative techniques to this usual technique exist. For example, monoclonal antibodies can be produced by expression of a nucleic acid cloned from a hybridoma. Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages that display V gene libraries at the phage surface ( for example, fUSE5 for E. coli, Scott, JK and Smith, GP, Science, 1990, 249: 386-390). Protocols for constructing these antibody libraries are described in Marks et al., 1991, J. Mol. Biol. 222: 581-597).

  The polyclonal antibodies can be obtained from the serum of an animal immunized against an antigen of peptide nature according to the usual operating procedures. The polyclonal antibodies thus obtained can, if necessary, and by exhaustion with BNP (77-108) and BNP1-76, according to techniques known to those skilled in the art such as, for example, column immunoadsorption. , to be made monospecific of the sequence Y7OTLRAPR76S77PKMVQGSG85 and / or of a peptide comprising the sequence RAPR76S77P, thus ensuring specificity with respect to proBNP (1-108), with the substantial exclusion of BNP (1-76) and BNP. (77-108).

  Capture and / or detection antibodies In sandwich technique, the capture antibody is selected so that it specifically recognizes an epitope, on the patient's natural antigen, while the detection antibody is chosen, preferably, but not necessarily so that it specifically recognizes another epitope, on the natural antigen of the patient.

  The biological sample may be optionally treated in a previous step, or directly in the presence of at least one capture antibody under conditions favoring the exposure of the epitope to be detected.

  The diagnostic method according to the invention can be carried out according to various formats well known to those skilled in the art: in the solid phase or in the homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples.

  According to a preferred embodiment, the capture antibody is immobilized on a solid phase. As non-limiting examples of solid phase, microplates, in particular polystyrene microplates, such as those sold by the company Nunc, Denmark, may be used. It is also possible to use solid particles or beads, paramagnetic beads, such as those supplied by Dynal or Merck-Eurolab (France) (under the trademark EstaporTM), or also test tubes made of polystyrene or polypropylene, etc. .

  An immunoassay format of sandwich sandwich between two antibodies (capture and detection) is particularly advantageous for the detection of antigens present in the biological sample.

  An immunoassay format for antigen detection by competition is also possible. Other immunoassay modalities are still conceivable and well known to those skilled in the art.

  ELISA assays, radioimmunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed.

  Kits Kits and reagents useful for the detection of proBNP (1108) in a biological fluid sample, in accordance with the method of the invention, can be provided for practicing the invention easily and applicable to many biological samples.

  Detection kits of proBNP (1-108) in a biological sample may contain at least one antibody as defined above. Other kits, containing as standard and / or control, at least one peptide of formula (I), (II) or a substituted peptide as defined above, may also be useful for the implementation of the invention.

  Another particular object of the invention is therefore a proBNP (1-108) detection kit in a biological fluid sample, comprising: at least one anti prol3NP (1-108) antibody specifically recognizing the peptide Y70TLRAPR76S77PKMVQGSG85 and or the RAPR76S77P peptide of proBNP (1-108) with the substantial exclusion of BNP (1-76) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples, and which is preferably a capture antibody of said proBNP (1108) present in the biological sample, and a labeled conjugate capable of binding specifically to the antigen-antibody complexes formed.

  As described above, the capture antibody may be advantageously presented in a form immobilized on a solid phase, such as a microplate, for example, but not exclusively.

  A preferred kit comprises at least: - an anti-proBNP capture antibody (1-108) specifically recognizing the peptide Y70TLRAPR75S77PKMVQGSG85 and / or the RAPR76S77P peptide of proBNP (1-108) to the substantial exclusion of BNP (1-76); ) and BNP (77-108), and having the ability to specifically recognize proBNP (1-108) circulating in human serum or plasma samples, said capture antibody being immobilized on a solid phase; and a labeled detection antibody directed against another intact epitope of proBNP (1-108), or optionally, a labeled detection antigen, which is a proBNP (1-108) peptide or proBNP. (1-108) himself.

  According to a particular embodiment, a proBNP (1-108) detection kit in a biological sample may contain: in a container, at least one antibody as defined above; - In another container at least one peptide as defined above, useful in particular as a standard and / or control.

  The following figures and examples illustrate the invention without limiting its scope.

  FIGURE 1 depicts the reactivity of protein A-Sepharose purified rabbit polyclonal antibodies with proBNP (1-108), BNP fragment (1-76), BNP (77-108), and GST (Glutathione-S-transferase, used as control protein) adsorbed on microplate at 5 μg / ml.

  FIG. 2 represents the reactivity of the polyclonal antibodies of the filtrate obtained after exhaustion on BNP-K3-NHS-sepharose resin. The polyclonal antibodies are prepared in the form of dilution ranges of 2 to 20 μg / ml, then tested on cups coated with proBNP (1-108), BNP (1-76) or BNP (77-108) polypeptide adsorbed at 5 μg. / ml.

  FIG. 3 represents the reactivity of the rabbit polyclonal serum eluate obtained after exhaustion on BNP-K3-NHS-sepharose resin.

  The polyclonal antibodies are prepared in the form of dilution ranges of 2 to 20 μg / ml, then tested on cups coated with proBNP (1-108), BNP (1-76) or BNP (77-108) polypeptide adsorbed at 5 μg. / ml.

  Figure 4 depicts an epitope analysis using the spot technique of rabbit polyclonal serum # 046 805 before depletion. (For this example, the background is 30.4 5.93 relative intensity units.) Figure 5 shows an epitope analysis using the spot technique of rabbit polyclonal serum # 046 805 after depletion on BNPK3-NHS-Sepharose resin. (For this example, the background is 31.3 6, 31 relative intensity units.) Figure 6 shows a calibration curve of the IRMA assay of proBNP (1-108).

  Figure 7 shows the results in cpm (counts per minute of radioactivity) of the IRMA proBNP (1-108) assay of samples from 6 healthy subjects and 7 patients with heart failure.

  Figure 8 shows a calibration curve of the proBNP ELISA assay (1-108).

  FIG. 9 represents an evaluation of the cross reaction with respect to BNP (1-76) and BNP (77-108), of the ELISA proBNP (1-108) assay combining the anti-BNP polyclonal antibody (77-108). ) with the non-depleted rabbit polyclonal antibody # 046 805 coupled with biotin.

  FIG. 10 represents the evaluation of the cross reaction with respect to BNP (1-76) and BNP (77-108), of the ELISA proBNP (1-108) assay combining a polyclonal anti-NT-proBNP antibody (1 -29) with the non-depleted rabbit polyclonal antibody # 046 805 coupled to biotin.

EXAMPLES

  Example 1 Peptide Synthesis for Immunization Synthetic peptides are produced by standard techniques well known to those skilled in the art. An example of this is Merrifield-type synthesis which is advantageous in view of its ease of implementation (Merrifield, (1963), R.C.Sheppard (1971), Atherton et al (1989)). As an automatic synthesizer, you can use Millipore's "9050 Plus Pep Synthesizer" synthesizer, Perspective's "Pioneer", or ABI's "433A" synthesizer. The peptides can also be obtained by homogeneous phase synthesis.

  The following syntheses were carried out on a Pioneer synthesizer, using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry: at each step the reagents (that is to say the protected amino acid and the coupling activators (TBTU (2 (1H-benzotriazol-1-yl) -1,1,3,2-tetramethyluronium tetrafluoroborate) / HOBt (N-hydroxybenzotriazol) are added in excess (in a ratio moles of reagents / moles of groups which can be substituted on the resin = 5) At the end of the synthesis, the peptide is separated from the resin by a solution based on trifluoroacetic acid (reagent K) The peptide is then precipitated in a cooled ether solution and then purified by HPLC .

  The inventors proceeded to the synthesis of peptides containing the following hinge region amino acid sequence R76S77: SEQ ID N 6: CGRAPRSP 3o SEQ ID N 7: Acetyl -CGRAPRSP SEQ ID N 8: CGRAPRSPK SEQ ID N 9: Acetyl -CGRAPRSPK SEQ ID NO: 10: CGRAPRSPKM-V SEQIDN 11: SEQ ID NO: SEQ ID NO: 13: SEQ ID NO: 14: SEQ ID NO: SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: SEQ ID NO: 18 ID N 19: SEQ ID NO: SEQ ID NO: 21: SEQ ID NO: 22

  C-G-R-A-P-R-S-P-K-M-V-Q-G-S-G R-A-P-R-S-P-G-C

  Acetyl-R-A-P-R-S-P-G-C Acetyl C-Y-T-L-R-A-P-R-S-P-K C-H-R-K-M-V-L-Y-T-LR-A-P-R-S-P-K C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-G-S-G (peptide Cl 3P30) C-E-T-LR-A-P-R-S-P-K-M-V-Q-G-S-G C-E-S-I-R-A-P-R-S-P-K-M-V-Q-G-S-G C-Y-T-L-R-A-PR-S-P-K-M-V-Q-G-S-F3A C-Y-T-L-R-A-P-R-S-P-K-M-V-Q-A-T-13A C-E-S-I-R-A-P-RS-P-K-M-V-Q-A-T-F3A C-E-S-I-R-A-P-R-S-P-A-L-A-S-G-T-A NOTE: 13A means beta-alanine.

  The subject of the invention is therefore any peptide selected from the group consisting of the sequences: SEQ ID N 6 to SEQ ID N 15 and SEQ ID N 17 to SEQ ID N 22 above.

  Example 2 Coupling of a Peptide to a Carrier Protein for Immunization The peptide is coupled to a protein carrier KLH (Keyhole Lympet Hemocyanin), thyroglobulin, BSA (Bovine Serum Albumin, via various functions (thiol, amine, aldehyde. ..) in order to make the peptide more immunogenic The coupling agent used to bind the peptide to the protein can be hetero or homo-bifunctional The most commonly used reagents are BS 3, sSMCC, SPDP One of the coupling techniques used is that which uses glutaraldehyde as a coupling agent and KLH as a carrier protein.The coupling of KLH with the peptide is carried out on the basis of the amine functional groups of the peptide. (N-terminal group and amine group carried by lysine mainly).

  A solution of the peptide C13P30 of Cys-YTLRAPRSPKMVQGSG-NH2 sequence (SEQ ID No. 16) at 5 mg / ml is prepared in 0.15M Dulbecco NaCl PBS buffer at pH 7.4. A vial of 20 mg of KLH lyophilized in PBS buffer (Pierce # 77600) is taken up in 2 ml of PPI water (water for injection), so as to obtain a solution of KLH at 10 mg / ml in PBS buffer.

  1 ml of the peptide solution (are 5 mg) is mixed with 1.25 ml of the KLH solution (ie 12.5 mg). Under an extractor hood, 2.25 ml of a 2% glutaraldehyde solution prepared extemporaneously (from 25% glutaldehyde, Sigma # G-5882) are added to the mixture. In order to avoid the formation of KLH complexes, the 2% glutaraldehyde solution is added dropwise and with stirring of the mixture. The conjugation reaction is carried out for 2 hours incubation at room temperature. The coupling reaction is stopped by adding a solution of sodium borohydride at 100 mg / ml so as to reach a final concentration of 10 mg / ml. The mixture is incubated overnight at 4 ° C. Finally, the solution is dialyzed overnight at 4 ° C. against Dulbecco's PBS buffer at pH 7.4. The solution is finally aliquoted and stored at -80 ° C.

  Example 3: Immunizations with peptides For the production of polyclonal antibodies, rabbits (New Zealand strain females) were immunized with the Cys-YTLRAPRSPKMVQGSG-NH2 peptide coupled to KLH according to Example 2. A first injection, a 1.5 ml emulsion of peptide coupled to KLH with 1.5 ml of complete Freund's adjuvant (Sigma # F-5881) is prepared, and 1 ml of this latter emulsion (ie 200 μg of peptide ) is injected intradermally to each of the rabbits. Two boosts are made at 20 days interval by intradermally injecting 1 ml of a peptide emulsion coupled to KLH (ie 200 μg of peptide) with incomplete Freund's adjuvant (Sigma 30 # F-5506). Twenty days after the second booster, a third booster is performed in the same manner as the previous boosters but by subcutaneous injection. Twenty days after this last booster, and after evaluation of the antibody titre obtained, the rabbits are bled. More particularly, the polyclonal rabbit antibodies identified by the number # 046 805 were used for further work.

  EXAMPLE 4 Obtaining and Purifying the Antibodies Before purification, the rabbit sera are centrifuged for 30 minutes at 4500 rpm at 4 ° C. After decantation, the serum is diluted by half in 1.5M glycine buffer at pH 8. 0 containing NaCl 1M.

  Example 4.a: Purification of IgGs on Protein A Sepharose Purification of the polyclonal antibodies is performed by affinity chromatography on a protein A-Sepharose gel (Amersham Biosciences # 17, 1279.02). Protein A extracted from Staphylococcus aureus specifically combines with the Fc fragment of IgG molecules. Then IgG, all subclasses, are eluted at pH 3.0.

  All buffers used are degassed for 15 minutes in an ultrasonic bath before passing through the column to prevent the formation of bubbles.

  A chromatography column is prepared using 12 ml of protein A-Sepharose. The gel column is equilibrated for 40 minutes with distilled and degassed water and then for 40 minutes with Dulbecco's PBS buffer pH 7.4 containing 0.5 M NaCl and finally with 1.5 M glycine buffer. pH 8.0 containing 1M NaCl, until a good baseline is obtained.

  Then, 10mL of rabbit serum diluted in half in 1.5M glycine buffer at pH 8.0 containing 1M NaCl, are passed through the column at a flow rate of 0.5ml / min. After appearance of the albumin peak, the serum IgGs are eluted with a 1M citric acid solution, brought to pH 3.0 with 0.1M citrate tris sodium buffer. The elution peak of IgG is recovered and rapidly dialyzed in Dulbecco's PBS buffer at pH 7.4 overnight at 4 C. The concentration of IgG is determined after dialysis by reading the optical density at 280 nm against PI3S buffer. .

  After purification on protein A, the reactivity of the polyclonal antibodies is tested on proBNP (1-108), E3NP (1-76), and BNP (77-108). The results obtained for rabbit polyclonal antibodies # 046805 are shown in FIG. 1. The rabbit polyclonal serum # 046805 is relatively well specific to proE3NP (1-108). However, we were able to note the presence of a low anti-BNP reactivity (77-108) at high concentrations, a reactivity that we were able to suppress by making the monospecific rabbit polyclonal by exhaustion on BNP-K3-NHS resin. Sepharose, according to the method described in Example 4.b.

  Example 4.b: Exhaustion of IgG on BNP-K3-NHS-Sepharose Resin The purpose of this operation is to eliminate the cross-reactivity observed with respect to BNP (77-108).

  The cross-reactivity of the obtained polyclonal antibody being located in the N-terminal position of the BNP molecule (77-108), it was important to present this region of the BNP (77-108) well to the immunoglobulins to be eliminated. For these purposes, BNP (77-108) was synthesized with an extension of 3 C-terminal lysine residues (BNP-K3) in order to promote its coupling to the NHS-Sepharose resin by its C-terminus.

  BNP-K3: S-P-K-M-V-Q-G-S-G-C-F-G-R-K-M-D-R-I-S-S-S-G-L-GC-K-V-L-R-R-H-K-K-K (SEQ ID No. 104) was synthesized on a Pioneer synthesizer, using the Fmoc (9-fluorenylmethyloxycarbonyl) chemistry cited above in Example 1.

  mg of BNP-K3 are dissolved using the 100mM NaHCO3 buffer, at pH 8.3, supplemented with 0.5M NaCl at a concentration of 10 mg / ml. 2 ml of NHS-Sepharose 4 Fast Flow resin (Amerscham Biosciences # 17, 0906.01) are centrifuged for 30 seconds at 1000 rpm at 4 C. The resin is washed with 15 ml of a cold 1 mM HCl solution. After centrifugation and removal of the HCl solution, the resin is mixed with the BNP-K3 ligand at 10 mg / ml. The mixture is incubated for 1 hour at room temperature with gentle stirring. After centrifugation and removal of the ligand solution, the non-reactive groups of the resin are blocked with 5 ml of 0.1M glycine buffer, pH 8.0. The mixture is incubated for 1 hour at room temperature with gentle stirring. After centrifugation and removal of the blocking buffer, the resin is taken up in 5 ml of 100mM NaHCO3 buffer, at pH 8.3, supplemented with 0.5M NaCl. Four washes using this buffer are made. At the last wash, the mixture is deposited on a chromatography column.

  After preparation of the chromatography column, 10 mg of rabbit IgG # 046 805 are deposited on the column. A peristaltic pump connected to the column makes it possible to circulate the IgG rabbit serum overnight at 4 C. The next day, the IgG solution is recovered (= filtrate). The bound IgG fraction on BNP-K3 is eluted (= eluate) using 20mM Tris buffer, pH 8.0, containing 5M urea. The eluate and the filtrate are then tested to ensure the effectiveness of the burnout. Figures 2 and 3 show the results of these tests carried out in ELISA with the filtrate and the eluate, respectively, of rabbit polyclonal serum # 046 805. As expected, the filtrate of rabbit polyclonal serum # 046 805 is specific for proBNP (1-108): no reactivity is observed on BNP (1-76) or on BNP (77-108). The eluate retains reactivity with respect to the important proBNP (1-108), but also a reactivity with BNP (77-108). These results confirm the effectiveness of the depletion of the rabbit polyclonal serum on the BNP-K3-NHS-sepharose resin.

  BNP (77-108) is from Sigma (# B-5900), while proBNP (1-108) and BNP (1-76) have been produced as recombinant proteins expressed after cloning in the pGEX-vector. 2T (Amersham Pharmacia Biotech) and transfection in E coli, by conventional techniques well known to those skilled in the art. The original vector was provided by Berlex Biosciences (Richmond, CA, USA) and its preparation is described by Yan et al. (PNAS 2000, 97, 8525-8529). The concentration of proBNP (1-108) and that of BNP (1-76) were determined by the Bradford method of colorimetric protein assay (Bradford M. Anal., Biochem., 1976; 72: 248-54).

  Example 5 Identification of the epitope recognized by the polyclonal serum of rabbit # 046 805 before and after exhaustion on BNP-K3-NHS-sepharose resin The polyclonal serum of rabbit # 046 805 before and after exhaustion on BNP-K3-NHS resin Sepharose was tested by the spot method to identify the epitope recognized by this polyclonal serum. This method, described by Frank (Tetrahedron 1992; 48: 9217-32), allows rapid nitrocellulose membrane synthesis of a large number of predefined sequence peptides. The protocols used are those described by Molina et al. (Pept Res 1996; 9: 151-5). On a sheet of paper are created circular areas (spots) of about 5 mm in diameter, having an amine function, which serve as anchor to the C-terminal amino acid of the synthetic peptide. The lengthening of the peptide chain is carried out by successive additions of Fmoc-activated amino acids. The side chains of the amino acids are blocked by suitable chemical groups. All peptides are synthesized with their N-terminal N-acetylated residue. At the end of the synthesis, the side chains are deprotected by the action of trifluoroacetic acid. This treatment does not affect the binding between the peptide and the cellulosic carrier, and the reactivity of the peptides can be evaluated by a colorimetric assay.

  The membrane-synthesized peptide series consists of 32 pentadecapeptides shifted by 3 amino acid residues representing the entire proBNP (1-108) sequence.

  Table I: Spot membrane consisting of 32 pentadecapeptides shifted from 3 to 3 amino acid residues representing the entire proBNP (1-108) sequence.

  Sequence N spot Sequence N spot HPLGSPGSASDLETS 1 GVWKSREVATEGIRG 17 (SEQ ID NO: 23) (SEQ ID NO: 39) GSPGSASDLETSGLQ 2 KSREVATEGIRGHRK 18 (SEQ ID NO: 24) (SEQ ID NO: 40) GSASDLETSGLQEQR 3 EVATEGIRGHRKMVL 19 (SEQ ID NO: 25) (SEQ ID NO. ID # 41) SDLETSGLQEQRNHL 4 TEGIRGHRKMVLYTL 20 (SEQ ID NO: 26) (SEQ ID NO: 42) ETSGLQEQRNHLQG 5 IRGHRKMVLYTLRAP 21 (SEQ ID NO: 27) (SEQ ID NO: 43) GLQEQRNHLQGKLSE 6 HRKMVLYTLRAPRSP 22 (SEQ ID NO: 28) (SEQ ID NO 44) (SEQ ID NO: 29) (SEQ ID NO: 45) NHLQGKLSELQVEQT 8 YTLRAPRSPKMVQGS 24 (SEQ ID NO: 30) (SEQ ID NO: 46) QGKLSELQVEQTSLE 47) LSELQVEQTSLEPLQ 10 RSPKMVQGSGCFGRK 26 (SEQ ID NO: 32) (SEQ ID NO: 48) LQVEQTSLEPLQESP 11 KMVQGSGCFGRKMDR 27 (SEQ ID NO: 33) (SEQ ID NO: 49) EQTSLEPLQESPRPT 12 QGSGCFGRKMDRISS 28 (SEQ ID NO: 34) (SEQ ID NO: 50) SLEPLQESPRPTGVW 13 GCFGRKMDRISSSSG 29 (SEQ ID NO: 35) (SEQ ID NO: 51) PLQESPRPTGVWKSR 14 G RKMDRISSSSGLGC 30 (SEQ ID NO: 36) (SEQ ID NO: 52) ESPRPTGVWKSREVA MDRISSSSGLGCKVL 31 (SEQ ID NO: 37) (SEQ ID NO: 53)

Claims (1)

(SEQ ID NO: 38) (SEQ ID NO: 54) After saturation of the membrane with TBS buffer at pH 7.0 supplemented with 0.1% Tween 20, 5% Blocking Buffer. % (Euromedex # SU-07-250), and 5% sucrose, the reactivity of rabbit polyclonal serum diluted to 10 μg / ml was tested (on an incubation of 1 hour 30 minutes at 37 ° C with shaking). After washing with the pH 7.0 TBS buffer supplemented with 0.1% Tween 20, the alkaline phosphatase conjugated anti-IgG conjugate (Sigma # A-8025) is incubated for 1 hour at room temperature under agitation. Finally, after a final series of washes, the revelation of the spots is carried out using the substrate composed of 30 ml of CBS buffer at pH 7.0 in which 120 μl of BCIP (5-bromo-4-chloro) is added. 3-lndolyl phosphate 0.15M), 15Opl of 1M MgCl 2 and 180 μl of MTT (Thiazolyl bliue tetrazolium bromide 0.1M). After scanning the membrane, the intensity of the spots of the membrane is evaluated (in relative units of intensity) using an image processing software. The background noise is calculated from spots undetected by the antiserum. The results of the epitope analysis of the rabbit polyclonal serum before exhaustion are shown in FIG. 4. Five peptides (spots 22 to 26) are detected by the polyclonal serum, and the sequence common to these five peptides is R76S77P. However, the reactivity is markedly increased when the RAP motif is added in the N-terminal position of the R76S77P motif. The results of the epitope analysis of the rabbit polyclonal serum # 046 805 after exhaustion on BNP-K3-NHS-sepharose resin are shown in FIG. 5. Four peptides (spots 22 to 25) are detected by the polyclonal serum, and the common sequence to these 4 peptides is RAPR76S77P. Contrary to what is observed for the polyclonal serum before exhaustion, no reactivity on the R76S77P motif alone is observed (spot 26). This explains that after exhaustion on BNP-K3-NHS-sepharose resin, the polyclonal serum no longer detects BNP (77-108) [as a reminder, the S77P motif corresponds to the first two amino acids of the BNP sequence (77- 108)]. In conclusion, the epitope recognized by the monospecific rabbit polyclonal serum # 046 805 is RAPR76S77P. Example 6 Identification of the Minimum Epitope The polyclonal serum of rabbit # 046 805, before and after exhaustion on BNP-K3-NHS-Sepharose resin, was tested by the spot method (described in Example 5) in order to identifying the minimum epitope, in the hinge peptide, allowing the specific recognition of the only proBNP (1-108) by the polyclonal serum. We synthesized a membrane consisting of 16 pentadecapeptides containing the sequence of the hinge peptide YTLRAPRSPKMVQGS, a successive substitution of each amino acid by an alanine residue (Alanine-scanning or Ala-scan) or glycocol Io (Table II). The results of the Ala-scan analysis of rabbit polyclonal serum # 046 805 before and after exhaustion on BNP-K3-NHS-Sepharose resin are shown in Table II. Table II: spot membrane consisting of 16 pentadecapeptides containing the sequence of the hinge peptide YTLRAPRSPKMVQGS with a successive substitution of each amino acid with an alanine (Alanine-scanning) or glycocolle residue. Reactivity of rabbit polyclonal serum # 046 805 before and after exhaustion on BNP-K3-NHS-sepharose resin. SEQ ID Sequence N spot Reactivity (in units Reactivity (in relative intensity units) relative intensity) polyclonal serum polyclonal serum rabbit # 046 805 non-rabbit # 046 805 sold out sold out SEQ ID No 46 YTLRAPRSPKMVQGS 716 84 69 SEQ ID No 55 ATLRAPRSPKMVQGS 717 90 68 SEQ ID No. 56 YALRAPRSPKMVQGS 7'18 101 71 SEQ ID NO: 57 YTARAPRSPKMVQGS 719 108 77 SEQ ID NO: 58 YTLAAPRSPKMVQGS 720 101 39 SEQ ID NO: 59 YTLRGPRSPKMVQGS 721 93 39 SEQ ID NO: 60 YTLRAARSPKMVQGS 722 103 96 SEQ ID NO. YTLRAPASPKMVQGS 723 50 23 SEQ ID NO: 62 YTLRAPRAPKMVQGS 7; 24 58 44 SEQ ID NO: 63 YTLRAPRSAKMVQGS 725 74 41 SEQ ID NO: 64 YTLRAPRSPAMVQGS 726 138 118 SEQ ID NO 65 YTLRAPRSPKAVQGS 727 98 77 SEQ ID No. 66 YTLRAPRSPKMAQGS 728 100 68 SEQ ID NO 67 YTLRAPRSPKMVAGS 729 95 71 SEQ ID NO 68 YTLRAPRSPKMVQAS 730 107 78 SEQ ID NO: 69 YTLRAPRSPKMVQGA 731 112 68 The critical amino acids in the recognition of the epitope recognized by rabbit polyclonal serum # 046 805 before exhaustion are R76S77P, whereas after exhaustion of rabbit polyclonal serum # 046 805 on resin BNP-K3-NHS-sepharose, in addition to the motif R76S77P, the RA motif becomes a contributor. These results therefore show that the obligatory minimum epitope in the specific recognition of proBNP (1-108) by rabbit polyclonal serum # 046 805 is RAPR76S77P.   Example 7: Mouse immunization with the proBNP recombinant protein (1108) BALB / c mice (6 week old females) were immunized with the recombinant proBNP (1-108) described in Example 4. b, by conventional techniques well known to those skilled in the art. At the first injection, an emulsion of 1 ml of proIBNP with 1 ml of complete Freund's adjuvant (Sigma # F-5881) is produced, and 300 μl of this emulsion (ie 100 μg of protein) are injected subcutaneously to each of the mice. Two boosts are carried out at 15-day intervals by intraperitoneal injection of 300 μl of a proBNP (1-108) emulsion (ie 100 μg of peptide) with incomplete Freund's adjuvant (Sigma # F-5506). Fifteen days after the second booster, a third booster is performed in the same manner as the previous boosters but by subcutaneous injection. Finally, 15 days after this last recall, blood samples are taken from the mice to analyze the immune response by the spot technique. The immune response of the mouse 12 has proved particularly interesting for further work.   Example 8 Identification of the Epitopes Recognized by the Mouse 12 Polyclonal Antibodies on the ProBNP (1-108) Sequence The principle of the spot technique used to identify the epitopes recognized by the mouse 12 polyclonal antibodies on the proBNP sequence (1-108) is described in Example 5.   The membrane-synthesized peptide series consists of 94 pentadecapeptides, shifted from an amino acid residue, representing the entire proBNP (1-108) sequence. The reactivity of the polyclonal serum of the mouse 12 diluted to 1 / 500th is tested on this membrane as described in Example 5. After scanning of the membrane, the intensity of the spots of the membrane is evaluated (in relative units of intensity) using image processing software. The background noise is calculated from spots undetected by the antiserum. The results of the epitope analysis of mouse polyclonal serum 12 are shown in Table III. Three regions located in the N-terminal position of the proBNP (1-108) sequence are particularly well detected by the antibodies of mouse antiserum 12: spots 1 to 12, 17 to 27, and 38 to 49. From Surprisingly, the antiserum of the mouse 12 also contains antibodies capable of recognizing spots whose peptide sequence comprises the motif RAPR76S77P: spots 64 to 68. The peptide sequences of these spots are described in Table III.   Table III: Epitopic analysis, using the spot technique, of the antiserum of the mouse 12.   N peptide sequence of the spot detected reactivity in N units peptide sequence of the spot spot intensity spot spot detected 1 HPLGSPGSASDLETS (SEQ ID No. 23) 177.0 38 LLPSPRPTGVWK (SEQ ID NO: 88) 2 PLGSPGSASDLETSG (SEQ ID No. 70) 170, 0 39 EPLQESPRPTGVWKS (SEQ ID NO: 89) 3 LGSPGSASDLETSGL (SEQ ID NO: 71) 145.7 40 PLQESPRPTGVWKSR (SEQ ID NO: 90) 4 GSPGSASDLETSGLQ (SEQ ID 24) 166.3 41 LQESPRPTGVWKSRE (SEQ ID NO: 91) SPGSASDLETSGLQE (SEQ ID 72) 185.0 42 QESPRPTGVWKSREV (SEQ ID NO: 92) 6 PGSASDLETSGLQEQ (SEQ ID 73) 169.3 43 ESPRPTGVWKSREVA (SEQ ID NO: 93) 7 GSASDLETSGLQEQR (SEQ ID 25) 155.2 44 ESPRPTGVWKSREVA (SEQ ID NO: 94) 8 SASDLETSGLQEQRN (SEQ ID NO: 74) 176.5 45 SPRPTGVWKSREVAT (SEQ ID NO: 95) 9 ASDLETSGLQEQRNH (SEQ ID 75) 108.0 46 PRPTGVWKSREVATE (SEQ ID NO: 96) SDLETSGLQEQRNHL (SEQ ID 76) 118.3 47 RPTGVWKSREVATEG (SEQ ID NO 16) 11 DLETSGLQEQRNHLQ (SEQ ID NO: 77) 121.0 48 PTGVWKSREVATEGI (SEQ ID NO: 97) 12 LETSGLQEQRNHLQG (SEQ ID NO: 78) 101.0 49 TGVWKSREVATEGIR (SEQ ID No. 98) 17 LQEQRNHLQGKLSEL (SEQ ID No. 79) 99.0 61 IRGHRKMVLYTLRAP (SEQ ID No. 99) 18 QEQRNHLQGKLSELQ (SEQ ID NO: 80) 179.8 62 RGHRKMVLYTLRAPR (SEQ ID NO: 100) _ EQRNHLQGKLSELQV (SEQ ID NO: 29) 189 , 6 63 GHRKMVLYTLRAPRS (SEQ ID NO: 101) _ QRNHLQGKLSELQVE (SEQ ID NO: 81) 211.8 64 HRKMVLYTLRAPRSP (SEQ ID NO: 53) _ RNHLQGKLSELQVEQ (SEQ ID NO: 82) 219.0 65 RKMVLYTLRAPRSPK (SEQ ID NO: 101) _ NHLQGKLSELQVEQT (SEQ ID No. 30) KMVLYTLRAPRSPKM 213.3 (SEQ ID No. 102) HLQGKLSELQVEQTS (SEQ ID NO: 83) 202.6 67 MVLYTLRAPRSPKMV (SEQ ID NO: 45) 24 LQGKLSELQVEQTSL (SEQ ID NO: 84) 189.5 68 VLYTLRAPRSPKMVQ ( SEQ ID NO: 103) QGKLSELQVEQTSLE (SEQ ID NO: 85) 187.3 26 GKLSELQVEQTSLEP (SEQ ID NO: 86) 36.6 27 KLSELQVEQTSLEPL (SEQ ID NO: 87) 69.9 Example 9: Obtaining monoclonal antibodies specifically recognizing the proBNP (1-108), with the substantial exclusion of BNP (1-76) and BNP (77-108). Three days before the completion of the lymphocyte fusion, mouse 12 has undergone hyper-immunization according to the protocol sui The total dose of immunogen, here 100 μg of proBNP (1-108) in sterile PBS buffer, is fractionated into 4 injections. The first and second injections each correspond to 1 / 10th of the total dose. These injections are performed subcutaneously at different locations and at 45 minutes intervals. The third injection, which corresponds to 2 / 10ths of the total dose, is performed 45 minutes after the second injection, subcutaneously. Thirty minutes after the third injection, an intraperitoneal injection of 100 μl of a 1 mg / ml promethazine solution in sterile PBS (Phenergan 2.5%, Laboratoires Medeva Parma) is performed to prevent anaphylactic shock. Finally 15 minutes later, the last injection corresponding to 6 / 10ths of the total dose is performed intraperitoneally.   The lymphocyte hybridization is carried out according to the method described by Koëhler and Milstein (Nature, 1975; 256: 495-97). It is carried out from the lymphocyte cells extracted from the spleen of the mouse and the myeloma cells (P3-X63-Ag8.653) previously cultured in RPMI 1640 medium (Bio-Whittaker # BE12-1671F), supplemented with a mixture L-glutamine, penicillin and streptomycin (Sigma # G-6784), supplemented with 10% previously decomplemented fetal calf serum (Bio-Whittaker # BE02701E) and 8-azaguanine (Sigma # A-8526). Lymphocyte cells and myeloma cells, previously placed in RPMI 1640 medium (Bio-Whittaker # BE12-167F) supplemented with a mixture of L-glutamine, penicillin and streptomycin (Sigma # G-6784) without addition of fetal calf serum, are mixed with 5 lymphocyte cells for 1 myeloma cell. After centrifugation of the mixture for 7 minutes at 900 rpm at room temperature and resuspension of the cell pellet, 1 ml of polyethylene glycol Hybri-Max (Sigma # P-7777) is added. After 1 minute of incubation in a water bath at 37 ° C., the cells are centrifuged for 1 minute and 30 seconds at 1000 rpm at room temperature. Finally, after incubation for 2 minutes in a water bath at 37 ° C., the pellet is resuspended and 6 ml of RPMI 1640 medium (Bio-Whittaker # BE12-167F) supplemented with a mixture of L-glutamine, penicillin and streptomycin (Sigma # G-6784) previously placed at 37 C, are added at a rate of 100 μl every 5 seconds, and 9 ml of this same medium are added at one time. After centrifugation for 10 minutes at 900 rpm at RT, and removal of the supernatant, the pellet is taken up by RPMI 1640 medium (Bio-Whittaker # BE12-167F), supplemented with a mixture of L-glutamine, penicillin and streptomycin. (Sigma # G-6784), supplemented with 15% fetal calf serum previously decomplemented (Bio-Whittaker # BE02701E) and HAT (Hypoxanthine, aminopterin, Sigma thymidine # H-0262), so as to distribute, under 100pI , 120,000 cells per well. The solution is deposited under 100 μl in the wells of the 96-well culture plates previously inoculated with murine macrophages. The plates are then placed in a CO2 incubator. Fifteen days after lymphocyte hybridization, the number of clones present in the fusion plates is estimated and expressed as a percentage of hybridoma development. Selection of hybridomas is carried out by ELISA on proBNP (1-108) and peptide comprising the sequence RAPR76S77P.   The selected hybridomas are maintained in culture and cloned in limiting dilution. The hybridomas thus cloned can then be used for the production of the monoclonal antibody in ascites fluid.   Example 10: Radioimmunometric assay of proBNP (1-108) Materials: 1) Solid phase: Maxisorp flat bottomed microplate and scored wells, Nunc (Denmark).   2) The capture antibody used is the polyclonal antibody obtained from the unexhausted rabbit serum # 046 805.   3) The conjugate used is an anti-BNP (77-108) antibody labeled with 1125 (anti-BNP-1125). This is the tracer antibody kit BNP Shionoria marketed by Shionogi.   4) Saturation buffer: Dulbecco PBS buffer pH 7.4 containing 1% serum bovine albumin (BSA, Sigma # A-7888) 5) Dilbecon dilution buffer: Dulbecco's pH 7.4 buffer containing 0.1% BSA and 0.1% Tween 20 (Sigma, # P-1379).   4) Wash buffer: Dulbecco PBS buffer pH 7.4 containing 0.1% tween 20.   5) ProBNP standard (1-108): Produced as a recombinant protein.   Protocol: The principle of the test used is based on the sandwich-type radioimmunoassay method, carried out in a flat-bottomed microplate and with scored wells. This is a test in a time when sample (or standard solution of proBNP (1-108)) and tracer are added one after the other without intermediate washing.   A sensitization solution is first prepared with rabbit polyclonal antibody diluted in Dulbecco's PBS buffer at pH 7.4 to 40 μg / ml. 300 μl of this solution are deposited in each well of the microplate.   The microplate is incubated overnight at 4 C.   After removal of the sensitization solution, the microplate is washed with Dulbecco's PBS buffer at pH 7.4 containing 0.1% Tween 20, and then saturated by the addition of 300 μl of Dulbecco's PBS buffer at pH 7. , 4 containing 1% BSA.   The microplate is then incubated for one hour at 37 ° C.   The microplate is then washed (3 times) with the washing solution (Dulbecco's PBS buffer, pH 7.4, supplemented with 0.1% Tween 20).   In each well, 100 μl of standard solution of proBNP (1-108), serum or plasma are deposited. ProBNP (1-108), if present, binds to the capture antibody retained on the solid phase.   200 μl of the ready-to-use anti-BNP-1125 tracer solution (extracted from the Shionogi BNP Shionoria kit) are added to each of the wells. 36   The reaction medium is incubated overnight (18-22h) at 4 C.   The following day, the microplate is washed (3 times) with 300 μl of the washing solution. At the last wash and after suction of the washing solution, each well is transferred to a previously identified tube.   The radioactivity present in each well and proportional to the amount of tracer fixed, therefore to the amount of proBNP (1-108) present in the sample, is measured using a gamma counter.   Figure 7 shows the results obtained using a standard range Io of proBNP (1-108).   Example 11: Results of the Human Sample Assay Using the IRMA proBNP (1-108) assay described in Example 10, 6 samples of healthy subjects and 7 samples of patients with heart failure were tested. Blood samples were all collected on tube containing EDTA. Figure 8 shows the results in cpm (counts per minute) of these sample assays. The results obtained on the samples of patients suffering from heart failure are significantly higher than those obtained on the samples of healthy subjects. These results highlight the presence of proBNP (1108) circulating in the samples of patients suffering from heart failure and constitute the first demonstration that serum proBNP (1-108) is a predictive marker of heart failure.   Example 12 Coupling of Rabbit Polyclonal Antibody # 046 805 to Biotin The coupling method utilizes a N-Hydroxy Succinimide (NHS) derivative of biotin that reacts with the primary amines of IgG to form an amide linkage.   A solution of (+) - Biotin N-succinimidyl ester (Fluka # 14405) at 100mM is prepared by dissolving Biotin in dimethylformamide. Biotinylation is carried out in a glass bottle. 500 μg of the polyclonal antibody of the previously purified but not depleted rabbit are deposited in the flask with 17 μl of the 100 mM biotin solution (the biotin / antibody mole ratio is 500). The reaction is carried out in Dulbecco's PBS buffer at pH 7.4. The reaction mixture is incubated for 1 hour 30 minutes at room temperature with gentle stirring. After coupling, the biotin is inactivated by adding a volume of 2M glycine buffer. The mixture is incubated for 10 minutes at room temperature with gentle stirring. Finally, the mixture is dialyzed overnight at 4 C against Dulbecco's PBS buffer at pH 7.4. The following day, a solution of sodium azide is added at 0.02% in final concentration. The conjugate is stored at 4 C.   Example 13 Immunoenzymometric Assay of proBNP (1-108) An immunoenzymometric assay was also developed.   Materials: 1) Solid phase: Maxisorp flat-bottomed microplate, Nunc (Denmark).   2) The uptake capture antibody is a polyclonal anti-BNP (77-108) antibody marketed by Strategic Biosolution (# B9105RA00-A0).   3) The conjugate used is the non-depleted rabbit polyclonal antibody coupled to biotin according to the method described in Example 12.   4) Streptavidin-peroxidase conjugate (Amersham Pharmacia Biotech # RPN1231V) 5) Buffer saturation: Dulbecco's PBS buffer pH 7.4 containing 1% serum bovine albumin (BSA, Sigma # A-7888) 6) Dilution buffer: Dulbecco PBS buffer pH 7.4 containing 0.1% BSA and 0.1% Tween 20.   7) Wash solution: Dulbecco PBS buffer pH 7.4 containing 0.1% Tween 20.   8) ProBNP standard (1-108): recombinant protein.   9) Revealing solution: the revealing solution is composed of: 9a) a substrate buffer: 0.01M citric acid solution, and 0.04M trisodium citrate containing 0.33% H2O2, pH final 5,6, and 9b) of a chromogen: OPD tablets (orthophenylenediamine). 1 OPD tablet to dissolve in 10m1 of substrate buffer. 10) Stop Solution: H2SO4 4N.   Protocol: The principle of the test used is based on the enzymoimmunological method of sandwich type, made in flat-bottom microplate. This is a two-step test where the sample (or standard solution) is first incubated with the capture antibody, then after incubation and washes, the detection antibody is added. .   A sensitization solution is first prepared with the anti-BNP polyclonal antibody (77-108) diluted in Dulbecco's PBS buffer at pH 7.4 to 10 μg / ml. 100 μl of this solution are deposited in each well of the microplate.   The microplate is incubated overnight at 4 C.   After removal of the sensitizing solution, the microplate is washed with Dulbecco's PBS buffer at pH 7.4 containing 0.1% Tween 20, and then saturated by the addition of 250 μl of Dulbecco's PBS buffer at pH 7. , 4 containing 1% BSA.   The microplate is then incubated for 1 hour at 37 ° C.   The microplate is then washed (3 times) with the washing solution.   In each well, 100 μl of standard solution of proBNP (1-108), serum or plasma are deposited. ProBNP (1-108), if present, binds to the capture antibody retained on the solid phase.   The reaction medium is incubated for 2 hours at room temperature. The microplate is then washed (5 washes) with 300 μl of the washing solution.   The conjugate, the polyclonal rabbit antibody coupled to biotin is added to the wells of the microplate at 6 μg / ml under 100 μl.   The reaction medium incubated for 2 hours at room temperature.   The microplate is then washed (5 washes) with 300 μl of the wash solution.   Finally, 100 μl streptavidin-POD conjugate diluted 1/1000 is added to each well of the microplate.   The reaction medium is incubated for 1 hour 30 at room temperature.   The plates are then washed (5 washes) with 300 μl of wash solution. In each well, 100 μl of the developing solution is dispensed. The reaction is allowed to proceed in the dark for 20 minutes at room temperature (18-24 C).   Then 50 μl of the stop solution is dispensed into each well.   After stopping the reaction, the reading of the optical density is carried out on a spectrophotometer at 4901'620 nm.   Figure 8 presents the results obtained using a standard range of proBNP (1-108).   EXAMPLE 14 Evaluation of the Cross-Reaction, with Respect to BNP (1-76) and BNP (77-108), of the ELISA ProBNP Assay (1-108) Combining the Anti-BNP Polyclonal Antibody (77-108) with the non-depleted rabbit polyclonal antibody # 046 805 coupled to biotin.   Using the ELISA proBNP assay (1-108) described in Example 13, the cross-reaction of the assay against BNP (1-76) and BNP (77108) was evaluated. Concentration ranges from 5 ng / ml to 100 ng / ml were performed with proBNP (1-108), E3NP (1-76) and BNP (77-108) and tested using the ProBNP ELISA assay ( 1-108) described in Example 13. The results of the change in optical density at 490 nm as a function of concentration are shown in Figure 9 for each of the proteins.   No cross reaction is observed with respect to BNP (1-76) or BNP (77-108): the signal obtained is equivalent to the background noise, regardless of the concentration tested. At concentrations of BNP (1-76) and BNP (77-108) usually found in patients (in the range of ng / ml for the highest concentrations), rabbit polyclonal antibody # 046 805 can be used in its version not exhausted without causing the appearance of cross reaction.   Example 15 Evaluation of the cross-reaction, with respect to BNP (1-76) and BNP (77-108), of the ELISA proBNP (1-108) assay combining a polyclonal anti-BNP antibody (1-76) with polyclonal rabbit antibody not depleted coupled with bioinin.   Using an ELISA assay using the protocol and the reagents described in Example 13, except that the anti-BNP polyclonal antibody (77-108) is here replaced by a polyclonal anti-NT-proBNP antibody. (1-29), cross-reactivity of the non-depleted biotinylated rabbit polyclonal antibody combined with an anti-BNP polyclonal (1-76) was evaluated. The polyclonal anti-NT-proBNP antibody (1-29) was produced according to the protocol described in Example 3, except that the peptide used as immunogen was the peptide NT-proBNP (1-29) coupled to the KLFI. Concentration ranges from 5 ng / ml to 100 ng / ml were carried out with proBNP (1-108), BNP (1-76) and BNP (77-108) and tested using the ProBNP ELISA assay ( 1-108). The results of the change in optical density at 490 nm as a function of concentration are shown in Figure 10 for each of the proteins.   No cross reaction is observed with BNP (1-76) or BNP (77-108), the signal obtained is equivalent to the background noise, regardless of the concentration tested. At concentrations of BNP (1-76) and BNP (77-108) usually found in patients (of the order of ng / ml), the rabbit polyclonal antibody # 046 805 can be used in its non-depleted version. without causing the appearance of a cross reaction. CLAIMS,   1. Peptide of formula: a1 - X1 RAPRSP X2 a2 (I) where a1 may be H or represent a function or a chemical group chosen from a thiol, alcohol, aminoxy, primary or secondary amine function, an amino-carboxyl group, a biotinyl group and an acetyl group, X1 represents a peptide sequence of 0 to 3 amino acids, derived from the natural sequence of proBNP (1-108) or not, X2 represents a peptide sequence of 0 to 8 amino acids, derived from the natural sequence of proBNP (1..108) or not, a2 may represent an OH, NH2 function or an alkoxyl group.   2. Peptide of formula: X-Y70TLRAPR76S77PKMVQGSG85-Z (II) where X can be H, or represent either an acetyl group or 1 to 3 amino acids not belonging to the proBNP (1-108) sequence, and Z can represent an OH function, or 1 to 3 amino acids not belonging to the proBNP sequence (1-108).   Peptide according to claim 2 having the sequence Y70TLRAPR76S77PKMVQGSG85.   4. Peptide comprising a sequence derived from the sequence XY70TLRAPR76S77PKMVQGSG85-Z (II) by substitution of one or more of the amino acids Y7), T71, L72, K79, M80, V81, Q82, G83, S84 and G85, with X which can be or represent either an acetyl group or 1-3 amino acids not belonging to the proBNP sequence (1-108), and where Z can be an OH function, or 1 to 3 amino acids not belonging to the proBNP sequence (1-108).   Peptide according to claim 1, having a sequence selected from the group consisting of SEQ ID N0 6: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 11 ID N 12: SEQ ID N 13: SEQ ID N 14: SEQ ID N 17: SEQ ID N 18: SEQ ID 19: SEQ ID N 20: following sequences C-G-R-A-P-R-S-P   Acetyl -CGRAPRSP CGRAPRSPK Acetyl-CGRAPRSPK CG-RA-PRSPKMV CGRAPRSPKMVQGSG RAPRSPGC acetyl -R-acetyl AP-RSPGC -CYTLRAPRSPK CFTLRAPRSPKMVQG-SG CFSIRAPRSPKMVQGSG CYTLRAPRSPKMVQGS-RA CY-TLRAPRSPKMVQAT- (3A SEQ ID NO 21: CFSIRAPRSPKM-VQ-A-T- 13A SEQ ID N 22: CFSIRAPRSPALASGTA 6. Peptide having the sequence CHRKMVLYTLRAPRSPK (SEQ ID N 15).   7. A proBNP (1-108) detection kit in a biological sample containing, as a standard and / or control, at least one peptide as defined in one of claims 1 to 6.