EP1723430A2

EP1723430A2 - Method for detecting the activatable free form of psa and the use thereof for diagnosing benign pathologies of the prostate and adenocarcinoma of the prostate

Info

Publication number: EP1723430A2
Application number: EP05739674A
Authority: EP
Inventors: Sandrine Busseret-Michel; Colette Jolivet-Reynaud
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2004-03-03
Filing date: 2005-03-01
Publication date: 2006-11-22
Also published as: WO2005085292A3; US20070254317A1; JP2008509379A; CN1926433A; WO2005085292A2

Abstract

The invention relates to an in vitro diagnostic method for diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate characterized by comprising a step for detecting the activatable free form of PSA in a biological sample from a patient suspected of having developed a benign pathology of the prostate or an adenocarcinoma of the prostate. This diagnostic method can be carried out by the following steps consisting of: i) bringing a binding partner capable of specifically binding to the activatable free PSA in contact with a biological sample from a patient suspected of having developed a benign pathology of the prostate or an adenocarcinoma of the prostate; ii) detecting the capture of the activatable free form of PSA by said binding partner; iii) calculating the ratio between the quantity of the activatable free form of PSA detected in step ii) and the quantity of a form of PSA other than the activatable free form present in a sample of the same type taken from the same subject, and; iv) determining if the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step iii) with a predetermined threshold value selected according to the type of ratio used and representative of the detection limit of each pathology.

Description

The present invention relates to the diagnosis of adenocarcinomas of the prostate. Method for detecting the activatable free form of PSA and its use for the diagnosis of benign prostate pathologies and of adenocarcinoma of the prostate. In particular, the subject of the invention is a method of diagnosing a benign pathology of the prostate or of an adenocarcinoma of the prostate in a patient suspected of being affected by such pathologies by using binding partners capable of recognizing this. specific prostate antigen (or PSA, for Prostate Specifies Antigen) specifically in its activatable free form. PSA is produced by the glandular epithelium of the human prostate, probably in an inactive zymogenic form (Lundwall et al. FEBS Lett. 1987), and is secreted in seminal fluid in its active form (Lilja, J. Clin Invest 1985 ). The biological activity of PSA in seminal fluid is linked to its limited proteolytic fragmentation of the predominant proteins secreted by the seminal vesicles (Lilja, J. Clin Invest 1985; Lilja et al. J. Clin Invest 1987; Me Gee et al. Biol . reprod. 1988). PSA is the main marker of prostate cancer, which will affect one in six men in the West during their lifetime. This protease of the kallikrein family, mainly secreted by the prostatic epithelium, is found at a concentration of 0, 5 to 5 mg / l in the seminal fluid and at a concentration a million times lower in the serum of a patient Thus, PSA is normally found at a concentration lower than 2.5 ng ml in the serum. However, this concentration increases in principle notably during prostate cancer and during benign alterations such as benign prostatic hyperplasia (BPH) or acute prostatitis. The protein sequence of PSA has been determined. It is a glycoprotein comprising 237 amino acids ("Molecular cloning of human prostate specifies antigen cDNA". Lundwall A., Lilja H., 1987. FEBS Lett 214: 317-322). A diagnostic method has been proposed which consists in measuring the concentration of serum PSA and in comparing it with a threshold value which is 4 ng / ml. However, it has been found that this diagnostic method wrongly suspects three out of four patients, which is detrimental. In addition, this method does not make it possible to diagnose 30 to 45% of cases of cancer confined to the gland, which however constitutes an early stage of the potentially curable disease, the diagnosis of which would therefore be particularly desirable. The unsatisfactory nature of this method is also demonstrated by the study reported in the article "Prostate Cancer Detection in Men With Serum PSA Concentrations of 2.6 to 4.0 ng / ml and Benign Prostate Examination '', Catalona et al, JAMA, May 14, 1997 - Issue 277, Νo. 18, which shows that the cut-off value must be less than 4 ng / ml for early detection of prostate cancer, and it has been shown that in serum, PSA were associated with protease inhibitors, such as rα-1-antichymouypsin (ACT), and α-2 macroglobulin (A2M). These associations lead to the inactivation of the chymotrypsic activity of PSA, as has been demonstrated in the article Εnzymatic activity of prostate specifies antigen and its reactions with ultracellular serine proteinase inhibitors "A. Christensson, CB Laurell, H. Lilja, 1990 Eur. J. Biochem 194: 755-763. This also made it possible to demonstrate that the PSA present in the serum was either in free form, that is to say unassociated, or in complexed form, that is to say associated. The use of the ratio of free PSA to total PSA was then proposed in order to improve the specificity of the diagnosis. Thus, patent application WO97 / 12245 describes a method making it possible to diagnose an adenocarcinoma of the prostate without biopsy. This method involves measuring the total amount of PSA in the patient's serum or blood. If this value is between 2.5 and 20 ng / ml, the concentration of free PSA is also measured. The free PSA to total PSA ratio is then calculated. If this ratio is less than 7%, the diagnosis is directed towards an adenocarcinoma of the prostate. However, the use of a 7% threshold for the diagnosis of prostate cancer is controversial by many authors, as shown in the publication by Lein et al. "Relation of free PSA / total PSA in serum for differentiating between patients with prostatic cancer and benign prostate hyperplasia: which cutoff should be used?". In this document published in the journal Cancer Investigation, 16 (1), 45-49, 1998, it has been shown that it is difficult, by means of this ratio, to systematically differentiate a prostate cancer from a BPH. For these reasons, the applicant is interested, in patent application WO00 / 02052, in the presence in the serum of cleaved forms of PSA among the free forms of PSA. The molecular forms of serum PSA from cancer patients or of BPH were mapped by two-dimensional electrophoresis, associated with clumioluminescence detection, in order to observe all of the forms of PSA, that is to say the free forms, including cleaved forms, and complexed forms. The electrophoresis profiles of sera from subjects suffering from prostate adenocarcinoma are relatively homogeneous, presenting essentially the non-cleaved free forms of PSA, while those of subjects suffering from BPH may comprise a relatively large proportion of cleaved free forms and slightly more basic spots without cleaved form. The increase in the ratio of serum free PSA to total serum PSA observed in patients with BPH is therefore essentially linked to the existence of cleaved free PSA, which could be enzymatically inactive and therefore incapable of binding to ACT, as well as 'to the presence of a slightly more basic form of free PSA than the active free PSA form, which could correspond to the zymogenic, inactive form of PSA. Based on these observations, the Applicant has described methods for diagnosing prostate adenocarcinoma comprising the quantification, after separation by two-dimensional electrophoresis, of the cleaved and / or uncleaved free PSA and the use of these values in order to establish a diagnostic. However, even if these methods have improved the diagnosis, they still require the implementation of two-dimensional electrophoresis. They are therefore quite expensive and require a lot of handling time. To avoid these manipulations, it was described in patent application WO01 / 77180 of new antibodies directed specifically against the inactive free PSA, as well as their use in a method of diagnosis of benign prostate pathology or adenocarcinoma of the. prostate. The Applicant has now discovered that there exists in patient biological samples, among the free forms of PSA, a new form of PSA which is not not the zymogenic form (ProPSA) but is also activatable, that is to say which potentially has the capacity to react with protease inhibitors, and that the use of the detection of such a free form of activatable PSA allowed a differential diagnosis between subjects suffering from benign prostate pathologies and subjects suffering from prostate adenocarcinoma with excellent specificity and sensitivity. Thus the subject of the present invention is a method of in vitro diagnosis of a benign pathology of the prostate or of an adenocarcinoma of the prostate, characterized in that it comprises the step of detection, in a biological sample derived from a patient suspected of having a benign prostate disease or an adenocarcinoma of the prostate, of the activatable free form of PSA. The different forms of PSA, present in biological fluids such as the seminal fluid or the serum of a patient have often had different names according to the authors. All agree that total PSA comes in two forms: the free form (free PSA) and the form complexed with this protease inhibitors such as TACT (complexed PSA). Free PSA can also be named according to its size: when it is produced in the form of ProPSA, it has additional amino acids (7 or 5 or 4 or 2). This is called zymogenic PSA or ProPSA-7, ProPSA-5, etc. This proPSA, after maturation and therefore loss of its additional amino acids, can be found either in intact form or in partial form. In the latter case, we speak of truncated or cleaved PSA. Free PSA in intact form can also be denatured (glycosylated or deglycosylated); we then speak of a distorted form. Thus, the free PSA forms include zymogenic PSA, intact PSA, denatured or not, and cleaved PSA. We also speak, for these different free or complexed forms of PSA, of active forms and inactive forms. By active form is meant the forms being capable of binding to protease inhibitors, such as TACT. By inactive form is meant the forms incapable of such a binding capacity. We know that complexed PSA is inactive since it has already linked to an inhibitor. Likewise, cleaved free PSA and denatured free PSA are inactive because they no longer have the capacity to bind to inhibitors. Finally, the forms of zymogenic PSA are inactive. The other free forms of PSA are therefore active. While in the seminal fluid or in supernatants of the LnCaP cell line, these active forms are present, it is commonly accepted that these active forms do not exist in serum because they are complexed with protease inhibitors. The Applicant has now demonstrated, against all expectations, that among these free forms exists, in the biological samples of the patients, an activatable free form, that is to say still having the capacity to bind to the protease inhibitors in serum, this binding being able to be implemented after activation of said activatable free form. It also demonstrated that the use of the assay of such a form of free activatable PSA allowed the diagnosis of patients with adenocarcinoma of the prostate or of BPH. Without wishing to be bound by a theory, the Applicant thinks that this activatable free form of PSA would, after activation, be equivalent to the form of PSA which complexes with ACT. This free activatable form would be a form of "closed" PSA, trapping the site of attachment to ACT, its activation making it possible to transform the "closed" form into an "open" form and thus release the site of fixation. The diagnostic method of the invention can be implemented either using a binding partner capable of binding specifically to activatable free PSA, or a binding partner capable of binding to non-specifically activatable free PSA. Thus, according to a first embodiment, the invention relates to a method of in vitro diagnosis of a benign prostate pathology or of an adenocarcinoma of the prostate, characterized in that it comprises the steps consisting in: i) contacting a binding partner capable of binding specifically to activatable free PSA with a biological sample from a patient suspected of having a benign prostate pathology or an adenocarcinoma of the prostate, i) putting in evidence the capture of the activatable free form of PSA by said binding partner, ϋi) calculating the ratio between the quantity of activatable free form of PSA detected in step ii) and the quantity of a form of PSA other than the activatable free form, present in a sample of the same kind taken from the same subject and iv) determine if the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step iϋ) with a predetermined threshold value, chosen according to the type of ratio used and representative of the limit of detection of each pathology. The Applicant has discovered that, unexpectedly, among the binding partners suitable for the purposes of the invention, those recognizing the epitope mimicked by the sequence SEQ ID No. 1 (DTPYPWGWLLDEGYD) were actually able to bind specifically in serum to this activatable free form of PSA without binding or to the denatured or cleaved free PSA, laughed at ProPSA, and that the use of this particular property made it possible to obtain a very sensitive and very specific prostate diagnostic method for pathologies . The biological samples in which the method of the invention is implemented are any biological sample likely to contain PSA. By way of example of such samples, mention may be made of seminal fluid, blood, serum, plasma and urine, serum and plasma being particularly preferred. Binding partners capable of specifically binding to activatable free PSA suitable for the purposes of the invention include, for example, antibodies, antibody fragments and mirnotopes, as well as any other partner known to those skilled in the art for having this. capacity. By antibody fragments is generally meant in the present application, any antibody fragment which has retained the specificity of the original antibody, in the present case the ability to bind specifically to the free activatable PSA, and in particular Fab and F (ab ') ₂ fragments. In the present application, the word "antibody" also denotes subsequently fragments of antibodies when the sense allows. The antibodies useful within the meaning of the invention include in particular purified polyclonal antibodies and monoclonal antibodies. The preparation of polyclonal antibodies and monoclonal antibodies is widely known to those skilled in the art and the principle of this preparation is recalled below. The polyclonal antibodies can be obtained by immunization of an animal with at least one target antigen of interest, followed by the recovery of the antibodies sought in purified form, by taking the serum from said animal, and separation of said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column on lacquer, an antigen specifically recognized by the antibodies is fixed, in particular a target antigen of interest. The monoclonal antibodies can be obtained by the hybridoma technique, the general principle of which is recalled below. First, an animal, generally a mouse, (or cells in culture as part of in vitro immunizations) is immunized with a double antigen of interest, the B lymphocytes of which are then capable of producing antibodies against said antigen. These antibody-producing lymphocytes are then fused with myeloma cells

"immortal" (murine in the example) to give er to hybridomas. From the heterogeneous mixture of cells thus obtained, a selection is then made of the cells capable of producing a particular antibody and of multiplying indefinitely. Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody whose Recognition properties vis-à-vis the tumor antigen of interest can be tested for example by ELISA, by immunotransfer in one or two dimensions, by immunofluorescence, or using a biosensor. The monoclonal antibodies thus selected are subsequently purified in particular according to the affinity chromatography technique described above. By mimotopes is meant any synthetic or recombinant peptide capable of mimicking a conformation which interacts specifically with said epitope. According to a preferred embodiment, the binding partner capable of binding specifically to the activatable free PSA fulfills at least one of the following conditions: - it is capable of recognizing the epitope mimicked by the sequence SEQ ID No. 1. - it is an antibody or an antibody fragment. The sequence SEQ ID No. 1 recognized by the appropriate binding partners for the purposes of the invention mimics a conformational epitope of PSA. An example of an antibody capable of recognizing the epitope mimicked by the sequence SEQ ID No. 1 suitable for the purposes of the invention is the antibody 5D3D11, as described in Michel S., et al., 1999, Clinical Chemistry, 45 (5): 638-650. The use of this particular antibody in the process of the invention is unexpected in the sense that this article indicates that this antibody is capable of inhibiting the enzymatic activity of PSA and therefore the binding of ACT, in other words that it is only capable of adhering to an active free form of "open" PSA. In the present case, the Applicant has also shown that it is capable of binding to the activatable free form, that is to say "closed". The conjugates consisting of the binding partner capable of binding specifically to the activatable free PSA and of the activatable free form of PSA are new and also constitute an object of the invention. According to one embodiment, said binding partner capable of binding specifically to the activatable free PSA of the conjugate of the invention is a binding partner capable of recognizing the epitope mimed by the sequence SEQ ID No. 1. The second step of the diagnostic method of the invention consists in demonstrating the capture of said activatable free form of PSA by said binding partner capable of specifically binding to the activatable free PSA. This step can be carried out directly by detecting the bond between said binding partner and said activatable free form of PSA, or else after elution of said activatable free form of PSA immunocaptured by said binding partner. The activatable free form of PSA immunocaptured by said hedgehog partner, then eluted will be called hereinafter.

Immunopurified activatable free PSA. The elution of the activatable free form of PSA immunocaptured by said hedge partner capable of binding specifically to the activatable free PSA can be implemented by any elution method known to those skilled in the art, such as a shock. pH. Preferably, an additive shock is used, for example using a 0.1 M glycine buffer, pH 2.8. The detection of the capture of said activatable free form of PSA, whether it is irrmunopurified or not, can be carried out by any detection means known in the field of immunoassays, such as direct detection and indirect detection. In the case of indirect detection, i.e. by means of a detection partner, a detection partner capable of adhering to the activatable free form of PSA is used. This detection partner binds on an epitope different from that used by said binding partner used in step i) when the activatable free PSA is not immunopurified. As a suitable detection partner for this purpose, there may be mentioned antibodies such as anti-total PSA antibodies, which constitutes a mode of reahsation of the invention. Examples of such anti-PSA antibodies capable of recognizing an epitope different from that used by said Haison partner used in step i) are described in the article by Michel S., et al (1999, supra). These detection partners can be previously marked. The term “marking” is intended to mean the attachment of a marker capable of directly or indirectly generating a detectable signal. A non-limiting Hste of these markers consists of:

• enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, acetylchoHne esterase, β-galactosidase, glucose-6-phosphate dehydrogenase,

• chromophores such as luminescent compounds, dyes,

• radioactive molecules such as ³² P, ³⁵ S or ¹²⁵ I,

• fluorescent molecules such as fluorescein, rhodomine, alexa or phycocyanins, and • particles such as gold particles, magnetic latex, Hposomes. Indirect labeling systems can also be used, such as, for example, through another Hgand anti-Hgand pair. The Hgand / anti-Hgand pairs are well known to those skilled in the art, and the following pairs may be mentioned, for example: biotin / streptavidin, hapten / antibody, antigen / antibody, pepti.de/ antibody, sugar / lectin, polynucleotide / complementary to the polynucleotide. In this case, it is the Hgand which is Hey to the partner of Haison. The anti-Hgand can be directly detectable by the markers described in the preceding paragraph or be itself detectable by a Hgand / anti-Hgand. These indirect systems can lead, under certain conditions, to an amplification of the signal. This signal amplification technique is well known to the person skilled in the art. etier, and reference may be made to earlier patent applications FR98 / 10084 or WO95 / 08000 from the Applicant or to article J. Histochem. Cytochem., (1997), 45: 481-491. Direct detection of the capture of the Hebre form activatable by said partner of Haison, that is to say without the intermediary of a detection partner, can be implementation for example by plasmon resonance or by cycH voltammetry on an electrode carrying a conductive polymer. Direct detection can also be implemented using the specific property of enzymatic activity of active forms of PSA. In this case, activation of the activatable Hebrew form of PSA should be carried out, if appropriate after its immunopurification. Indeed, the activation of this activatable Hbre form makes it possible to Hbb the Haison site to the enzyme substrate, which can then be reacted with an enzyme substrate. Thus, the detection is carried out by determining the enzymatic activity of the Hbre form of the immunopurified PSA and activated, which constitutes a particular embodiment of the invention. Examples of enzymatic substrates which are suitable for the purposes of the invention include all the substrates revealing the chymotrypsic type protease activity widely known to those skilled in the art. Such substrates are available for example from

Enzyme System Products. They are composed of a recognized peptide sequence and cHvée by PSA, this sequence being coupled to a chromophore or fluorophore group. The activation of the activatable Hebre form of PSA can be implemented by at least one of the following methods:

- bringing said activatable form into contact with a miHeu of high saline concentration, of at least 0.15M, preferably of at least 1M, more preferably of at most 2M, - Season of PSA Hbre activable i munopurified to an antibody capable of increasing the enzymatic activity of PSA, these two methods being able to be implemented separately simultaneously or successively in any order. Mention may be made, as miHeu of high saline concentration, of miHeux containing 1.5M NaCl and as an antibody capable of increasing the enzymatic activity of PSA, there may be mentioned the antibody 8G8F5 (bioMérieux, France). Due to the particular property of these antibodies capable of increasing the enzymatic activity of PSA, their use in the process of the invention makes it possible to improve the sensitivity of said process. Thus, according to one embodiment, the method of the invention is characterized in that it uses, in addition to the Haison partner capable of specifically targeting Her activable PSA, an antibody capable of increasing the enzymatic activity of PSA , and in particular the antibody 8G8F5. This antibody capable of increasing the enzymatic activity of PSA can be used as a capture partner in an ELISA type assay when the Haison partner capable of specifically targeting Her activatable Hebre PSA is used to immunopurify the activatable Hebre PSA. It can also be used as a detection partner, in particular when the Haison partner capable of specifically targeting the activatable Hebre PSA is used as capture partner. The conjugates consisting of the Haison partner capable of specifically Herb activating the PSA Hbre, such as a Haison partner capable of recognizing the epitope mimicked by the sequence SEQ ID No. 1, and of the activable Hbre form of the PSA, lacquer is activated, are also new and constitute a particular embodiment of the invention. In the process of the invention, the Haison partners capable of being Her specifically to activatable PSA Hbre, being specifically haunted to activable PSA Hbre, can be used as such or in particular in the form fixed on a solid support and / or Hes to a marker. The fixation of these Haison partners on a solid support is well known. The support can be reacted with any solid, biological or synthetic material, endowed with adsorbent properties or capable of fixing a coupling agent. Materials are known and described in the literature. Among the solid materials capable of fixing these partners of Haison by adsorption, one will quote for example polystyrene, polypropylene, latex, etc. Among the materials allowing to fix these partners of Haison by oovalence using a coupling agent, there may be mentioned in particular dextran, ceUulose, etc. The support can be presented, for example, in the form of discs, tubes, tubes, cones or plates, in particular microtiter plates. The Haison of Haison partners with a marker will also allow direct detection, as described above. This marker is as previously described. When the Haison partner is not connected to a support or when the activatable Hbre PSA is immunopurified, the method of the invention can also use a capture partner. In the first case, this capture partner is able to link to an epitope different from that recognized by the partner of Haison capabb to link to the PSA Hbre activable specifically. Examples of capture partner include anti-PSA antibodies, as described above. Insofar as both a capture partner and both a detection partner are used in the method of the invention to assay the Pbre HSA these partners can be activated with different epitopes, themselves being different from the epitope recognized by the Haison partner capable of specifically activating with PSA Hbre activable when PSA

He activatable was not immunopurified. The methods implementing the various partners of the activatable form of PSA for the demonstration of the capture of the activatable Hbre form of PSA by said partner of Haison are widely known to those skilled in the art. By way of example, mention may be made of the sandwich type methods such as the ELISA method, and the competitive assay methods. Step Hi) of the method of the invention consists in calculating the ratio between the quantity of activable Hebre form of PSA detected in stage H) and the quantity of a form of PSA other than said activatable form of the invention , present in a sample of the same nature taken from the same subject By the expression "sample of the same nature taken from the same subject", we mean either two fractions of the same sample, or two samples from two different samples but which must be of the same kind, for example serum samples. The forms other than the activatable Hbre form of PSA are in particular complexed PSA, total PSA, total PSA Hbre, zymogenic PSA Hbre, denatured PSA Hbre, PSA Hbre cHve and their associations, that is to say all active or inactive forms, Hbre or complexed, of PSA. The assay of each of these different forms, in particular using specific antibodies, is known. The complexed PSA is assayed for example using antibodies described in patent application WO98 / 22509. The total PSA is assayed for example using antibodies described by H. Nagasaki et al. (1999), Clin. Chem. 45: 4486-496. Other antibodies capable of binding to various forms of Hbre PSA, including total PSA Hbre, are marketed by Chugai (Japan), BiosPacific (EmeryviHe, CA) and Euromedex US Biological (Swampscott, MA). The quantification is carried out in a known manner from the methods for demonstrating the detection of immunological reactions as described above (sandwich, etc.), for example by determining the quantity of label when labeling is used. Of course, when the quantities measured are intended to be compared, these values should be comparable. In other words, the measured values should relate to the same dilution or possible concentration of the sample as well as to the same volume. The different ratios which can be calculated in step Hi) of the process of the invention can be chosen from the following: - amount of activatable Hebre PSA / amount of total PSA

- quantity of activable PSA Hbre / quantity of total PSA Hbre,

- amount of activatable Hebre PSA / amount of complexed PSA,

- amount of activable PSA Hbre / amount of PSA Hbre cHve,

- amount of activatable Hebre PSA / amount of free zymogenic PSA, - quantity of activable PSA Hbre / quantity of denatured PSA Hbre, - quantity of activatable PSA Hbre / quantity of (denatured PSA Hbre + zymogenic PSA Hbre),

- amount of activable PSA Hbre / amount of (denatured PSA Hbre + PSA Hbre cHve),

- quantity of activable PSA Hbre / quantity of inactive PSA Hbre (zymogen, denatured and cHve) - the inverse of these ratios, or

- Combinations of these ratios, the preferred ratios being relative to the quantity of PSA Hbre activatable over the quantity of (denatured PSA Hbre + PSA Hbre cHve) or the quantity of PSA Hbre activatable over the quantity of PSA Hbre inactive. According to a particular embodiment of the invention, the form of PSA other than the activatable Hebre form used to calculate the ratio in step (in) of the process of the invention is the form of denatured Hebre PSA + the form of PSA Hbre chvé or the form of PSA

Inactive. The implementation of this calculation is obviously done as follows: - the quantity of activatable PSA Hbre is evaluated in a biological sample taken from a subject by using the partner of Haison capable of being specifically Her to activable PSA Hbre, as described above

one of the quantities chosen from the quantity of total PSA, the quantity of total PSA Hbre, the quantity of complexed PSA, the quantity of zymogenic PSA Hbre, the quantity of PSA Hbre cHve, the quantity of denatured PSA H is evaluated, the amount of denatured PSA Hbre + PSA Hbre cHve, the amount of denatured PSA Hbre + zymogenic PSA Hbre and the amount of inactive PSA Hbre, or their associations, on a similar sample taken from the same subject, and

- Said ratio or said inverse ratio, or said combination of ratios is determined. Step iv) of the method of the invention consists in determining whether the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step Hi) with a value- predetermined threshold, chosen according to the type of report used and representative of the detection limit for each pathology. We know that, in general, the results of immunological tests depend largely the characteristics of specificity and affinity of the antibodies used, and that these characteristics influence the values measured with these antibodies. It is therefore understandable that it is not possible to give precise threshold values and that threshold values adapted to each antibody used can be determined in each case by simple routine experiments. It should be clearly understood that here a threshold value is called either a discrete value, or an interval of values corresponding to a zone of determination. Obviously, when the measured value is included in the indeterminacy interval, or is very close to the threshold value in the case of a discrete value, one cannot definitively conclude and additional investigations should be carried out. Of course, when a threshold value has been determined for a given type of report, it can be deduced therefrom threshold values corresponding to other types of reports. In the case where we consider a report PSA Hbre activable / (denatured PSA Hbre + PSA Hbre cHve) or a report PSA Hbre activable / PSA Hbre inactive, we will diagnose an adenocarcinoma of the prostate for the patients having a report higher than the value- threshold obtained from said report, and a BPH or other non-cancerous prostatic pathology ("normal patients") will be diagnosed for patients with a ratio below this threshold value. Generally, when the amount of activatable PSA Hbre is found in the numerator of the calculated ratio, a prostate adenocarcinoma will be diagnosed for patients with a ratio greater than the threshold value obtained from said ratio, and a diagnosis will be made. BPH or other non-cancerous prostatic pathology (“normal patients”) for patients with a ratio below this threshold value. On the other hand, when the amount of activatable PSA Hbre is found at the denominator of the calculated ratio, a prostate adenocarcinoma will be diagnosed for patients with a ratio below the threshold value obtained from said ratio, and a BPH or other diagnosis will be diagnosed. non-cancerous prostate pathology (“normal patients”) for patients with a ratio greater than this threshold value. To implement the method of the invention, the invention also relates to a diagnostic kit making it possible to diagnose an adenocarcinoma of the prostate or a benign pathology of the prostate, said kit comprising:

a Haison partner capable of specifically targeting Her activable Hbre PSA, preferably a Haison partner capable of recognizing the epitope mimicked by the sequence SEQ ID No. 1, more preferably an antibody or an antibody fragment, and,

- Means for assaying forms of PSA other than the activatable Hbre form, preferably antibodies or antibody fragments. Such means which can be used in said kit are as described above for the determination of the form of PSA which it is desired to quantify. The method of the invention can also be implemented by using a partner of Haison capable of getting Her to the PSA Hbre non-specifically activatable. Such partners can be partners capable of integrating with total PSA, including in particular activatable Hebre PSA. By way of example of such partners, one can determine the antibodies, the antibody fragments and the mimotopes, as well as any other partner known to a person skilled in the art for having this capacity, as described above and in particular the antibody 11E5C6. (Michel et al., 1999, supra), the conformational epitope of which overlaps the C-terminal region of the PSA (Michel S et al., 2001, J. Mol. Recognit, 14: 406-413). This Haison partner capable of being linked to non-specifically activable Hbre PSA can be used in the process of the invention as described above with the Haison partner capable of being specifically activated to activable Hbre PSA, namely as capture partner. or to immunopurify PSA Hbre. However, in this particular embodiment, the detection of the activatable Hebre form of PSA must be implemented using the particular property of enzymatic activity of the active forms of PSA. In this case, the activatable Hbre form of PSA should be activated. In fact, the activation of this activatable free form makes it possible to Hbb the Haison site to the enzyme substrate, which can then be reacted with an enzyme substrate as described above. Thus, the detection is implemented by determining the enzymatic activity of the Hbre form of the activated PSA. Thus, according to this embodiment, the invention relates to a diagnostic method a benign prostate pathology or an adenocarcinoma of the prostate according to claim 1, characterized in that it comprises the steps consisting in: i) bringing into contact a partner of Haison capable of being Her with PSA Hbre activatable in a non-specific manner, with a biological sample from a patient suspected of having a benign prostate pathology or an adenocarcinoma of the prostate, ϋ) highlight the capture of the activatable Hebre form of PSA by said partner of Haison by determination of the enzymatic activity of the Hbre form of activated PSA, after activation of the activable Hbre form of PSA ϋi) calculate the ratio between the quantity of activable Hbre form of PSA detected in step H) and the quantity of a form of PSA other than the activatable Hbre form, present in a similar sample taken from the same subject and iv) determining whether the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step iH) to a predetermined threshold value, chosen according to the type of ratio used and representative of the limit of detection of each pathology. The activation of the activatable Hebre form of PSA can be implemented by at least one of the following methods:

bringing said activatable form into contact with a miHeu of high saline concentration, of at least 0.15M, preferably of at least 1M, more preferably of at most 2M, as described above,

- Haison PSA Haison activatable with an antibody capable of increasing the enzymatic activity of PSA, and in particular the antibody 8G8F5, these two methods can be implemented separately, simultaneously or successively, in any order. Due to the particular property of these antibodies capable of increasing the enzymatic activity of PSA, their use in the process of the invention makes it possible to improve the sensitivity of said process, which constitutes a preferred mode of reaction. Thus, according to this embodiment, the method of the invention is characterized in that it uses, in addition to the Haison partner capable of taking up the PSA Hbre activatable so non-specific, an antibody capable of increasing the enzymatic activity of PSA, and in particular the antibody 8G8F5. This antibody capable of increasing the enzymatic activity of PSA can be used as such, in an assay, as a capture partner for activable PSA Hebre previously immunopurified in a specific or non-specific manner from biological samples. The

HSA PSA thus activated after its capture by the antibody will be detected by its enzymatic activity which will be measured via kinetics of hydrolysis of a fluorescent substrate. The slope of the kinetics will be expressed in fluorescence units. The correspondence in quantities of PSA will be obtained thanks to a standard curve curve with quantities of active PSA, between With the exception of the above, steps i) to iv) of this particular embodiment are as described above. the present invention will be better understood with the aid of the following examples given solely by way of illustration and without limitation, as well as with the aid of the appended FIGS. 1 to 6, on which: - FIG. 1 shows the photograph of 3 gels of two-dimensional electrophoresis obtained from 0.5 μg of PSA derived from the culture supernatant of LNCaP cells immunopurified with the antibodies 5D3D11 (photograph A), 6C8D8 (photograph B) and 11E5C6 (photograph

VS),

FIG. 2 represents a standardization graph giving the fluorescence as a function of the PSA concentration obtained by using seminal PSA previously in-tmunopurified with the antibody 5D3D11 and assayed in total PSA,

- Figure 3 is a graphical representation giving the values of the PSA activable / (PSA Hbre cHve + PSA Denatured PSA) ratios obtained with the method of the invention for serum from patients with prostate cancer and treated by radiotherapy (RT ), by hormone therapy (HT) or by prostatectomy (PR), of patients with cancer but whose treatment is not known (others), of patients with benign hyperplasia (BPH) and of normal patients (PN) (part A of the graph), as well as the values of the PSA Hbre PSA total ratios obtained according to a process of the prior art (part B of the graph) with these same sera,

- Figure 4 is a graphical representation giving the values of the PSA ratios activable / (PSA Hbre cHve + PSA Hbre denatured) obtained with the method of the invention in sera having a PSA Hbre / PSA total ratio of between 0.15 and 0.25, the sera being from patients suffering from cancer prostate and treated with radiotherapy (RT), hormone therapy (HT) or prostatectomy (PR), patients with cancer but whose treatment is unknown (others), patients with benign hyperplasia (BPH) and of normal patients (PN), - Figure 5 is a graphical representation giving the values of the PSA activable / (PSA Hbre cHve + PSA Denatured PSA) ratios for the sera of patients with prostate cancer and patients with hyperplasia benign (BPH), with a total PSA level of less than 2.5 ng / ml, and

FIG. 6 is a graphical representation giving the values of the activatable PSA / (PSA Hbre cHve + PSA Hbre denatured) ratios for the sera of patients with prostate cancer and patients with benign hyperplasia (BPH), obtained with the antibody 5D5A5 or the antibody 11E5C6 as anticoips for detection of activatable PSA.

Example 1; Detection of the activatable free form of PSA from LNCaP supernatants 1.1 Preparation of LNCaP supernatants PSA was synthesized by the LNCaP eye strain according to the technique described in the articles "LNCaP Produces both putative zymogen and inactive, free form of prostate specifies antigen "E. Corey et al., 1998, Prostate 35: 135-143," Androgen-Sensitive human prostate cancer ceH, LNCaP, Produce both N-terminaUy mature and tuncated prostate specifies antigen isoform "To Herrala et al., 1997, Eur. J. Biochem. 255: 329-335, "Production of iHgram concentration of free prostate specifies antigen (fPSA) from LNCaP ceH culture: Difference between fPSA from LNCaP ceU and seminal plasma." JT Wu et al., 1998, J. CHn. Lab. Anal. 12: 6-13. The PSA thus produced by the eye LNCaP is composed of PSA Hbre. In addition, as the culture of Hgnea is reahsed in the presence of fetal calf serum, which contains ACT, a proportion of PSA-ACT complex is thus also present. 1.2 Binding of the Hbre Forms of the PSA of the Supernatant by Haison Antibodies We used either the antibody 5D3D11, which is a Haison partner recognizing the epitope mimed by the sequence SEQ ID No. 1, or the antibody 6D8C8 which is a Haison partner not recognizing this epitope (Michel S., et al., 1999, supra). The procedure was carried out as follows: The antibodies 5D3D11 or 6C8D8 were fixed on a support of Sepharose resin activated with CNBr (Pharmacia) according to the conventional protocol provided by the manufacturer, well known to those skilled in the art. The coupling ratio used was 5 mg of antibody per 1 ml of swollen resin. 1 ml of resin having fixed 5 mg of antibody was brought into contact with 200 ml of a pool of ceNular supernatants of LNCaP containing approximately 500 μg of total PSA assayed using the Vidas detection device (PSA kit total, bioMérieux, France). After 2 passages on the affinity column, the fraction containing the unbound PSA was kept (designated FI), and the PSA fixed by the antibodies was eluted with a 0.1 M glycine buffer pH 2.8 then neutralized at pH 7.2 with 2 M Tris pH 8. There were thus obtained the Hebrew forms of PSA immunopurified either with the antibody 5D3D 11 or F antibody 6C8D8. 1.3 Determination of the Hbre forms of PSA of the supernatant after immunopurification with the antibodies 5D3D11 and 6C8D8 1.3.1. Analysis by two-dimensional electrophoresis The PSA contained in the various fractions resulting from immunopurification was analyzed by two-dimensional electrophoresis and Western-Blot During the separation according to the first dimension, the proteins migrate according to their isoelectric point (pi) by isoelectrofocaHsation ( EEF) on a pH gradient immobilized on deposit strips (HnmobUine Dry-strip pH 3-10, 18 cm, non-linear, Pharmacia) under denaturing and reducing conditions. 0.5 μg of PSA were added to 10 μl of a solution containing 10% SDS and 2.3% DIT (dithio-1,4-threitol), and the mixture was heated for 5 min at 96 ° C. . Then the sample was made up to 500 μl with the rehydration solution (8.3 M urea, 2 M thiourea, CHAPS 4% (3 - [(3-cholarrήdopropyl) αimethylammonio] -l-piOpane sulfonate), DTT 100 mM, Servalyt 4-9 2%, Orange G 1 mg / ml), and brought into contact with the strips in a glass test tube 20 cm long. The whole was then covered with oil paraffin, and incubated overnight. The separation was carried out under a voltage increasing linearly from 100 to 3500 N in 8 h, and a step of focusing at 6000 N was carried out for 80 to 100 kVh. The proteins having focahsed at their isoelectric point were then separated in a second dimension according to their size thanks to SDS -PAGE electrophoresis, on a large homogeneous 12% acrylamide gel at 40 mA per gel, for 5-6 h, according to the conventional protein electrophoresis technique. The gels obtained after separation according to the second dimension were transferred onto a PVDF membrane (polyvinyhdene trifluoride; MiUipore) in CAPS / methanol buffer (add 3- [cyclohexilamino] -l-propanesulfonic), for 1 night under a current of 1 A, maintaining the temperature at 15 ° C. The membranes were saturated overnight at 4 ° C. in TBS (Tris Buffer Saline, Tris 15 mM pH 8, ΝaCl 140 mM) containing 0.05% of Tween 20 and 5% of dehydrated skimmed milk. The anti-PSA 13C9E9 antibody, previously identified to detect all forms of PSA (Charrier JP, et al., 2001, Electrophoresis, 22, 1861- 1866), was diluted in the saturation solution to 10 μg / l, and was added to the membranes. After an incubation of 1 h at 37 ° C, the membranes were washed three times (5 min) with a saturation solution, and brought into contact with the anti-mouse antibody conjugate coupled to peroxidase (Jackson ImmunoResearch, West Grove , United States of America), diluted to l / 5000 ^th in the saturation solution. They were then incubated for 1 h at room temperature, washed three times in saturation solution. The immunoreactivity was detected by using a chemHuminescent substrate (Pierce), by incubating the membranes in Fluor S, and by making an acquisition of the image (between 1 min. And 1 h depending on the sample). The images obtained were then processed using the Multi-Analyst software (Biorad). The photographs of these gels are reproduced in FIG. 1 where the photograph A corresponds to the use of the antibody 5D3D11, the photograph B corresponds to the use of the antibody 6C8D8 and the photograph C corresponds to the antibody 11E5C6 (Michel S. et al., 1999, supra) which is a total anti-PSA antibody used for comparison. This figure shows that, in comparison with PSA immunopurified with the anti-total PSA antibody 11E5C6, PSA immunopurified with the antibody 5D3D11 has very little chvee or truncated forms. On the other hand, these forms are present in a much greater proportion in PSA in-u unopurified with the antibody 6C8D8. 1.3.3 Characterization of the zymogenic and mature forms of immunopurified PSA by N-terminal sequencing of PSA 5 to 8 μg of cellular PSA immunopurified with the antibodies 5D3D11 or 6C8D8 were subjected to separation on SDS -PAGE gel under reduced conditions and transferred to polyvinyHdene difluoride (PVDF) membrane according to techniques well known to those skilled in the art. The PSA bands were stained with a 0.25% solution of Rouge Ponceau S in 3% TCA (trichloroacetic acid), and the band corresponding to mature PSA (approximately 30 kDa) was cut out and subjected to degradation of 'Edman on a Procise 292A protein sequencer (AppHed Biosystems). The results obtained showed that the PSA immunopurified by the antibody 5D3D11 contained only the mature form (GG ...), while the PSA immunopurified by the antibody 6C8D8 contained the mature form, as well as the proPSA (-7) and proPSA (-5). 1.3.4 Measurement of the enzymatic activity of the immunopurified cellular PSA The wells of a black Nunc Maxisorp ELISA plate (compatible with fluorescence detection) were coated for 2 hours at 37 ° C. with 100 μl of a streptavidin solution at 10 μg / ml in carbonate buffer pH 9.6 and blocked for 2 hours at 37 ° C with TBS (50 mM Tris-HCl pH 7.5, 0.15 M NaCl) containing 2 mg / ml of BSA (Bovine Serum Albumin) After 3 washing of the wells in 0.05% TBS-Tween, 100 μl of biotinylated total anti-PSA antibody diluted to 10 μg / ml in TBS-BSA were introduced. The total anti-PSA antibody can be either the 11E5C6 antibody which does not modify the enzymatic activity of PSA (Michel S. et al., 1999, supra), or the 8G8F5 antibody (bioMérieux, France). After 2 hours of incubation at 37 ° C, the wells were washed with 0.05% TBS-Tween, and the PSA immunopurified by antibodies 5D3D11 or 6C8D8, diluted in TBS-BSA to 2.5 μg ml was introduced and incubated overnight at 4 ° C. The wells were then washed again in 0.05% TBS-Tween and incubated for 15 min. either with the TBS-BSA described above (containing 0.15 M NaCl), or with TBS-BSA whose NaCl concentration was raised to 1.5 M. The wells were then emptied and brought into contact with 100 μl of the fluorescent substrate Mu- HSSKLQ-AFC (Enzyme System Products, ICN fihale) diluted to 300 μM in TBS-BSA containing either 0.15 M or 1.5 M NaCl. The enzymatic kinetics were followed at 37 ° C for 2 hours (exdtation 390 nm, emission 510 n; 1 measurement / minute) with the Fluoroskan reader (Thermolabs Systems). The enzymatic activity of PSA corresponds to the slope of the curve obtained. The results are shown in Table 1 below: Table 1

The enzymatic activity is expressed in fluorescence units x 1000 / min. This table shows that the PSA immunopurified by the antibody 5D3D11 has a detectable enzymatic activity. This activity is increased in the presence of 1.5 M NaCl. The capture of PSA by the antibody 8G8F5 also increases the enzymatic activity of PSA. PSA in-tmunopurified by the antibody 6C8D8, meanwhile, has only a residual activity detectable in the presence of 1.5 M NaCl and the antibody 8G8F5, showing that this form of PSA immunopurified is not activated. This example demonstrates that, among the active Hbre forms of PSA in the LNCaP cells, there is the activatable Hbre form which is detectable according to the method of the invention.

Example 2; Detection of the activatable free form of PSA from serum from patients with prostate adenocarcinoma The serum pool used is composed of 30 sera from patients with prostate cancer. PSA concentration of this pool: total PSA: 71 ng / ml; PSA Hbre: 12 ng / ml, determined using FappareU Vidas. 2.1. Immunopurification of serum The resin described in paragraph 1.2 is used as a solid support having fixed the antibodies 5D3D11 and 6C8D8. 50 μl of these resins are brought into contact with 1 ml of the serum pool described above overnight at 4 ° C. with stirring. The tubes are then centrifuged, the fraction containing the unbound PSA was kept, the resin was washed 3 times with 0.5% PBS-Tween (the washing fractions are combined and stored), then the PSA fixed by the antibody 5D3D11 or 6C8D8 was eluted for 5 min with 200 μl of 0.1 M Glycine PH 2.2 containing 1 mg / ml of BSA and neutralized at pH 7.2 with 2 M Tris pH 8. 2.1. Measurement of the enzyme activity of immunopurified PSA from serum The wells of a black Nunc Maxisorp ELISA plate (compatible with fluorescence detection) were “coated” for 2 hours at 37 ° C. with 100 μl of a solution of streptavidin at 10 μg / ml in carbonate buffer pH 9.6 and blocked for 2 hours at 37 ° C with TBS (Tris-HCl 50 ΠM pH 7.5, 0.15 M NaCl) containing 2 mg / ml of BSA (Bovine Serum Albumin) . After 3 washes of the wells in 0.05% TBS-Tween, 100 μl of biotinylated anti-PSA total anticoips diluted to 10 μg / ml in TBS-BSA were introduced. The total anti-PSA antibody can be: o either the 11E5C6 antibody, which does not modify the enzymatic activity of PSA, o or the 8G8F5 antibody which increases the enzymatic activity of the captured PSA. After 2 hours of incubation at 37 ° C, the wells were washed with 0.05% TBS-Tween, and 100 μl of PSA immunopurified from serum with the antibodies 5D3D11 or 6C8D8 were introduced and incubated overnight at 4 ° C. The wells were then washed again in 0.05% TBS-Tween and incubated for 15 min with TBS-BSA 2 mg / ml, the NaCl concentration of which was raised to 1.5 M. The wells were then emptied and brought into contact with 100 μl of the fluorescent substrate Mu-KGISSQY-AFC (Enzyme System Products, an ICN subsidiary) diluted to 400 μM in TBS-BSA containing 1.5 M NaCl. The enzymatic kinetics were followed at 37 ° C. for 2 hours (excitation 390 nm, emission 510 nm; 1 measurement / minute) with the Fluoroskan reader (Thermolabs Systems). The enzymatic activity of PSA corresponds to the slope of the curve obtained. The results are shown in Table 2 below: Table 2

The enzymatic activity is expressed in fluorescence units x 1000 / minute. * The activity measured directly from the serum (without immunopurification) is underestimated, because the activated PSA immediately complexes with serum FACT. This table shows that we are able to measure an enzymatic activity on PSA immunopurified from serum: the serum therefore contains activatable PSA Hbre. There is always a significant difference in activity between the PSA immunopurified by the antibody 5D3D11 and that immunopurified by the antibody 6C8D8, showing that the antibody 5D3D11 specifically recognizes the activatable PSA Hbre.

Example 3; Use of the antibody 5D3D11 in a diagnostic test for the discrimination between a BPH and adenocarcinoma of the prostate For the assay, the Vidas apparatus was used, adapted to include the appropriate assay reagents, but used according to the instructions of the supplier (bioMérieux, France). 3.1 Determination of activatable PSA in patient sera The antibody 5D3D11 is used as capture antibody in a sandwich type test. The PSA captured is detected by the total anti-PSA antibody 5D5A5 coupled with alkaline phosphatase. The protocol is as described above. The standardization of the test was carried out according to the supplier's recommendations using seminal PSA immunopurified with the antibody 5D3D11 and assayed in total PSA. The detection limit of the test is 0.05 ng / ml of active PSA / ml. The standardization curve in the concentration range from 0 to 1.5 ng / ml is shown in Figure 2. This curve, which is close to a straight line, shows that the assay with a Haison partner recognizing the epitope mimicked by the sequence SEQ ID No. 1 makes it possible to detect PSA Hbre activatable with good sensitivity. 3.2 Determination of (PSA Hbre cHve + PSA Hbre denatured) in patient sera The procedure described above was repeated, except that the anti-PSA Hbre 6C8D8 antibody was used as capture antibody for capture in the serum of the inactive PSA Hbre forms, as well as the total anti-PSA antibody 11E5C6 coupled with alkaline phosphatase as detection antibody, which in the present case recognizes the forms of PSA Hbre cHve and Hbre denatured. 3.3 Use of the activatable PSA / cPSve Hbre + denatured PSA hbre ratio) for better discrimination between prostate cancer and BPH A retrospective study was carried out with 177 sera from patients containing

PSA. These sera correspond to:

- 89 prostate cancers well identified with total PSA levels, tumor grades, Gleason scores and any information on treatments (radiotherapy, hormone therapy, prostatectomy or unknown) carried out subsequently - 65 benign hyperplasias,

- 23 normal prostates (with PSA levels> 2.5ng / ml but negative biopsies). On these sera, the activable PSA Hbre / / ratio (PS A denatured Hbre + PSA Hbre cHve) obtained using the previous assays was calculated and these values were reproduced on the graph of FIG. 3, part A As well as the shows part A of FIG. 3, using a threshold of 0.66 for sera having a total PSA level of between 2.5 and 10 ng / ml, the PSA activatable / / (PSA Hebre cHve + PSA Hebre denatured) ratio ) characterizes 22/33 cancers (67%), 19/19 normal prostates (100%) and 27/28 BPH (96%). For comparison, the PSA Hbre to total PSA ratio was calculated using two Vidas kits (Kit PSA total and Kit PSA Hbre, bioMérieux, France) according to the supplier's recommendations. The results are reproduced on part B of the graph in FIG. 3. This part B shows that U exists an uncertainty zone for values from 0.15 to 0.25. 3.4 Sensitivity of the process of the invention 3.4.1 For the sera included in the uncertainty zones of the processes of the prior art The values of the activable PSA Hbre / / (denatured PSA Hbre + PSA Hbre cHve) ratios obtained according to the method of the invention for the 28 sera the area of uncertainty obtained with the method of the prior art has been reproduced in FIG. 4. This figure shows that, for sera whose PSA Hbre / total PSA ratio is between 0.15 and 0.25, sera in which the known methods do not make it possible to discriminate a cancer from a BPH, the activatable PSA / ratio (PSA Hbre cHve + PSA Hbre denatured) makes it possible to characterize 13/15 cancers (80%), 7/7 BPH (100 %) and 6/6 normal prostates (100%). 3.4.2 For sera before a PSA level lower than 2.5ng / ml The 20 sera from cancer patients (11) or BPH (9) having a PSA level lower than 2.5 ng / were selected ml and the values of the PSA activable Hbre / PSA ratios (denatured PSA Hbre + PSA Hbre cHve) are reproduced in FIG. 5. This figure shows that the discrimination between prostate cancer and BPH using the activatable PSA ratio / (Denatured PSA Hbre + PSA Hbre cHve) also remains valid for sera having a PSA level of less than 2.5 ng / ml. 3.5 Modification of the detection antibody The above procedure was repeated with 8 sera, 5 sera from patients with prostate cancer and 3 sera from patients with BPH, except that the antibody was used. 11E5C6 (Michel S., et al., 1999, supra) as detection antibodies. The results are indicated in FIG. 6 on which the values of the activable PSA / shabre PSA (denatured PSA shabre + PSA ibre cHve) using the antibody 5D5A5 as detection antibody for activatable PSA are reproduced in part A of the curve, and the values of the PSA Hbre activatable / (PSA Hbre denatured + PSA Hbre cHve) ratios obtained with the same sera but with the antibody 11E5C6 as detection antibody are reproduced in part B. This figure shows that U does exist a threshold value regardless of the detection antibody used and that the threshold value depends on the antibody used in detection. Example 4; Detection of the activatable free form of PSA from a binding partner capable of binding in a non-specific manner to the activatable free PSA in a biological sample 1. Samples used Nine individual sera were used having total PSA concentrations varying from 157 at 3.8 ng / ml and coming from the CHU of Liège, that is to say from the hospital of the Armies, Desgenettes, located in Lyon. 2. PSA immunopurification using the antibody 11E5C6 2.1. Coupling of the antibody with beads 10 ⁸ beads previously sensitized to streptavidin (Dynabeads® M-280 Streptavidin, Dynal), ie 150 μl of biUes solution, were introduced into tubes and washed 3 times with 500 μl of 0.1 M phosphate buffer pH 7.4, 0.15 M NaCl, 0.1% BSA supplemented with 0.5% Tween 20 (D + T buffer 0.5%). Each wash was carried out at room temperature for 5 min. on the wheel so that the solution remains homogeneous. The 10 ⁸ biUes were coupled with 500 μl of 11E5C6 antibody (bioMérieux,

Marcy, France) at 20 μgml, i.e. 10 μg of antibody, in 0.05% D -l- T buffer at room temperature, for 30 min on the wheel. After coupling, 5 μl of 10 mM biotin were added to the 500 μl of antibody solution and incubated for 30 min. at room temperature on the wheel. To remove excess biotin and antibodies, the biUes were then washed 5 times with 500 μl of 0.5% D + T buffer on the wheel for 5 min at room temperature. 2.2. Fixation of PSA 1 ml of sample containing PSA (either seminal PSA used as a control or serum) was introduced into the tubes containing the 10 ⁸ biUes and was incubated overnight at 4 ° C. After this incubation period, the unbound PSA was collected and assayed to determine the amount of PSA residue. Three washes were then carried out, in order to unhook the fixed proteins from nonspecifically, with 500 μl of buffer D having a Tween 20 concentration of 0.5%. 2.3. Elution The PSA was eluted for 5 min. at room temperature with 100 μl of 0.2 M Tris-glycine pH 2.2 supplemented with BSA at 1 mg / ml. The acidic pH leads to the breakdown of Haison PSA-Ac. The recovered eluate was neutralized with 11 μl of 0.1 M Tris pH 9.6 in order to avoid degradation of the protein by the acidity of the solution and to preserve its enzymatic activity. 3. Assay of samples in PSA Hbre and total PSA This assay was carried out using the VIDAS device (bioMérieux, Marcy, France), using assay kits measuring total PSA (TPSA) and PSA Hbre (FPSA) , according to the recommendations of the supplier and according to the following protocol: Specific antibodies directed respectively against total PSA or PSA Hbre are fixed on the cones. For the two tests, the detection antibody is the 11E5C6 antibody coupled to alkaline phophatase, which catalyzes the reaction of hydrolysis of the substrate into a product whose emitted fluorescence is measured at 450 nm. Each test requires 200 μl of sample which are placed in the bar and the results are available in lh. The TPSA test has a sensitivity of 0.07 ng / ml PSA and can detect up to 100 ng / ml PSA. The FPSA test has a sensitivity of 0.05 ng / ml and can detect up to 10 ng / ml of PSA. 4. Measurement of the enzymatic activity of PSA 4.1. Fixation of capture antibodies The wells of an ELISA plate were coated with 100 μl of streptavin (Hluée at 10 μg / ml in carbonate buffer pH 9.6, and incubated for 2 h at 37 ° C. The wells were then saturated with 250 μl of 50 mM Tris buffer + 0.15 M NaCl pH 7.5 (TBS) supplemented with BSA at 2 mg / ml (TBS + BSA), in order to avoid non-specific attachments and therefore to reduce noise After 3 washes in TBS containing 0.05% Tween 20 (TBS + T 0.05%), 100 μl of 8G8F5 antibody (bioMérieux, Marcy, France) biotinylated at 10 μg / ml were then added and incubated for 2 h at 37 ° C. This step was followed by 3 washes in TBS + 0.05% 4.2 4.2 Incubation of PSA 85 μl of PSA diluted in TBS + BSA (seminal PSA or immunopurified serum) were introduced into the wells and incubated overnight at 4 ° C. The wells were then washed 3 times in TBS + 0.05% T, which makes it possible to remove the unbound PSA. The enzyme was then incubated for 15 min at 37 ° C, with 100 μl of the TBS + BSA substrate supplemented with 1.5 M NaCl. 4.3. Development and determination The GISSQY-AFC substrate was diluted in TBS + BSA supplemented with 1.5 M NaCl. 100 μl of this diluted substrate were then added to the wells and incubated at 37 ° C. The fluorescence emitted was measured every minute for 2 h at 37 ° C., by the Fluoroskan apparatus (ThermoLabsystem). The enzyme activity of the enzyme was determined by calculating the slope of the kinetics and expressed in fluorescent units * 1000 / min. 5. Results. The results are shown in Table 3 below.

Table 3

Among these sera, sera 1 and 9 come from patients confirmed as having prostate cancer, which is also confirmed by the method of the invention.

Claims

1. Method for in vitro diagnosis of a benign pathology of the prostate or of an adenocarcinoma of the prostate, characterized in that U comprises the step of detection, in a biological sample obtained from a patient suspected of being suffering from a benign pathology of the prostate or from an adenocarcinoma of the prostate, of the activatable Hebre form of PSA.

2. A method of diagnosing a benign pathology of the prostate or of an adenocarcinoma of the prostate according to claim characterized in that U comprises the steps consisting in: i) bringing into contact a partner of Haison able to specifically Her at PSA

Hebre activable with a biological sample from a patient suspected of having a benign prostate pathology or an adenocarcinoma of the prostate, ii) highlight the capture of the Hebre activable form of PSA by said partner binding agent, Si) calculate the ratio between the quantity of activable Hbre form of PSA detected in step H) and the quantity of a form of PSA other than the activatable Hbre form, present in a similar sample taken from the same subject, and iv) determine if the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step Hi) with a predetermined threshold value, chosen according to the type of report used and representative of the limit of detection of each pathology.

3. A method of diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate according to claim 2, characterized in that said partner of Haison used in step i) is capable of recognizing the epitope mimicked by the sequence SEQ ID N ° 1.

4. Method for diagnosing a benign prostate pathology or a prostate adenocarcinoma according to one of claims 2 or 3, characterized in that said Haison partner used in step i) is an antibody or an antibody fragment.

5. A method of diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate according to any one of claims 2 to 4, characterized in that the demonstration of the capture of the activatable Hebre form of PSA by said partner of Haison is implemented by indirect detection via a detection partner, preferably by the use of a total anti-PSA antibody.

6. A method of diagnosing a benign pathology of the prostate or of an adenocarcinoma of the prostate according to any one of claims 2 to 4, characterized in that the demonstration of the capture of the activatable Hebre form of PSA by said partner of Haison is implemented by determining the enzymatic activity of the activatable Hebre form of the immunopurified and activated PSA.

7. A method of diagnosing a benign pathology of the prostate or of an adenocarcinoma of the prostate according to any one of claims 2 to 6, characterized in that U uses, in addition to the partner of Haison capable of being Her specifically to PSA Hbre activatable, an antibody capable of increasing the enzymatic activity of PSA

8. A method of diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate according to any one of claims 2 to 7, characterized in that the form of PSA other than the activable Hbre form used to calculate the ratio is the inactive Hebrew form of PSA or the chbre and denatured Hebrew forms of PSA.

9. Diagnostic kit for diagnosing an adenocarcinoma of the prostate or a benign pathology of the prostate, characterized in that U comprises:

- a partner of Haison able to specifically Her to PSA Hbre activable and

- Means for dosing forms of PSA other than the activatable Hebre form.

10. Diagnostic kit according to claim 9, characterized in that said partner of Haison is capable of recognizing the epitope imitated by the sequence SEQ ID No. 1, preferably an antibody or an antibody fragment.

11. Diagnostic kit according to one of claims 9 or 10, characterized in that said means are antibodies or antibody fragments.

12. Conjugate consisting of a Haison partner capable of specifically Herb activating the PSA Hbre and the activatable Hbre form of PSA.

_13.. Conjugate according to Claim 12, characterized in that the said Haison partner is capable of recognizing the epitope iriimated by the sequence SEQ ID No.1.

14. Conjugate according to one of claims 12 or 13, characterized in that the activatable Hebre form of PSA is activated.

15. A method of diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate according to claim 1, characterized in that U comprises the steps consisting in: i) bringing into contact a partner of Haison capable of Her to PSA Hbre activable in a non-specific way, with a biological sample from a patient suspected of having a benign prostate pathology or an adenocarcinoma of the prostate, Ë) highlight the capture of the activable Hebrew form of PSA by said partner of Haison by determination of the enzymatic activity of the activated Hebrew form of PSA, after activation of the activatable Hebrew form of PSA, m) calculate the ratio between the quantity of activated Hebrew form of PSA in step H) and the quantity of a form of PSA other than the activatable Hbre form, present in a sample of the same nature taken from the same subject, and iv) determine whether the patients are affected by an adenocarcinoma of the prostate or by a benign pathology of the prostate by comparing the value of the ratio determined in step ni) with a predetermined threshold value, chosen according to the type of ratio used and representative of the limit of detection of each pathology.

16. A method of diagnosing a benign pathology of the prostate or an adenocarcinoma of the prostate according to claim 15, characterized in that the activation of the activatable Hebre form of PSA is carried out by use of an antibody capable to increase the enzymatic activity of PSA.