Method for diagnosing a transmissible spongiform subacute encephalopathy caused by an unconventional transmissible agent strain in a biological sample

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 1 PROCESS FOR DIAGNOSING A TSE CAUSED BY A STRAIN OF ATNC IN A BIOLOGICAL SAMPLE The present invention relates to a method for diagnosing a TSE caused by a strain of ATNC in a biological sample by detection of PrPres as well as its use in the context of a differential diagnosis of the different strains of ATNC in a biological sample.

Subacute transmissible spongiform encephalopathies (TSEs) are caused by unconventional transmissible agents (UTAs), also called prions, the precise nature of which remains disputed to this day.

TSEs mainly include Creutzfeldt-Jakob disease in humans (CJD or CJD for Creutzfeldt-Jakob disease), scrapie in sheep and goats and bovine spongiform encephalopathy (BSE or BSE for bovine spongiform encephalopathy) in cattle; other encephalopathies have been demonstrated in felids, mink or certain wild animals, such as deer or elk.

These diseases are constantly fatal and there is currently no effective treatment.

In TSE, there is an accumulation of a host protein, PrP (or prion protein), in an abnormal form (PrPres), mainly in the central nervous system; PrPres copurifies with infectivity and its accumulation precedes the appearance of histological lesions.

In vitro, it is toxic to cultures of neurons.

The two isoforms of PrP have the same amino acid sequence (see figure 1), but differ in their secondary structure: PrPres has a significantly higher content in folded R-sheets, whereas normal PrP (PrPsens) has a higher large number of propellers a.

Two biochemical properties generally make it possible to distinguish these two isoforms.

- PrPres is partially resistant to proteases, in particular to proteinase K (PK), which causes cleavage of its N-terminal end.

After the action of PK, PrPres is often called PrP27-30 because of the molecular weight appa CA 02390891 2011-01-24 VO 01/35104 PCT / FR00 / 03159 rent of the biglycosylated form; it is generally accepted that the PrPres cleavage site lies between amino acids 89 and 90 (Prusiner et al, Cell, 1984) for usual strains.

- PrPres is insoluble in non-ionic detergents, such as TritonTM X100 or TritonTM 114.

The normal form of the prion protein (PrPsens) is in principle completely degraded by proteases and is perfectly soluble in the presence of nonionic detergents.

The peptide sequences of hamster, human, bovine and ovine PrPsens are shown in FIG. 1; they include in particular an octapeptide unit (P (H / Q) GGG (- / T) WGQ), repeated 4 or 5 times, depending on the species.

This repeated octapeptide motif corresponds to amino acids 51-91 of PrP (human PrP sequence numbering) (B.

Oesch et al., 199 1).

To detect the presence of the infectious agent, the most recent methods are based on the selective detection of abnormal PrP (PrPres), bound to the infectious agent, taking advantage of its partial resistance to proteases.

We can distinguish :

- Western blot methods which are based on the immunological detection of PrPres in a tissue extract, after treatment of the extract with a protease so as to destroy the normal isoform of PrP (PrPsens), separation of proteins from the extract by electrophoresis, transfer onto a polymer membrane, and detection by a specific antibody recognizing PrP (Schaller 0. et al., 1999), - ELISA type tests, which also involve treatment of tissue extracts with a protease.

Among these various tests, which involve treatment of the tissue extracts with a protease, there may be mentioned:

- that described by Serban et al. (Neurology, 1990, 40, 110), who developed a test for the detection of PrPres which includes immobilization of proteins on a nitrocellulose membrane, followed by protease digestion, denaturation and immunodetection with monoclonal antibodies.

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 3 - that described by Oesch et al. (Biochemistry, 1994, 33, 5926-5931), who have proposed, to quantify the amount of PrPres, an immunofiltration test for the purification of PrPres (ELIFA or enzyme-linked immunofiltration assay).

- that described by Gratwohl et al., 1997, which propose an ELISA type assay.

After treatment of the samples with proteinase K and purification of the PrPres by centrifugation, the latter is adsorbed on microtiter plates and detected using polyclonal rabbit antibodies.

- that described by Safar et al., 1998, which does not use proteinase K and compares the immunoreactivity of PrPres immobilized on a solid support depending on whether or not it has been subjected to a denaturing treatment.

In general, these various tests have the drawback of lacking sensitivity and thus of causing false-negatives.

Other methods propose treatments of the sample with denaturing products (Oesch et al., 1994 and 1999; WO 00/22438, The Regents of the University of California), a treatment limited to proteinase K (WO 00 / 29850, Wallac Oy et al.) Or treatment with a metallopeptidase (WO 00/22438) which makes hidden antigenic sites accessible, detectable by the 3F4 monoclonal antibody which recognizes the 109-112 region of PrP (WO 00/29850 or WO 00/22438).

The method, described in WO 00/29850, Wallac Oy et al., Has the major drawback of lacking specificity, in particular due to incomplete elimination of PrPsens, under the recommended treatment conditions, whereas the method described in WO 00/22438, The Redents of the University of California lacks sensitivity, due to the use of the revealing antibody 3F4 (WO 00/22438), which only binds to a single motif on the protein .

The Applicant recently proposed a test for the quantitative detection of PrPres, which comprises a purification step which leads to a significantly more sensitive detection and which represents a great advance for the health surveillance and the determination of PrPres in slaughterhouses. .

This process is described in particular in PCT International Application WO 99/41280 and in a preliminary report by Directorate General XXIV of the European Commission CA 02390891 2002-05-09 WO 01/35104 PCT / FROO / 03159 4 (Consumer policy and protection of their health http://europa.eu.int/comm/da24/health/).

However, given the need to have a particularly reliable diagnostic test for TSEs, the Applicant has continued its work.

In particular, he established that in order to obtain a detection test:

(i) sensitive, i.e. having the ability to correctly identify uninfected animals, (ii) specific, i.e. having the ability to correctly identify infected animals showing clinical symptoms, (iii) exhibiting the lowest possible detection limit, i.e. capable of allowing the detection of small amounts of PrPres, (detection of PrPres before the onset of clinical symptoms) and (iv) reproducible, the treatment of the biological sample to be analyzed must be carried out under conditions which make it possible to preserve all or part of the octa-peptide units exclusively in the PrPres.

In particular, he found that in order to effectively meet the four conditions (i) - (iv) stated above, it is necessary to define precise processing conditions for the sample to be analyzed which completely eliminate the PrPsense from the sample, while allowing specific capture of PrPres, under the defined conditions.

The present invention relates to a method for diagnosing (Method A) of a TSE or prion disease caused by a strain of ATNC by detection of PrPres in a biological sample, characterized in that it comprises:

(1) treating said sample with at least one proteinase K (PK), so as to completely degrade PrPsens while retaining all or part of the repeated octa-peptide units of PrPres, whatever the strain of ATNC;

preferably, said PK treatment is carried out either at a concentration of between 30 g / ml and 200 g / ml for 10 minutes at 37 C for a 10% homogenate (biological sample in the form of a homogenate in a buffer suitable), or for a period and at a concentration equivalent to the concentration CA 02390891 2002-05-09 WO 01/35104 PCT / FROO / 03159 of between 30 .tg / ml and 200 g / ml for 10 minutes at 37 C for a 10% homogenate, (2) bringing said treated sample into contact with a ligand of said octapeptide units, in particular antibodies directed against said repeated octapeptide units and (3) detecting the possible presence of the repeated octapeptide unit-ligand complex.

According to an advantageous embodiment of said method, the treatment with said proteinase K is between 30 seconds and 2 hours, at a temperature below 80 ° C., preferably between 10 minutes and 30 minutes at 37 ° C., at the aforementioned concentrations, and even more preferably for 10 minutes at a concentration of between 30 tg / ml and 200 .ig / ml for a 10% homogenate (final concentration) or for 30 minutes at a concentration of between 10 .tg / ml and 70 g / ml for a 10% homogenate (final concentration) or at a concentration between 25 g / ml to 175 .tg / ml for a 25% homogenate (final concentration).

It should be noted that a certain number of parameters are closely related to each other: the proteinase K concentration depends directly on the duration of treatment (incubation time) of the sample: it can be considered that 37 C for example, a 10 minute incubation with a PK at a concentration between 30 and 200 µg / ml of 10% homogenate is equivalent to a 30 minute incubation with a PK at a concentration between 10 µg / ml. / ml and 70 g / ml of 10% homogenate.

For example, a 30 minute incubation with PK at pg / ml is equivalent to a 10 minute incubation with PK at a concentration of 75 µg / ml.

Whatever the PK concentration and the incubation time, the treated sample must no longer contain undegraded PrPsens, while the PrPres, possibly present, has retained all or part of the octa-peptide units.

Other parameters can intervene to a lesser degree (that is to say in a non-essential way), in the establishment of the active concentration of PK: it is in particular the buffer in which said PK is placed in solution;

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 6 when the PK is dissolved in the sample homogenization buffer, depending on the homogenization buffer used, the minimum PK concentration may vary , within the range of concentrations specified above - in glucose buffer or in guanidine (or one of its salts) the minimum PK concentration is preferably 25 1g / ml (incubation 30 minutes) or 75 g / ml (10 minutes incubation) - in PBS buffer, the minimum PK concentration is preferably 50 g / ml (30 minutes incubation) or 150 g / ml (10 minutes incubation).

Advantageously, the PK can be carried out in a buffer different from the homogenization buffer; such a buffer advantageously comprises at least one surfactant and / or at least one chaotropic agent and / or at least one salt.

Also advantageously, after the treatment (or incubation) of the sample with proteinase K, the suspension obtained is advantageously treated under the conditions defined in international application 99/41280, namely:

- addition to said suspension of a buffer B, as defined in this international application 99/41280 and in particular C3-C6 alcohols and alcohol mixtures whose mean theoretical dielectric constant is between 10 and 25, - the centrifugation of the suspension obtained and - solubilization of the pellet in a buffer comprising at least one surfactant and / or at least one chaotropic agent, under the conditions defined in said international application 99/41280.

Such a test has the advantage of being particularly suitable for the differential diagnosis of TSE in the same host, caused by different strains of ATNC, and in particular for the differential diagnosis of BSE strains compared to the usual strains of scrapie in sheep. .

It has, for example, been shown that the BSE strain has infected humans in Great Britain by analysis of the electrophoretic profiles of PrPres, which showed differences in migration depending on the strains of ATNC; a characteristic CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 7 profile (type 4) was found in patients developing a new variant of Creutzfeldt-Jakob disease (vCJD) which appears to be linked to the passage in human food for cattle infected with the BSE agent (Collinge et al., Nature, 1996).

A similar profile was observed in a macaque infected with the BSE agent (Lasmezas et al., 1996) and a cat presumably contaminated with this agent (Priola et al., Nature Med., 1996).

However, this is a long and delicate method to implement if reproducible results are to be obtained, which is probably part of the controversy that exists concerning the classification of the different types of PrPres and the use. of this method (Parchi et al., Nature, 1997).

There is also a strong suspicion of contamination of sheep with the BSE agent (Butler, Nature, 1998).

These animals are in fact sensitive to this agent, whereas there is an endemic disease in sheep, scrapie (Anglo-Saxon scrapie), another TSE, which has similar clinical and histological characteristics that cannot be differentiated.

The only reference method for a differential diagnosis between the agent of BSE and scrapie, is inoculation in mice and study of the lesion profile, which requires approximately two years of waiting and which does not was carried out in Great Britain only on 9 sheep for a population of several tens of millions of heads.

The first tests were carried out to try to distinguish the different strains of NCTA in a sample; Kuczius T. et al., 1999 considered that the variations observed between the different strains of ESST (scrapie, ESB and CJD) by the technique of glycotyping do not make it possible to distinguish each strain; they selected other biochemical and biological markers of PrPres which make it possible to better distinguish between them the different strains of prions.

The analysis parameters they used are: long term resistance to proteinase K, molecular weight of PrPres, topology and amount of PrPres deposits.

The results obtained show that the PrPres of different BSE and scrapie strains show significant differences in their long-term resistance to proteinase K.

Depending on the strain of scrapie, resistance to PK varies:

low resistance: Chandler strain; intermediate resistance: strain 22A; relative stability:

strain 87V.

Under the same conditions, the BSE strains exhibit intermediate resistance CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 8.

While glycotyping does not distinguish between the 87V scrapie strain and BSE strains, these two types of strains are clearly distinguishable after long-term PK treatment.

However, these are not sufficiently reliable protocols that can be used on a large scale and in the field.

In this context, it is important to have a method for detecting PrPres that is reliable, sensitive, allowing differential diagnosis, inexpensive and easy to carry out, from a biological sample, such as a tissue sample.

The present invention therefore also relates to a method for differential diagnosis (method B) of TSEs caused by strains of ATNC, in a biological sample, by detection of the PrPres associated with the different strains of ATNC, characterized in that that it includes:

(a) the detection of PrPres in a first fraction of said sample, in accordance with steps (1) to (3) of the method for diagnosing a strain of ATNC as defined above (method A), then:

(b) for each sample for which the presence of an octa-peptide repeating unit-ligand complex is detected in step (a):

- the treatment of a second fraction of said sample with at least one proteinase K (PK), so that the majority of the repeated octapeptide units is eliminated for the PrPres associated with at least one strain of interest, in particular the strain of BSE and where all of the PrPres associated with the other ATNC strains retain all or part of said repeated octa-peptide units, under said conditions; preferably, said treatment is carried out under the same conditions as those defined in step (1) or (a), but at a higher PK concentration than that used in step (1) or (a), - the bringing said second fraction of said treated sample into contact with a ligand capable of specifically recognizing said repeated octapeptide units and - detecting the possible presence of the repeated octapeptide units-ligand complex.

The present invention also relates to a method for differential diagnosis (variant of method B) of TSEs caused by strains of ATNC CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 9 in a biological sample considered as containing PrPres (detection test carried out by any method), characterized in that it comprises for each sample for which the presence of PrPres has been detected:

- the treatment of another fraction of said biological sample in accordance with step (b) as defined above, - the contacting of said second fraction of said treated sample, with a ligand capable of specifically recognizing said patterns repeated octapeptide, and - the detection of the possible presence of the repeated octapeptide units-ligand complex.

As part of the differential diagnosis (method B), in addition to the PK concentration, other conditions may possibly affect the degradation of the octa-peptide units, depending on the strain: in fact, when the PK is used in a buffer different from the homogenization buffer, such as in a buffer which advantageously comprises at least one surfactant and / or at least one chaotropic agent and / or at least one salt, the number of degraded octapeptide units may vary depending on the composition of said buffer and the concentration of the various agents.

The term “strain of interest” means the strain (s) of ATNC, the strain of BSE in particular, for which it is desired to eliminate the repeated octapeptide units, for example.

One of the main characteristics of methods A and B is to play on the conditions under which the protease treatment is carried out so as to control the degradation of the repeated octapeptide units.

Depending on the intended use, we will ensure that these patterns are preserved (method A) or destroyed (method B):

- in the context of method A, this check will aim to ensure that all or part of the repeated octapeptide units are retained with the aim of promoting very sensitive detection of PrPres.

- in the context of method B, the control of degradation by the protease will aim to ensure that the majority and possibly all of the repeated octapeptide units are degraded for the PrPres corresponding to the strain (s) of interest, whatever either the species in which it is expressed, under conditions or all or CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 part of the repeated octa-peptide units are kept for the PrPres corresponding to all the other strains of ESST.

In this case, the advantage of the invention is to allow a differential diagnosis of the strain of interest compared to the other strains of TNA.

5 Indeed :

Surprisingly, under the conditions according to step (1) or step (a), the PK does not cause the cleavage of all or part of the repeated octapeptide units of the PrPres associated with all the strains of ATNC or prions. , while the conditions of step (b) cause the cleavage of the repeated octa-peptide motifs of the PrPres associated with the strain of interest, while all of the PrPres associated with the other strains of ATNC retain all or part of said repeated octa-peptide units.

Also surprisingly, such a test, in particular when the ligand is an antibody, has the following advantages:

high detection sensitivity because the antibodies directed against these repeated octa-peptide motifs have a much greater affinity than the antibodies directed against other regions of PrPres 27-30 and are more resistant to the buffers used ;. in fact, the recognition of a repeated motif promotes high affinity interactions and offers the possibility of binding several antibody molecules on a single PrP molecule;

the possibility of a differential diagnosis of the strains of ATNC or prions since it is possible, depending on the conditions used, to obtain or not the digestion of these patterns; it is in particular possible to eliminate them for all the diseases linked to the strain of interest, the BSE agent for example, while it is possible to keep it for the other so-called prion diseases.

As a result, the methods according to the invention provide a simple test for the differential diagnosis of BSE and / or of another strain of interest compared to the other strains by using two different conditions (step (1) or (a) and step (b)), the conditions of step (1) or (a) (retaining the repeated octa-peptide motifs of the PrPres associated with the set of NCTA strains) allowing the detection of all the strains and the conditions of step (b ) (eliminating these patterns only in the PrPres associated with a strain of interest) revealing only the other strains.

CA 02390891 2011-01-24 WO 01/35104 PCT / FR00 / 03159 II Such tests therefore find particular application in the search for contamination of sheep by the strain of BSE which is not usually known to distinguish from conventional strains of scrapie.

According to an advantageous embodiment of said methods, the PK is dissolved in a buffer which preferably comprises:

at. at least one surfactant, selected from the group consisting of:

- anionic surfactants, such as SDS (sodium dodecylsulphate), sarkosyl (lauroyl-sarcosine), sodium cholate, sodium deoxycholate, sodium taurocholate;

- zwitterionic surfactants, such as SB 3-10 (decyl-sulfobetaine), SB 3-12 (dodecyl-sulfobetaine), SB 3-14, SB 3-16 (hexadecylsulfobetaine), CHAPS and deoxvCHAPS;

- nonionic surfactants, such as C12ES (dodecyl-octaethylenealycol), TritonTM X100, TritonTM X114, TweenTM 20, TweenTM 80, MEGATM 9 (nonanoyl-methyl (2lucamine), ocrylglucoside, LDAO (dodecyldimethylamine oxide) or NP40 or - mixtures of surfactants such as a mixture of an ionic surfactant and a nonionic surfactant, a mixture of two ionic surfactants or a mixture of an ionic surfactant and a zwitterionic surfactant and / or, b. at least one chaotropic agent selected from the group consisting of urea and guanidine or a mixture thereof and / or c. at least one salt selected from alkali metal salts or not.

According to an advantageous arrangement of this embodiment, said buffer comprises at least 5% of anionic surfactant, preferably sarkosyl, optionally combined with SDS.

According to another advantageous embodiment of said methods, the ligand is selected from the group consisting of aptamers and antibodies capable of binding specifically to the region of the repeated octapeptide units.

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 12 The present invention also relates to a diagnostic kit for implementing the methods according to the invention, characterized in that it comprises in combination with at least one surfactant and / or at least one chaotropic agent and / or at least one salt and one protease as defined above.

The repeated octa-peptide-antibody complex (in the case where the ligand is an antibody) is detected by standard immunological methods.

In addition to the foregoing arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention as well as to the accompanying drawings, in which :

FIG. 1 represents the different sequences of PrP: human, ovine, bovine, murine and cricetidienne;

FIG. 2 represents the detection of PrPres by Western blot;

- Figures 3 to 6 correspond to different conditions according to step (1) or (a) on the one hand and step (b) on the other hand, in humans (Figures 3 and 4) and in ruminants (Figures 5 and 6), in the case where the presence of the repeated octa-peptide units is detected by a two-site immunometric assay.

- Figures 7-12 illustrate the influence of buffers in the differential detection of BSE and scrapie.

FIGS. 13 and 14 illustrate the influence of the composition of the buffers and of the proteinase K (PK) concentration for the detection of the different types of CJD.

FIG. 15 illustrates the results obtained by direct digestion of brain homogenates with proteinase K.

FIG. 16 illustrates the differences in resistance of PrPres to proteinase K as a function of the types of CJD.

FIG. 17 illustrates the detection by Western blot of PrPres digested with PK and purified in the form of SAF.

It should be understood, however, that these examples are given only by way of illustration of the object of the invention, of which they in no way constitute a limitation.

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 13 EXAMPLE 1 Obtaining and characterization of monoclonal antibodies specific for the repeating octapeptide unit.

- Synthesis and labeling of the peptide.

A peptide representative of the repeated octa-peptide motif of PrP, for example the motif GGWGQPHGGGWGQG- (NH2), corresponding to the sequence 79-92 of human PrP, was synthesized using an automatic synthesizer (Milligen 9050 , Waters, Milford, MI).

The peptide was covalently coupled with acetylcholinesterase (AchE) via a heterobifunctional reagent, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Calbiochem, France), as previously described. for other peptides or proteins (McLaughlin et al., 1987, Grassi et al., 1989).

This method involves the reaction of a thiol group introduced into the peptide with the maleimide function which has been attached to AchE by reaction with SMCC.

The thiol group was introduced into the peptide by reaction with N-succinimidyl S-acetylthioacetate (SATA) as described previously (McLaughlin et al., 1987).

Coupling was obtained by reacting AChE-SMCC with an excess of thiolated peptide.

- Immunization and preparation of monoclonal antibodies A preparation of "Scrapie-associated fibr-they" (SAFs, preparation of PrPres) was obtained from infected hamster brains (scrapie strain 263K) as described previously (Lasmezas et al., 1997).

This preparation was inactivated by treatment with formic acid before immunization of the mice.

Mice knocked out (knockout) for the PrP gene (PrP 10 mice) were immunized with these SAF preparations and hybridoma cells were prepared as described previously (Grassi et al., 1988, 1989).

Screening of culture supernatants was performed as described above. 57 hybridomas were identified and stabilized; they were called SAF-1 through SAF-90.

All of these antibodies were found to recognize SAFs immobilized on microtiter plates while a minority of them demonstrated an ability to recognize peptideAchE conjugates.

Of these, seven clearly recognize the repeated octa-peptide motif (peptide 79-92); these are the antibodies SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF35, SAF-37.

The list of antibodies obtained as well as their main characteristics are presented in the table below.

After cloning and expansion in the form of CA 02390891 2002-05-09 WO 01/35104 PCT / FROO / 03159 14 ascitic fluid, the monoclonal antibodies were purified by affinity chromatography on a Protein A Sepharose column and stored at -20 C until use.

The isotype of the antibodies was determined by radial immunodiffusion according to the Ouchterlony technique.

- Screening of hybridoma culture supernatants The presence of antibodies specific for PrP in hybridoma culture supernatants was demonstrated in two ways, by testing either their ability to bind peptide-AchE conjugates or SAFs hamster.

In the first case, the screening was carried out in plates containing an anti-mouse IgG antibody immobilized as described previously (Creminon et al., 1993, Frobert et al., 1991).

Briefly, 100 µl of culture supernatants and 100 µl of Peptide-AchE conjugate were reacted overnight at +4 C in plates containing immobilized goat anti-mouse IgG antibodies.

After washing the plates, 200 μl of Ellman's reagent (Ellman et al. 1961) were added to the wells in order to detect the presence of AchE fixed on the solid phase.

In the second case, plates containing an immobilized SAF preparation were prepared by reacting 50 µl of a 2 g / ml solution in 0.05 M phosphate buffer, pH 7.4 overnight at room temperature.

After washing, the plates were saturated with EIA buffer (100 mM phosphate buffer, pH 7.4 containing 150 mM NaCl, 0.1% bovine serum albumin (BSA) and 0.01 sodium azide. %) overnight at +4 C and were stored at this temperature until use.

The binding of the monoclonal antibodies to the immobilized SAFs was demonstrated using goat anti-mouse IgG antibodies labeled with AchE as described previously (Negroni et al., 1998).

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 Peptide Peptide mAbs Isotype Recognized mAbs Isotype recognized SAF 1 IgM X SAF 54 * IgG2b 142-160 SAF 2? A SAF56 IgM A SAF 3 IgG2a X SAF 58 IgM A SAF 4 IgG2a A SAF 59 IgM A SAF5 IgG1 X SAF 60 * IgG2b 142-160 SAF7 IgG2a X SAF 61 IgG2a 142-160 SAF 8 IgG 1 A SAF 63 IgM A SAF9 IgG 1 A SAF 65 1c-G1 (?) A SAF 10 IgGI A SAF 66 IgG2a 142-160 SAF 11 IgG2a X SAF 67 IgG 1 X SAF 12 IgM A SAF 68 IgG2a A SAF 13 IgM A SAF 69 * IgG2b 142-160 SAF 14 IgGI A SAF 70 * IgG2b 142-160 SAF 15 * IgG3 79-92 SAF 72 IgG2b A SAF 21 IgGI X SAF 73 IgGI X SAF 22? A SAF 75 IgG2a 142-160 SAF 23 IgGI X SAF 76 IgG2a 142-160 SAF 24 IgGI A SAF 77 IgGI X SAF 31 * IgG2b 79-92 SAF 80 IgGI X SAF 32 * IgG2b 79-92 SAF 81 IgM A SAF 33 * IgG2b 79-92 SAF 82 IgG 1 A SAF 34 * IgG2a 79-92 SAF 83 * IgG 1 A SAF 35 * IgG2b 79-92 SAF84 * IgG2b A SAF 37 * IgG2b 79-92 SAF 85 IgM A SAF 42 ILG1 A SAF 91 IgM A SAF 44 Ig MA SAF 94 IgM A SAF 50 IgM A SAF 95 IgG 1 A SAF51 IgM A SAF96 IgM A SAF 53 * IgG2a X Table: Monoclonal Antibodies Produced Against Hamster FAS Preparation.

79-92: antibody recognizing the peptide 79-92 5 142-160: antibody recognizing the peptide 142-160 Epitope X: antibody which recognizes only the immobilized SAFs Epitope A: antibody which recognizes the peptide 126-164 but does not bind the peptide 142-160.

10 * monoclonal antibodies which have demonstrated their ability to recognize the PrPsens of at least one species tested during this study (human, bovine, ovine, mouse and hamster) in the context of an immunometric assay.

CA 02390891 2011-01-24 WO 01/35104 PCT / FRO0103159 16 EXAMPLE 2: Detection of PrPres by Western blot 1.

Treatment of the sample (i) Preparation of a tissue homogenate, from the different biological samples - BSE cow, scrapie sheep, viCJD human (type 4), sporadic CJD human (type 1) and corresponding negative controls 350 are taken mg of bovine brain: it is crushed and homogenized at 20% (w / v) in a 5% glucose solution.

To achieve homogenization, the brain sample (350 mg) and 1.4 ml of glucose solution are introduced into tubes comprising ceramic beads, and vigorously shaken (Ribolyser Hybaid).

Positive samples are diluted in homogenate from healthy brains of the corresponding species as follows:

for the chickweed: at 1 / 100cmc. for the cow: at 1150`m` for the man: type 1 or 4: at 1140`m, type 3: at 1 / 80cm`; type 2 at 1 / 20`m '(ii) conditions of step (1) A first fraction (500 l) of 20% homogenate obtained in (i) is incubated with 500 l of a buffer comprising sarkosyl 10% (SKI 0), 10% TritoüTm X100 (T10), 2M Puree (U2) and proteinase K (PK) at 60 g / m1 of buffer (PK1) for 10 minutes at 37 C (corresponding at 30 .ig / m1 final for a homogenate at 10%).

In Figures 3-6, PK3 corresponds to a PK concentration of 180 g / ml of buffer and PK6 corresponds to a PK concentration of 360 µg / ml of buffer.

(iii) 500 l of a buffer consisting of 1-butanol (corresponding to buffers B, as described in international application WO 99/41280) are added; then centrifuged at 15,000 rpm for 5 min. (approximately 17,000 g).

(iv) the centrifugation pellet which contains the PrPres is taken up in 80-100 l of a buffer C as described in international application WO 99/41280, preferably a Laemmli buffer containing 4% SDS and heated to CA 02390891 2002-05-10 i3 Prtnttd 28-a5-2002 4uk vtvr ~~ C F009 ~. .. _ .. , 23-01-2002 F-1-C 17 100 C for 5 min., To carry out a Western blot or taken again, to carry out an immunometric assay, successively in a C1 buffer comprising 6M urea and 0.5 sarkosyl %, followed by heating for 5 min. at 100 ° C., then in a C2 buffer comprising 2M guanidine, followed by heating for 5 min. at 100 C.

(v) conditions of step (b) A second fraction (500 l) of 20% homogenate obtained in (i) is incubated with 500 l of a buffer comprising 5% sarkosyl (SK5), SDS at 5% (SDS5), 1M urea (IU) and proteinase K (PK) at 360 g / ml (PK6) for 10 min. at 37 ° C., then steps (iii) and (iv) above are carried out.

II.

Western blot The samples obtained are used to carry out SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane under the conditions set out in example 1 and in example 3 of the aforementioned international application WO 99/41280.

The immunodetection of PrPres is carried out with the monoclonal antibodies SAF70 and SAF37 as described in Example 1, above and goat anti-rabbit Ig conjugated to peroxidase (1/2500). The immunoreactivity is revealed by chemiluminescence (ECL, Amlersham), quantified and visualized on autoradiographic films, as illustrated in FIG. 2.

To this figure:

* tracks 1-5 correspond to conditions according to step (1) or (a) (SK10 + T10 + U2 + PK1):

Track 1: negative control Track 2: BSE cow Track 3: scrapie sheep Track 4: vMCJ man (type 4) Track 5: male CJD (type 1) and * tracks 6-10 correspond to conditions depending on the stage ( b) (SK10 + SDS5 + U1 + PK6):

Track 1: negative control Track 2: BSE cow 1 MODIFIED SHEET s3 CA 02390891 2002-05-09 WO 01/35104 PCT / FROO / 03159 18 Track 3: scrapie sheep Track 4: vCJD man (type 4) Track 5: male CJD (type 1) and The results obtained show that - in step (1) or (a) (lanes 1 to 5), the PrPsens is systematically destroyed, while the signal obtained with the PrPres is systematically greater, with the antibody directed against the repeated octa-peptide units, to the signal obtained on the same samples with an antibody directed against the 94-230 region of PrP;

- in step (b) (lanes 6 to 10), the PrPsens is systematically destroyed, while the signal obtained on lanes 8 and 10 (in the presence of the antibody directed against the repeated octa-peptide units) is similar or higher than the signal obtained on the same samples with an antibody directed against the 94-230 region of PrP, while it is lower and even undetectable on lanes 7 and 9 (PrPres of BSE).

EXAMPLE 3 Detection of PrPres with a two-site immunometric assay using as capture antibody a monoclonal antibody recognizing the repeated octa-peptide motifs.

To carry out an immunometric assay at two sites, the pellet obtained in (iv) in Example 2 is for example dissolved in a buffer comprising sarkosyl (0.25-1%) and urea (0.25-8 M) or SDS (0.25-1%) and urea (0.25-1 M); the sample obtained will preferably be diluted (to '/ 4 or to V), after heating with a buffer containing albumin leading to a final albumin concentration of between 0.1 and 1% (w / v) or with a buffer containing 1% deoxy cholate).

The two-site immunometric assay is carried out in microtiter plates containing an antibody immobilized under the conditions already described for other proteins (Grassi et al., 1989).

Its principle is as follows:

the analyzed PrP is recognized by an antibody fixed on the solid support (capture antibody) and by a second antibody, recognizing another part of the molecule, which is labeled with an enzyme (here acetylcholinesterase, tracer antibody), at 5 Ellman units / ml.

In the context of the invention, the capture antibody is directed against the repeated octa-peptide motifs and the tracer antibody recognizes a sequence comprised CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 19 in the region 94-230 of PrP, for example the zone 142-160 of PrP.

After washing the solid phase, the enzymatic activity fixed on the plate is proportional to the amount of PrPres having the octa-peptide units repeated initially in the sample analyzed.

In practice, the assay is carried out as follows. 100 l of the PrP solution to be analyzed are deposited in the wells of the microtiter plate containing the antibody recognizing the repeated octa-peptide patterns.

After 3 hours of reaction at room temperature, the plate is washed before adding 100 l of a solution of the tracer antibody (5 Ellman units / ml).

After reaction overnight at +4 C, the plates are washed again before adding 200 l of a substrate solution (reagent from Ellman, Grassi et al., 1989) which will make it possible to measure the activity of l. acetylcholinesterase fixed on the solid phase.

After 30 minutes of enzymatic reaction, the absorbance (OD at 414 nm) of each well is measured.

EXAMPLE 4 Comparative study of different conditions: step (1) or (a) and step (b).

The samples are prepared as described in Example 2.

The homogenates are prepared as in Example 2.

- in humans Figures 3 and 4 illustrate the results obtained with different buffers:

* conditions according to step (1) or (a) of the process according to the invention I: homogenate at 20% + SK10 + T10 + U2 + PK1 III ': homogenate at 10% + SK5 + T5 + U1 + PK0.5 (Figure 4) * conditions according to step (b) of the method according to the invention:

II: 20% homogenate + SK10 + T10 + U2 + PK3 III: 20% homogenate + SK10 + T10 + U2 + PK6 III ": 10% homogenate ++ SK5 + T5 + Ul + PK6 (figure 4) IV: 20% homogenate + SK20 + PK3 V: 20% homogenate + SK20 + Ul + PK3 VI: 20% homogenate + SK20 + U2 + PK3 VII: 10% homogenate + SK20 + PK3 VIII: 10% homogenate + SK20 + U 1 + PK3 CA 02390891 2002-05-11 20 IX: homogenate at 10% + SK20 + U2 + PK3 X: homogenate at 10% + SK5 + SDS5 + U1 + PK6 X ': homogenate at 10% a + SK2 , 5 + SDS2.5 + U2 + PK6 (figure 4) XI: 20% homogenate + SK20 + PK6 XII: 20% homogenate + SK20 + U1 + PK6 XIII: 20% homogenate + SK20 + U2 + PK6 XIV: 10% homogenate + SK20 + PK6 XV 10% homogenate + SK20 + U1 + PK6 XVI: 10% homogenate + SK20 + U2 + PK6 The amount of PrPres having retained the repeated octa-peptide motif is measured using the two-site immunometric assay (absorbance or OD at 414 nm, see Example 3; the appearance of a coloration is measured. yellow, Ellman's reagent): negative control (D). Sporadic type 1 (D) CJD and type 4 (a) vCJD).

- in ruminants Figures 5 and 6 illustrate the results obtained with different buffers:

= * conditions according to step (1) of the method according to the invention:

I: 20% homogenate + SK10 + T10 + U2 + PK1 III ': 10% homogenate + SK5 + T5 + U1 + PKO, 5 (figure 6) * conditions according to step (b) of the process according to the invention :

II: 20% homogenate + SK10 + T10 + U2 + PK3 III: 20% homogenate + SK10 + T10 + U2 + PK6 III ": 10% homogenate ++ SK5 + T5 + U1 + PK3 (figure 6) IV: 20% homogenate + SK20 + PK3 V: 20% homogenate + SK20 + U1 + PK3 VI: 20% homogenate + SK20 + U2 + PK3 VII: 10% homogenate + SK20 + PK3 VIII: 10% homogenate + SK20 + U1 + PK3 IX: homogenate at 10% + SK20 + U2 + PK3 X: homogenate at 10% + SK5 + SDS5 + UI. + PK6 X ': homogenate at 10% + SK5 + SDS5 + U1 + PK3 (figure 5) 2 MODIFIED SHEET _ rt ~ CA 02390891 2011-01-24 WO 01/35104 PCT / FR00 / 03159 21 XI: 20% homogenate + SK20 + PK6 XII: 20% homogenate + SK20 + UI + PK6 XIII: 20% homogenate + SK20 + U2 + PK6 XIV: 10% homogenate -; - SK20 + PK6 XV: 10% homogenate + SK20 + U l + PK6 XVI: homogenate at I0% + SK20 + U2 + PK6 The amount of PrPres before the repeated ocra-peptide motif is preserved is measured using the two-site immunometric assay (absorbarice or OD at 414 nm , see example '): negative control (11), bovine ATNC and sheep ATNC (a).

- Figures 7 (Western blot) and 8 (two-site immunometric assay):

The comparison is carried out using homogenates at 20% of brains of healthy sheep, of sheep affected by scrapie and of bovine animals affected by BSE, obtained under the conditions set out in Example 2.

.

Sample processing:

A: sarkosyl 10% + TritonTM 1 0% + urea 2M + proteinase K 60 .Lg / ml, 10 minutes B: sarkosyl 10% + urea 2M + proteinase K 240 ug, iml, 10 minutes C: sarkosyl 10 / o + proteinase K 240 gg / ml, 10 minutes. Detection by Western block (FIG. 7): Saf 37 and Saf 84 antibody (see example 1) by immunometry (FIG. 8): capture by Saf 37 and visualization by an antibody directed against the 94-230 region of PrP.

Figures 9 (Western blot) and 10 (two-site immunometric assay):

The comparison is carried out using homogenates at 20% of brains of healthy sheep, of sheep affected by scrapie and of bovine animals affected by BSE, obtained under the conditions set out in Example 2.

Sample processing:

A: sarkosyl 10% + TritonTM 1 0% + urea 2M + proteinase K 60 g / m1, 10 minutes B: SDS 10% + TritonTM 5% + urea 2M + proteinase K180.tg/ml, 10 minutes CA 02390891 2011-01 -24 WO 01 / 3510.3 PCT / FROO / 03159 12 Detection under the same conditions as above.

Increasing the PK dose alone makes it possible to reveal a differential sensitivity of the region of repeated ocra-peptide motifs, between BSE and scrapie in sheep but not in humans (between type 1 and type 4).

On the other hand, by also modifying the composition of surfactant and of chaotropic agent, this makes it possible to show a differential sensitivity of the region of the repeated ocra-peptide units in all cases.

EXAMPLE 5 Influence of the composition of the buffers on the differential detection of BSE and of scrapie - Figures 11 (Western block) and 12 (two-site immunometric assay) The comparison is carried out using homogenates at 10% of brains of healthy mice or of mice experimentally infected with the strain C506M3 (scrapie) or with a strain 6PBI (ESB), obtained. under the conditions set out in Example 2.

.

Sample processing A: sarkosyl 10% + TritonTM 1 0% + urea 2M + proteinase K 60 tg / ml, 10 minutes B: sarkosyl 10% + TritonTM 10% + urea 2M + proteinase K60 g / ml, 10 minutes C: sarkosyl 10% + TritonTM 10% + urea 2M + proteinase K 180 pg / ml, 10 minutes D: sarkosyl 10% + TritonTM 1 0% + urea 2M + proteinase K3 60 .tg / m1, 10 minutes E: sarkosyl 10% -urea 2M + proteinase K 180 µg / ml, 10 minutes.

. Detection by Western blot (FIG. 11): Saf 37 and Saf 70 antibody (see example 1) by immunometry (FIG. 12): capture by Saf 37 and visualization by an antibody directed against the 94-230 region of PrP.

The results obtained show that the increase in the PK dose alone makes it possible to reveal a differential sensitivity of the region of the repeated octa-peptide units between BSE and scrapie, in mice.

CA 02390891 2011-01-24 \ VO 01/35104 PCT / FR00 / 03159 23 EXAMPLE 6: Influence of buffers and of the proteinase K concentration on the detection of different CJD.

Figures 13 (Western blot) and 14 (two-site immunometric assay) The comparison is made using 10% homogenates of brains of healthy men and men with CJD (types 1, 2, 3 and 4) , obtained under the conditions set out in Example 2.

.

Sample processing A: sarkosyl 10% + TritonTM 10% + urea 2M + proteinase K30 .tg / ml, 10 minutes B: sarkosyl 10% + TritonTM 10% + urea 2M + proteinase K180 .tg / ml, 10 minutes C: sarkosyl 10% -proteinase K 30 ug / ml, 10 minutes D: sarkosyl 10% + proteinase K 60 ug / ml, 10 minutes E: sarkosy! 10% + proteinase K 180 µg / ml, 10 minutes F: sarkosyl 10% + proteinase K 360 µg / m1, 10 minutes.

. Detection by Western blot (FIG. 13): Saf 37 and Saf 70 antibody (see example 1) by immunometry (FIG. 14): capture by Saf 37 and visualization by an antibody directed against the 94-230 region of PrP.

- Figures 15 (Western block) and 16 (Immunometry) The comparison is made using 10% homogenates of brains of healthy men and men with CJD (types 1, 2, 3 and 4), obtained in the conditions set out in Example 2.

Sample processing Figure 15: Proteinase K digestion (150 µg / ml), 10 minutes.

Figure 16: 10% sarkosyl + 2M urea + proteinase K (30 to 360 µg / ml, for 10 minutes.

. Detection by Western blot (FIG. 15): Saf 37 antibody and an antibody directed against the 94-230 region of PrP (see example 1) CA 02390891 2002-05-09 WO 01/35104 PCT / FROO / 03159 24 by immunometry ( FIG. 16): capture by Saf 37 and visualization by an antibody directed against the 94-230 region of PrP.

The results obtained show that the increase in the PK dose makes it possible to reveal a differential sensitivity of the region of the repeated octapeptide units in the different types of CJD.

Under these conditions, there is no significant difference between type 1 and type 4; the fact of changing the composition of surfactant and chaotropic agent, at the same dose of PK (E, figures 13 and 14) makes it possible to destroy the octa-peptides in type 4 while keeping them in type 1.

Moreover, FIG. 16 makes it possible to show the difference in sensitivity of the PrPres obtained from different strains, for the same buffer, as a function of the PK dose.

Furthermore, FIG. 15 shows that direct treatment of the homogenate with PK demonstrates another type of sensitivity of PrPres to degradation.

EXAMPLE 7 Detection by Western Blot of the Digested and Purified PrPres in the Form of SAF.

Homogenates, obtained under the conditions according to Example 2, are treated as follows:

Homogenate 20% (500 41) + NaCl 20% (500 l) + [sarkosyl 20% + SB314 2%] (500 l) + PK (20 g / ml final) for 1 hour.

Figure 17 illustrates the betd results:

lanes 1-7: results obtained with different dilutions of scrapie strain C506M3 in mice (dilutions 1/20, 1150, 1/250, 1/500, 1/1000 and 1/2000) lane 8: molecular weights lanes 9 -15: results obtained with different dilutions of EBS strain in mice (dilutions from 1/1000 to 1/10).

It is possible to completely eliminate the ESB signal.

CA 02390891 2002-05-09 WO 01/35104 PCT / FR00 / 03159 a and c: the results obtained confirm that there is a significant increase in the signal in the presence of antibodies directed against the repeated octapeptide units.

e: this figure shows that it is possible to obtain a decrease in signal with BSE in both monkeys and humans (lanes 4 and 7).

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As emerges from the foregoing, the invention is in no way limited to those of its embodiments, embodiments and applications which have just been described more explicitly; on the contrary, it embraces all the variants which may occur to the technician in the field, without departing from the framework or the scope of the present invention.