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

IS 2 280 263 T3

DESCRIPTION

Diagnostic procedure for a transmissible subacute spongiform encephalopathy (ESST) caused by a strain of CNTA in a biological sample.

The present invention relates to a diagnostic method of a transmissible subacute spongiform encephalopathy (ESST) caused by a strain of ATNC in a biological sample by detecting PrPres as well as its use within the framework of a differential diagnosis of the different strains of ATNC in a biological sample.

Transmissible spongiform subacute encephalopathies (ESST) are caused by unconventional transmissible agents (CNTA), also called prions, the precise nature of which is contested on this date. ESST encephalopathies essentially comprise Creutzfeldt-Jakob disease in man (MCJ or CJD for Creutzfeldt-Jakob disease), scrapie in sheep and goat, and coil spongiform encephalopathy (ESB or BSE for coil spongiform encephalopathy) in cattle; other encephalopathies were evidenced in felids, as in the case of mink or some wild animals, such as the brain or elk.

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

In ESST encephalopathies 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 neuron cultures.

The two isoforms of PrP have the same amino acid sequence (see Figure 1), but are different in their secondary structure: PrPres has a significantly higher content in pleated β sheets, while normal PrP (PrPsens) has a higher content. number of propellers a.

Two biochemical properties allow, in general, to distinguish these two isoforms:

- PrPres is partially resistant to proteases, in particular to proteinase K (PK), which entails a separation of its N-terminal end. After the action of PK, PrPres is frequently referred to as Prp27-30 because of the apparent molecular weight of the blycosylated form; It is generally accepted that the PrPres cleavage site is between amino acids 89 and 90 (Prusiner et al, Cell 1984) for common strains

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

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

The peptide sequences of PrPsens from the hamster, humans, bovines and sheep are represented in Figure 1; they allow, in particular, an octa-peptide motif (P (H / Q) GGG (- / T) WGQ), repeated 4 or 5 times, depending on the species. This octa-peptide repeat motif corresponds to amino acids 51-91 of PrP (human PrP sequence numbering) (B. Oesch et al, 1991).

To detect the presence of the infectious agent, the most recent procedures are based on a selective detection of abnormal PrP (Prpsens), bound to the infectious agent, benefiting from its partial resistance to proteases.

It can be distinguished:

- Western-blot procedures based on the immunological detection of PrPres in a tissue extract, after treatment of the extract by a protease in such a way as to destroy the normal isoform of PrP (PrPsens), separation of proteins of the extract by electrophoresis, transfer on a polymer membrane and detection by a specific antibody that recognizes PrP (Schaller O. Et al 1999).

- ELISA-type tests, which also involve a treatment of tissue extracts by a protease.

Among these different tests, which involve a treatment of tissue extracts by a protease, the following can be mentioned:

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

IS 2 280 263 T3

- the one described by Oesch et al (Biochemistry, 1994, 33, 5926-5931) who have proposed, to quantify the amount of PrPres, an immunofiltration assay for the purification of PrPres (ELIFA or enzyme-linked immunofiltration assay) .

- that described by Gratwohl et al, 1997, which proposes an ELISA type dosage. After treatment of the samples with proteinase K and purification of PrPres by centrifugation, it is absorbed on microtitration plates and detected with the help of rabbit polyclonal antibodies.

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

In general, these different tests have the disadvantage of lack of sensitivity and, therefore, of carrying false negatives.

Other methods propose treatments of the sample by 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 a treatment by a methalopeptidase (WO 00/22438) that make hidden antigenic sites accessible, detectable by the monoclonal antibody 3F4 that recognizes the region 109-102 of PrP (WO 00/29850 or WO 00/22438).

The method, described in WO 00/29850, Wallac Oy et al, has the significant drawback of lacking specificity, in particular because of an incomplete elimination of PrPsens, under the recommended treatment conditions, while the method described in document WO 00/22438, The Regents of the University of California lacks sensitivity, due to the use of the detection antibody 3F4 (WO 00/22438), which is only bound by a single motif in the protein.

The applicant has recently proposed a test for the quantitative detection of PrPres, which comprises a purification step that leads to a significantly more sensitive detection and which represents a breakthrough for the health surveillance and dosing of PrPres in slaughterhouses. This procedure is described, in particular, in the PCT international patent application WO 99/41280 and in a preliminary report of the Directorate General XXIV of the European Commission (Consumer policy and protection of your health; http: // europa. eu.int/comm/dg24/health/).

However, given the need for a particularly reliable diagnostic test for ESSTs, the applicant has continued its work.

In particular, it has established that to obtain a screening test:

(i) sensitive, that is, it has the ability to correctly identify uninfected animals;

(ii) specific, that is, it has the ability to correctly identify infected animals that show clinical symptoms;

(iii) that has a detection limit as low as possible, that is, adequate to allow the detection of small amounts of PrPres (detection of PrPres before the appearance of clinical symptoms) and (iv) reproducible.

The treatment of the biological sample to be analyzed must be carried out under conditions that allow all or part of the octa-peptide motifs to be preserved exclusively in the PrPres.

In particular, it has been found that in order to respond effectively to the four conditions (i) - (iv) stated above, it is necessary to define precise treatment conditions for the sample to be analyzed that totally eliminate the PrPsens from the sample, allowing a specific capture. of the PrPres under the defined conditions.

The present invention has as its object a diagnostic method (method A) of an ESST or prion disease caused by a strain of ATNC by detection of PrPres in a biological sample, characterized in that it comprises:

(1) the treatment of said sample with at least one proteinase K, either with a concentration between 30 pg / ml and 200 pg / ml for 10 minutes at 37 ° C for a 10% homogenate, either for a period and at a concentration equivalent to the concentration between 30 pg / ml and 200 pg / ml under the conditions mentioned above and even more preferably for 10 minutes at a concentration between 30 pg / ml and 200 pg / ml for a 10% homogenate or for 30 minutes at a concentration between 10 pg / ml and 70 pg / mL, for a 10% homogenate, said proteinase K being put in solution in a buffer solution selected from the group consisting of homogenization buffers of the biological sample and buffer solutions comprising at least one of the following agents: at least one surfactant and / or at least one chaotrope agent and / or at least one salt, so that the PrPsens is completely degraded, preserving all or part of the repeated octa-peptide motifs of the PrPres, whatever the ATNC strain

ES 2 280 263 T3 (2) the contacting of said treated sample with a ligand of said octa-peptide motifs and (3) the detection of the occasional presence of the complex of repeating octa-peptide motifs-ligand.

It must be borne in mind that a certain number of parameters are closely linked to each other: the proteinase K concentration directly depends on the duration of treatment (incubation time) of the sample. It can be considered that at 37 ° C for example, a 10 minute incubation with a PK at a concentration between 30 and 200 pg / ml of 10% homogenate is equivalent to a 30 minute incubation with a PK with a concentration comprised between 10 pg / ml 170 pg / ml of 10% homogenate. For example, a 30 minute incubation with a PK at 25 pg / ml is equivalent to a 10 minute incubation with a PK at a concentration of 75 pg / ml.

Regardless of the PK concentration and the duration of incubation, the treated sample must not contain undegraded PrPsens, while the PrPres, occasionally present, has retained all or part of the octapeptide motifs.

Other parameters may 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 solution in which said PK is put into solution; When PK is put in the sample homogenization buffer solution, depending on the homogenization buffer used, the minimum concentration of PK may vary, within the range of previously specified concentrations:

- in a glucose or guanidide buffer solution (or one of its salts), the minimum concentration of PK is preferably 25 pg / ml (30-minute incubation) or 75 pg / ml (10-minute incubation ).

- in a PBS buffer solution, the minimum PK concentration is preferably 50 pg / ml (30 minute incubation) or 150 pg / ml (10 minute incubation).

Advantageously, PK can be carried out in a buffer solution other than homogenization; Such a buffer solution comprises, in a preferred embodiment, at least one surfactant and / or at least one chaotropic agent and / or at least one room.

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

- addition to said suspension of a buffer solution B, as defined in this international patent application 99/41280 and in particular, the alcohols in C₃-C₆ and alcohol mixtures, whose medical theoretical dielectric constant is between 10 and 25

- centrifugation of the suspension obtained and

- the solubilization of the residue in a buffer solution comprising at least one surfactant and / or a chaotropic agent, under the conditions defined in said international patent application 99/41280.

Such a test has the advantage of being particularly adapted to the differential diagnosis of ESST in the same host, caused by different strains of ATNC and in particular, adapted to the differential diagnosis of ESB strains with respect to the usual strains of scrapie in the sheep.

For example, the ESB strain has been shown to have infected humans in Great Britain by analysis of the electrophoretic profiles of PrPres which has shown migration differences as a function of the ATNC strains; A characteristic profile (type 4) has been found in patients who develop a new variant of Creutzfeldt-Jakob disease (vMCJ) that seems to be linked to the passage to human feeding of bovines infected by the ESB agent (Colligne et al, Nature, 1996). A similar profile was observed in the case of a macaque infected by the ESB agent (Lasmezas et al, 1996) and a cat truly contaminated by this agent (Priola et al, Nature Med., 1996).

However, it is a long and delicate method to implement if reproducible results are to be obtained, which is really an element of controversy that exists regarding 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 by the ESB agent (Butler, Nature, 1998). These animals are, in fact, sensitive to this agent, whereas there is an endemic disease in sheep, scrapie (scrapie for Anglo-Saxons), another ESST, which presents similar and indistinguishable clinical and histological characteristics. The only reference method for a differential diagnosis between the ESB agent and scrapie is inoculation in the mouse and the study of the lesion profile, which requires approximately two years of waiting and which was carried out in Great Britain more than in the United Kingdom. 9 sheep for a population of several tens of millions of heads.

IS 2 280 263 T3

First tests were carried out to try to distinguish the different strains of ATNC in a sample; Kuczius T, et al 1999 have considered that the variations observed between the different ESST strains (scrapie, ESB and CJD) by means of the glycotyping technique do not allow each strain to be distinguished; have selected other biochemical and biological markers of PrPres that allow better distinguishing between them the different strains of prions. The analysis parameters used are the following: long-term resistance to proteinase K, molecular mass of PrPres, topology and quantity of PrPres deposits. The results obtained show that the PrPres of different strains of ESB and scrapie show significant differences in their long-term resistance to proteinase K. Depending on the strain of scrapie, resistance to PK varies: small resistance: strain Chandler; intermediate resistance: strain 22A; relative stability: strain 87V. under the same conditions, ESB strains show intermediate resistance, while glycotyping does not allow to distinguish between scrapie strain 87V and ESB strains, these two types of strains being clearly distinguishable after long-term treatment with PK . However, these are not sufficiently reliable and usable protocols on a large scale and in the field.

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

Consequently, the present invention also has as its object a procedure for the differential diagnosis (procedure B) of the ESSTs caused by strains of ATNC, in a biological sample by detecting the PrPres associated with the different strains of ATNC, characterized in that it comprises:

(a) the detection of the PrPres of an ATNC strain in a first fraction of said sample, in accordance with steps (1) to (3) below:

(1) the treatment of said sample with at least one proteinase K, so that it completely degrades the PrPsens, preserving all or part of the octa-peptide repeating motifs of the PrPres, whatever the strain of ATNC (2) the bringing said treated sample into contact with a ligand of said octa-peptide motifs ^Y (3) detection of the occasional presence of the octa-peptide repeat-ligand motif complex and then:

(b) for each sample for which the presence of a complete octa-peptide repeat-ligand motif is detected in step (a):

- the treatment of a second fraction of said sample with at least one proteinase K, so that most of the repeated octa-peptide motifs are eliminated for the PrPres associated with at least one strain of interest, in particular the ESB strain and where the set of PrPres associated with the other ATNC strains conserve all or part of said repeated octa-peptide motifs

- the putting in contact of said second fraction of said treated sample, with a ligand of said repeating octa-peptide motifs and

- detection of the occasional presence of the octa-peptide repeat-ligand motif complex

The present invention also has as its object a differential diagnosis procedure (variant of procedure B) of ESSTs caused by strains of ATNC in a biological sample considered to contain PrPres (detection test carried out by any method), characterized in that comprises, for each sample for which the presence of a PrPres was detected, the implementation of step (b) as defined above.

- the contacting of said second fraction of said treated sample with a ligand of said repeated octapeptide motifs and

- detection of the occasional presence of the octa-peptide repeat-ligand motif complex.

According to an advantageous implementation mode of said process, the treatment with said proteinase K according to step (a) or (b) is carried out for 30 seconds to 2 hours, at a temperature below 80 ° C, preferably between 10 minutes. and 30 minutes.

According to an advantageous arrangement of this mode of implementation, the treatment with said proteinase K is carried out at a concentration comprised between 30 pg / ml and 200 pg / ml for 10 minutes at 37 ° C for a 10% homogenization, or during a period and at a concentration equivalent to the concentration comprised between 30 pg / ml and 200 pg / ml under the conditions mentioned above and even more preferably, for 10 minutes at a concentration between 10 pg / ml and 70 pg / ml for a 10% homogenate.

IS 2 280 263 T3

According to another advantageous way of putting this process into practice, the proteinase K is put into solution in a buffer solution selected from the group consisting of the homogenization buffers of the biological sample and of the buffer solutions that comprise at least one of the following agents: at least one surfactant and / or at least one chaotropic agent and / or at least one salt.

Within the framework of differential diagnosis (procedure B), in addition to the PK concentration, other conditions may occasionally intervene on the degradation of the octa-peptide motifs, depending on the strain: Indeed, when PK is put into practice in a buffer solution other than the homogenization request, such as in a buffer solution comprising, in an advantageous embodiment, at least one surfactant and / or at least one chaotrope agent and / or at least one salt, the number of degraded octa-peptide motifs can vary depending on the composition of said buffer solution and the concentration of the different agents.

Strain of interest is understood to be the ATNC strain (s), the ESB strain in particular, for which it is desired to eliminate the repeating octa-peptide motifs, for example.

One of the main characteristics of procedures A and B is to consider the different conditions in which the protease treatment is carried out so as to control the degradation of the repeating octa-peptide motifs. Depending on the use considered, it will be carried out in such a way that these reasons are preserved (procedure A) or destroyed (procedure B):

- Within the framework of procedure A, this control will consider the possibility that all or part of the repeated octa-peptide motifs are conserved in order to favor a very sensitive detection of PrPres.

- within the framework of procedure B, the purpose of controlling degradation by protease will be that the majority, and occasionally all, of the octa-peptide repeating motifs are degraded for the PrPres corresponding to the strains of interest, whatever it may be the species in which it is expressed, under conditions in which all or part of the repeating octa-peptide motifs are conserved for the PrPres corresponding to all other ESST strains. In this case, the interest of the invention is to allow a differential diagnosis of the strain of interest with respect to other strains of ATNC.

Indeed:

Surprisingly, under the conditions according to stage (1) or stage (a), PK does not bring about the discrepancy of all or part of the octa-peptide repeating motifs, of the PrPres associated with all strains of ATNC or prions, while the conditions of step (b) bring about the discrepancy of the repeated octa-peptide motifs of the PrPres associated with the strain of interest, while the set of PrPres associated with the other ATNC strains conserve all or part of said repeated octa-peptide motifs.

Also, surprisingly, such an assay, particularly when the ligand is an antibody, has the following advantages:

- high detection sensitivity since antibodies directed against these repeating octa-peptide motifs have a much higher affinity than antibodies directed against other areas of PrPres 27-30 and are more resistant to the buffers used; indeed, the recognition of a repeated motif favors high affinity interactions and offers the possibility of binding several antibody molecules to a single PrP molecule;

- the possibility of a differential diagnosis of the ATNC strains or prions, since it is possible, depending on the conditions put into practice, to obtain, or not, the digestion of these motifs; in particular, it is possible to eliminate them for all diseases linked to the strain of interest, the ESB agent for example, while it is possible to conserve it for the other so-called prion diseases. Consequently, the methods according to the invention propose a simple test for the differential diagnosis of ESB and / or of another strain of interest with respect to the other strains by putting into practice two different conditions step (1) or (a) and step (b) ), the conditions of step (1) or (a) (preserving the repeated octa-peptide motifs of the PrPres associated with the set of ATNC strains) that allow the detection of all strains and the conditions of step (b) (eliminating these motifs only in the PrPres associated with a strain of interest) that only reveals the other strains.

Therefore, such tests find, in particular, application in the investigation of contamination of sheep by the ESB strain that is not usually known to distinguish from the classic scrapie strains.

According to an advantageous way of putting these procedures into practice, the PK is put into the buffer solution that preferably comprises:

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

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

IS 2 280 263 T3

- zwitterionic surfactants, such as SB 3-10 (decyl-sulfobetaine), SB 3-12 (dodecyl-sulfobetaine), SB 3-14, SB 3-16 (hexadecyl-sulfobetaine), CHAPS and deoxyCHAPS ,

- nonionic surfactants, such as C12E8 (dodecyl-octa-ethylene glycol), Triton ^® X100, the Triton ^® X114, Tween ® 20, Tween ® 80, MEGA 9 (nonanoyl-methylglucamine), octiglucoside, LDAO (dodecyl-dimethylamine oxide) or NP40 or

- surfactant mixtures such as a mixture of an ionic surfactant and a non-ionic surfactant, a mixture of two ionic surfactants or a mixture of an ionic surfactant and a zwitterionic surfactant and / or

b. at least one chaotrope agent selected from the group consisting of urea and guanidine or a mixture of them and / or at least one salt selected from alkali metal salts or not.

According to an advantageous arrangement of this mode of implementation, said buffer solution comprises at least 5% anionic surfactant, preferably sarkosyl, occasionally associated with SDS.

According to another advantageous way of carrying out said methods, the ligand is selected from the group consisting of aptaremos and antibodies that bind to the region of the repeating octa-peptide motifs.

According to another advantageous way of putting said procedures into practice, the treatment of step (b) is carried out under the same conditions as those defined in step (a) (1), but at a PK concentration higher than that used in step (a) (1).

The present invention also has as its object a diagnostic device for the implementation of the methods according to the invention, characterized in that it comprises, in combination, at least one surfactant and / or at least one chaotropic agent and / or at least a salt and a protease, as defined above.

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

In addition to the previous provisions, the invention also includes other provisions, which will be deduced from the description given below, which refers to examples of application of the method object of the present invention as well as the attached drawings, in which:

- Figure 1 represents the different PrP sequences: human, ovine, bovine and cricetidian *;

- Figure 2 represents the detection of PrPres by Western blot;

- Figures 3 to 6 correspond to different conditions according to stage (1) or (a) on the one hand and stage (b) on the other hand, in the case of man (Figures 3 and 4) and in the case of ruminants (Figures 5 and 6), in the event that the presence of the repeated octa-peptide motifs is detected by an immunometric dose in two places

- Figures 7 to 12 illustrate the influence of buffers on the differential detection of ESB and scrapie

Figures 13 and 14 illustrate the difference in the composition of the buffer solutions and the concentration of proteinase K (PK) for the detection of the different types of MCJ.

- Figure 15 illustrates the results obtained by a direct digestion of the brain homogenates by proteinase K

- Figure 16 illustrates the differences in resistance of PrPres to proteinase K as a function of MCJ types

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

However, it should be understood that these examples are provided for illustrative purposes only of the subject of the invention, of which they do not constitute, in any way, a limitation.

IS 2 280 263 T3

Example 1

Obtaining and characterizing monoclonal antibodies specific for the repetitive octa-peptide motif

- Synthesis and labeling of the peptide

A representative peptide of the octa-repeating peptide motif of PrP, for example the GGWGQP-HG-GGWGQG (NH2) motif, which corresponds to the sequence 79-92 of human PrP, was synthesized with the aid of an automated synthesizer (Milligen 9050, Waters, Milford, MI). The peptide was covalently linked to acetylcholinestarase (AchE) via a heterobifunctional reagent, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Calbiochem, France), as previously described for others. peptide or proteins (McLaughlin et al 1987, Grassi et al 1989). This method involves the reaction of a thiol cluster introduced into the peptide with the maleimide function that was fixed on the AchE by reaction with the SMCC. The thiol cluster was introduced into the peptide by reaction with N-succinimidyl-S-acetylthioacetate (SATA) as previously described (McLaughlin, et al, 1987). The binding was obtained by reacting AchE-SMCC with an excess of thiolated peptide.

- Immunization and preparation of monoclonal antibodies

A preparation of "Scrapie-associated fibrils" (SAFs, PrPres preparation) was obtained from infected hamster brains (scrapie strain 263K) as previously described (Lasmezas et al 1997). This preparation was inactivated by treatment with formic acid before immunization of the mice. Knock-out mice for the PrP gene (PrP mouse^0/00) were immunized in these SAF preparations and hybridoma cells were prepared as described above (Grassi et al, 1988, 1989). Screening of culture supernatants was performed as described above. 57 hybridomas have been identified and stabilized; they are designated SAF-1 through SAF-90. All of these antibodies have been shown to recognize SAFs immobilized on microtitration plates, while a minority of them have shown an ability to recognize peptide-AchE conjugates. Among the latter, 7 of them clearly recognize the octa-repeating peptide motif (peptide 79-92); These are the antibodies SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35 and SAF-37. The list of the antibodies obtained, as well as their main characteristics, are presented in the following table. After cloning and expansion in the form of ascite liquid, 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 PrP-specific antibody in hybridoma culture supernatants was demonstrated in two ways, proving its ability to bind peptide-AchE conjugates or hamster SAFs. In the first case, the screening was performed on plates containing an immobilized anti-mouse IgG antibody as previously described (Créminon 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 goat anti-IgG antibodies from immobilized mice. After washing the plates, 200 µl of Eilman's reagent (Eilman et al 1961) was 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 solution with 20 pg / ml in a 0.05 M phosphate buffer, pH 7.4, overnight at room temperature. After washing, the plates were saturated with EIA buffer solution (100 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, 0.1% serum albumin coil (BSA) and sodium azide 0 , 01%) overnight at + 4 ° C and were stored at this temperature until use. The binding of monoclonal antibodies in immobilized SAFs was evidenced with the aid of AchE-labeled goat anti-mouse IgG as described above (Negroni et al 1998).

IS 2 280 263 T3

PICTURE

Monoclonal antibodies raised against a hamster SAF preparation

<td colspan="6"></td>

<td>AcMs</td><td>Isotype</td><td>Peptide recognized</td><td>AcMs</td><td>Isotype</td><td>Peptide recognized</td>

<td>SAF 1</td><td>IgM</td><td>X</td><td>SAF 54 *</td><td>IgG2b</td><td> 142 - 160</td>

<td>SAF 2</td><td> ¿</td><td>TO</td><td>SAF 56</td><td>IgM</td><td>TO</td>

<td>SAF 3</td><td>IgG2a</td><td>X</td><td>SAF 58</td><td>IgM</td><td>TO</td>

<td>SAF 4</td><td>IgG2a</td><td>TO</td><td>SAF 59</td><td>IgM</td><td>TO</td>

<td>SAF 5</td><td>IgGl</td><td>X</td><td>SAF 60 *</td><td>IgG2b</td><td> 142 - 160</td>

<td>SAF 7</td><td>IgG2a</td><td>X</td><td>SAF 61</td><td>IgG2a</td><td> 142 - 160</td>

<td>SAF 8</td><td>IgGl</td><td>TO</td><td>SAF 63</td><td>IgM</td><td>TO</td>

<td>SAF 9</td><td>IgGl</td><td>TO</td><td>SAF 65</td><td>IgGl (?)</td><td>TO</td>

<td>SAF 10</td><td>IgGl</td><td>TO</td><td>SAF 66</td><td>IgG2a</td><td> 142 - 160</td>

<td>SAF 11</td><td>IgG2a</td><td>X</td><td>SAF 67</td><td>IgGl</td><td>X</td>

<td>SAF 12</td><td>IgM</td><td>TO</td><td>SAF 68</td><td>IgH2a</td><td>TO</td>

<td>SAF 13</td><td>IgM</td><td>TO</td><td>SAF 69 *</td><td>IgG2b</td><td> 142 - 160</td>

<td>SAF 14</td><td>IgGl</td><td>TO</td><td>SAF 70 *</td><td>IgG2b</td><td> 142 - 160</td>

<td>SAF 15 *</td><td>IgG3</td><td> 79 - 92</td><td>SAF 72</td><td>IgG2b</td><td>TO</td>

<td>SAF 21</td><td>IgGl</td><td>X</td><td>SAF 73</td><td>IgGl</td><td>X</td>

<td>SAF 22</td><td> ¿</td><td>TO</td><td>SAF 75</td><td>IgG2a</td><td> 142 - 160</td>

<td>SAF 23</td><td>IgGl</td><td>X</td><td>SAF 76</td><td>IgG2a</td><td> 142 - 160</td>

<td>SAF 24</td><td>IgGl</td><td>TO</td><td>SAF 77</td><td>IeGl</td><td>X</td>

<td>SAF 31 *</td><td>IgG2b</td><td> 79 - 92</td><td>SAF 80</td><td>IgGl</td><td>X</td>

<td>SAF 32 *</td><td>IgG2b</td><td> 79 - 92</td><td>SAF 81</td><td>IgM</td><td>TO</td>

<td>SAF 33 *</td><td>IgG2b</td><td> 79 - 92</td><td>SAF 82</td><td>IgGl</td><td>TO</td>

<td>SAF 34 *</td><td>IgG2a</td><td> 79 - 92</td><td>SAF 83 *</td><td>IgGl</td><td>TO</td>

<td>SAF 35 *</td><td>IgG2b</td><td> 79 - 92</td><td>SAF 84 *</td><td>IgG2b</td><td>TO</td>

<td>SAF 37 *</td><td>IgG2b</td><td> 79 - 92</td><td>SAF 85</td><td>IgM</td><td>TO</td>

<td>SAF 42</td><td>IgGl</td><td>TO</td><td>SAF 91</td><td>IgM</td><td>TO</td>

<td>SAF 44</td><td>IgM</td><td>TO</td><td>SAF 94</td><td>IgM</td><td>TO</td>

<td>SAF 50</td><td>IgM</td><td>TO</td><td>SAF 95</td><td>IGgl</td><td>TO</td>

<td>SAF 51</td><td>IgM</td><td>TO</td><td>SAF 96</td><td>IgM</td><td>TO</td>

<td>SAF 53 *</td><td>IgG2a</td><td>X</td><td></td><td></td><td></td>

- 92: antibody that recognizes peptide 79-92

142 - 160: antibody that recognizes peptide 142 - 160 Epitope X: antibody that only recognizes immobilized SAFs Epitope A: antibodies that recognize peptide 126 - 164 but do not bind Peptide J - 160 ♦ monoclonal antibodies that have demonstrated their ability to recognize the PrPsi of at least one species tested during this study (human, bovine, ovine, hamster rat) within the framework of an immunometric dosage.

Example 2

Detection of PrPres by Western blot

I. Treatment of the sample (i) Preparation of a tissue homogenate from the different biological samples

- ESB cow, sheep scrapie, vMCJ male (type 4), sporadic MCJ male (type 1) and corresponding negative controls

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350 mg of bovine brain was extracted: it is crushed and homogenized at 20% (p / v) in a 5% glucose solution

To perform homogenization, the brain extraction (350 mg) and 1.4 mL of glucose solution are introduced into tubes containing ceramic balls and shaken vigorously (Ribolyser Hybaid).

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

• For sheep: 1/100 • For cow: 1/50 • For man: type 1 or 4: 1/40; type 3: 1/80; type 2 to 1/20 (ii) conditions of stage (1)

A fraction (500 jul) of the 20% homogenate obtained in (i) is incubated with 500 µl of a buffer solution comprising 10% sarkosil (SK 10) of the 10% Triton ® X100 (T10), of the urea 2 M (U2) and proteinase K (PK) at 60 jug / ml of buffer solution (PK 1) for 10 minutes at 37 ° C (corresponding to 30 jug / ml final for a 10% homogenate).

In Figures 3 to 6, PK 3 corresponds to a PK concentration of 180 jug / ml of buffer solution and PK 6 which corresponds to a PK concentration of 360 jug / ml of buffer solution.

(iii) 500 µl of a buffer solution consisting of butanol-1 (corresponding to buffer solutions B, as described in the international patent application WO 99/41280) is added; it is then centrifuged at 15,000 rpm for 5 minutes (approximately 17,000 g).

(iv) the centrifugation residue, containing PrPres, is recovered in 80-100 µl of a buffer solution C, as described in the international patent application WO 99/441280, preferably a Laemmli buffer solution containing 4% of SDS and heated at 100 ° C for 5 minutes to perform a Western blot or recovered, to carry out an immunometric dosage successively in a C1 buffer solution comprising 6 M urea and 0.5% sarkosil, followed by heating for 5 minutes at 100 ° C and then, in a C2 buffer solution comprising 2M guanidine, followed by heating for 5 minutes at 100 ° C.

(v) conditions of step (b)

A second fraction (500 jul) of the 20% homogenate obtained in (i) is incubated with 500 µl of a buffer solution containing 5% sarkosil (SK5), 5% SDS (SDS 5), 1 M urea (U1 ) and proteinase K (PK) at 360 jug / ml (PK 6) for 10 minutes at 37 ° C and then, steps (iii) and (iv) above are performed.

II. Western blot

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

Immunodetection of PrPres is carried out with the monoclonal antibodies SAF 70 and SAF 37 such as those described in example 1 above and the presence of the goat anti-rabbit IGs conjugated with the peroxidase (1/2500). Immunoreactivity is detected by chemiluminescence (ECL. Amlersham), quantified and visualized on autoradiographic films, as illustrated in Figure 2.

In this Figure:

* tracks 1-5 correspond to conditions according to stage (1) or (a) (Sk10 + T10 + U2 + PK 1):

Track 1: negative control

Track 2: ESB cow

Track 3: scrapie sheep Track 4: vMCJ man (type 4)

Track 5: MCJ man (type 1) and

ES 2 280 263 T3 * Tracks 6 to 10 correspond to conditions according to stage (b) (SK 10 + SDS 5 + U1 + PK 6):

Lane 1: negative control Lane 2: ESB cow

Track 3: scrapie sheep Track 4: vMCJ man (type 4)

Track 5: MCJ man (type 1) and

The results obtained show that:

- in stage (1) or (a) (lanes 1 to 5), the PrPsens is systematically destroyed, while the signal obtained with the PrPres is systematically higher, with the antibody directed against the repeated octa-peptide motifs, than signal obtained in the same samples with an antibody directed against the area 142-160 of PrP;

- in step (b) (lanes 6 to 10), PrPsens is systematically destroyed while the signal obtained in lanes 8 and 10 (in the presence of the antibody directed against the octa-peptide repeating motifs) is similar to or greater than that signal obtained in the same samples with an antibody directed against the area 142-160 of PrP, while it is lower and even undetectable in lanes 7 and 9 (PrPres of the ESB).

Example 3

Detection of PrPres with a two-site immunometric dosing using a monoclonal antibody that recognizes repeating octa-peptide motifs as capture antibody

To carry out an immunometric dosing in two places, the residue obtained in (iv) in example 2 is, for example, dissolved in a buffer solution containing sarkosil (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 (1/4 or 1/2), after heating with a buffer solution containing albumin that leads to a final albumin concentration between 0.1 and 1% (p / v) or with a buffer solution containing 1% deoxycholate).

Immunometric dosing in two places is carried out in microtitration plates containing an immobilized antibody under the conditions already described for other proteins (Grassi et al 1989). Its principle is as follows: the PrP analyzed is recognized by an antibody fixed on the solid support (capture antibody) and by a second antibody, which recognizes another part of the molecule, which is labeled with an enzyme (in this case, the acetylcholinestarase, tracer antibody), in 5 Eilman units / ml (1 Eilman unit = 7.35 x 10 ² enzyme units).

Within the framework of the invention, the capture antibody is directed against the repeating octa-peptide motifs and the traced antibody recognizes a sequence within the 94-230 region of PrP, for example, the 142-160 region of PrP. PrP. After washing the solid phase, the enzymatic activity fixed on the plate is proportional to the amount of PrPres possessing the octa-peptide motifs initially repeated in the analyzed sample.

In practice, the dosage is carried out as follows:

100 pl of the PrP solution, to be analyzed, are deposited in the wells of the microtitration plate containing the antibody that recognizes the repeating octa-peptide motifs. After 3 hours of reaction at room temperature, the plate is washed with the addition of 100 µl of a solution of the tracer antibody (5 Eilman units / ml (1 Eilman unit = 7.35 x 10² enzyme units). After one night of reaction at + 4 ° C, the plates are washed again before the addition of 200 μl of a substrate solution (reagent of Eilman, Grassi et al 1989) that will allow to measure the activity of acetyl - choline esterase 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: stage (1) or (a) and stage (b) The samples are prepared as described in example 2.

The homogenates are prepared as described in example 2. - in the case of man

ES 2 280 263 T3 Figures 3 and 4 illustrate the results obtained with different buffer solutions:

* conditions according to step (1) or (a) of the process according to the invention:

I: 20% homogenized + SK 10 + T10 + T10 + U2 + PK 1

III ': 10% homogenate + SK 5 + T5 + U1 + PK 0.5 (Figure 4) * 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 'homogenate at 10% + SK5 + T5 + U1 + PF6 (Figure 4)

IV: 20% homogenate + SK20 + PK3

V: homogenized at 20% + SK20 + U1 + PK3

VI: 20% homogenate + SK20 + U2 + PK3

VII: 10% homogenate + SK20 + PK3

VIII: 10% homogenate + SK20 + U1 + PK6

X: 10% homogenate + SK5 + SDS5 + U1 + PK6

X ': 10% homogenate + SK2.5 + SDS 2.5 + U2 + PK6 (Figure 4)

XI: 20% homogenized + SK20 + PK6

XII: homogenate at 20% + SK20 + U1 + PK6 XIII: homogenate at 20% + SK20 + U2 + PK6 XIV: homogenate at 10% + SK20 + PK6 XV: homogenate at 10% + SK20 + U1 + PK6 XVI: homogenate at 10 % + SK20 + U2 + PK6

The amount of PrPres that has conserved the octa-repeating peptide motif is measured with the aid of the immunometric dose in two places (absorbance or OD at 414 nm, see example 3; the appearance of a yellow coloration is measured, Eilman's reagent) : negative control (□), sporadic MCJ type 1 (□) and vMCJ type 4 ().

- in the case of ruminants

Figures 5 and 6 illustrate the results obtained with different buffer solutions:

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

I: 20% homogenized + SK10 + T10 + U2 + PK1

III ': 10% homogenate + SK5 + T5 + U1 + PK0.5 (Figure 6) * conditions according to step (b) of the process according to the invention:

II: 20% homogenate + SL10 + T10 + U2 + PK3

III: 20% homogenate + SK10 + T10 + U2 + PK6

III ”: 10% homogenate + Sk5 + t5 + u1 + PK3 (Figure 6)

IV: 20% homogenate + SK20 + PK3

IS 2 280 263 T3

V: homogenized at 20% + SK20 + U1 + PK3

VI: 20% homogenate + SK20 + U2 + PK3

VII: 10% homogenate + Sk20 + PK3

VIII: 10% homogenate + SK20 + U1 + PK3

IX: 10% homogenate + SK20 - U2 + PK3

X: 10% homogenate + SK5 - SDS5 + U1 - PK6

X ': 10% homogenate + SK5 + SDS5 + U1 + PK3 (Figure 5)

XI: 20% homogenized + SK20 + PK6

XII: 20% homogenate + SK20 + U1 + PK6 XIII: 20% homogenate + SK20 + U2 + PK6 XIV: 10% homogenate + SK 20 + PK6 XV: 10% homogenate + 'SK20 + U1 + PK6 XVI: 10% homogenized + SK20 + U2 + PK6

The amount of PrPres that has conserved the octa-repeating peptide motif is measured with the help of the immunometric dose in two places (absorbance or OD at 414 nm, see example 3 negative control (□) bovine CNTA (□) and ovine CNTA ( ).

- Figures 7 (Western blot) and 8 (immunometric dosing in two places):

• The comparison is made from 20% homogenates of healthy sheep brains, sheep awaiting scrapie and ESB bovine brains, obtained under the conditions set out in example 2. • Treatment of the samples:

A: 10% sarkosil + 10% Triton + 2 M urea + 60 pg / mL proteinase K, 10 minutes B: 10% sarkosil + 2 M urea + 240 pg / mL proteinase K, 10 minutes C: 10% sarkosil + proteinase K 240 pg / ml, 10 minutes • Detection

By Western blot (Figure 7): SAF 37 and SAF 84 antibody (see example 1)

By immunometric (Figure 8): capture by SAF 37 and revelation by an antibody directed against the 94-230 zone of a PrP.

- Figures 9 (Western blot) and 10 (immunometric dosing in two places):

The comparison is made from the 20% homogenates of healthy sheep brains, sheep waiting for scrapie and ESB bovine brains, obtained, under the conditions set out in example 2:

Sample treatment:

A: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 60 pg / ml, 10 minutes

B: 10% SDS + 5% Triton® + 2M urea + 180 pg / ml proteinase K, 10 minutes.

• Detection in the same conditions as above

The increase only in the dose of PK allows a differential sensitivity to appear in the area of ​​the repeated octa-peptide motifs, between ESB and scrapie in the case of sheep, but not in the case of man (between type 1 and type 4).

IS 2 280 263 T3

On the contrary, by also modifying the composition in surfactant and chaotropic agent, this allows a differential sensitivity to appear in the area of ​​the octa-peptide motifs repeated in all cases. Example 5

Influence of the composition of the buffer solutions on the differential detection of ESB and scrapie

- Figures 11 (Western blot) and 12 (immunometric dosing in two places):

The comparison is made from the 10% homogenates of brains of healthy mice or mice experimentally infected with the strain C506M3 (scrapie) or with the strain 6PB1 (ESB) obtained, under the conditions indicated in example 3.

Sample treatment:

A: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 30 pg / ml, 10 minutes

B: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 60 pg / ml, 10 minutes

C: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 180 pg / ml, 10 minutes

D: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 360 pg / ml, 10 minutes

E: 10% sarkosil + 2M urea + proteinase K 180 pg / ml, 10 minutes

Detection

By Western blot (Figure 11): SAF 37 and SAF 70 antibody (see example 1)

By immunometry (Figure 12): capture by SAF 37 and revelation by an antibody directed against the 94-230 region of PrP.

The results obtained show that increasing the dose only in PK allows a differential sensitivity to appear in the area of ​​the octa-peptide repeating motifs between ESB and scrapie, in the case of mice.

Example 6

Influence of buffer solutions and proteinase K concentration on the detection of the different MCJ

- Figures 13 (Western blot) and 14 (immunometric dosing in two places):

The comparison is made from 10% homogenates of brains of healthy men and of men reached by MCJ (types 1, 2, 3 and 4) obtained under the conditions set out in example 3.

Sample treatment:

A: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 30 pg / ml 10 minutes

B: sarkosil 10% + Triton® 10% + urea 2 M + proteinase K 180 pg / ml 10 minutes

C: sarkosil 10% + proteinase K 30 pg / ml 10 minutes

D: sarkosil 10% + proteinase K 60 pg / ml 10 minutes

E: sarkosil 10% + proteinase K 180 pg / ml 10 minutes

F: sarkosil 10% + proteinase K 360 pg / ml 10 minutes

Detection

By Western blot (Figure 13): SAF 37 and SAF 70 antibody (see example 1)

By immunometry (Figure 14): capture by SAF 37 and revelation by an antibody directed against the 94-230 region of PrP.

IS 2 280 263 T3

- Figures 15 (Western blot) and 16 (immunometry)

The comparison is made from 10% homogenates of brains of healthy men and of men reached by MCJ (types 1, 2, 3 and 4) obtained under the conditions set out in example 2.

Sample treatment:

Figure 15: digestion by means of proteinase K (150 // g / ml) 10 minutes

Figure 16: 10% sarkosil + 2M urea + proteinase K (30 to 360 jug / ml for 10 minutes)

Detection

By Western blot (Figure 15): SAF 37 antibody and an antibody directed against the 94-230 area of ​​PrP (see example 1)

By immunometry (Figure 16): capture by SAF 37 and revelation by an antibody directed against the 94-230 region of PrP.

The results obtained show that increasing the dose in PK allows a differential sensitivity to appear in the area of ​​the octa-peptide repeating motifs in the different types of MCJ. Under these conditions, there is no significant difference between type 1 and type 4; changing the composition into surfactant and attentive chaotrope, with the same dose of PK (E, Figures 13 and 14) allows the octa-peptide to be destroyed in type 4 while preserving them in type 1.

Furthermore, Figure 16 shows the difference in sensitivity of PrPres from different strains, for the same buffer solution, as a function of the dose in PK.

On the other hand, Figure 15 shows that a direct treatment of the homogenate using PK reveals another type of sensitivity of PrPres to degradation.

Example 7

Western blot detection of digested and purified PrPres in the form of SAF

The homogenates, obtained under the conditions according to example 2, are treated as follows:

Homogenate 20% (500 Jul) + NaCl 20% (500 //.l) + [Sarkosil 20% + SB314 2%] (500 Jul) + PK (20 jug / ml final) for 1 hour.

Figure 17 illustrates the results: byd:

lanes 1-7: results obtained with different dilutions of scrapie strains C506M3 in the case of mice (dilutions 1/20, 1/50, 1/250, 1/500, 1/1000 and 1/2000)

Track 8: molecular weights

Lanes 9-15: results obtained with different dilutions of strain ESB in the case of mice (dilutions from 1/1000 to 1/10).

It is possible to completely eliminate the ESB a and c signal: the results obtained confirm that there is a significant increase in the signal in the presence of antibodies directed against the repeating octa-peptide motifs.

e: this Figure shows that it is possible to obtain a decrease in the signal with ESB in both monkeys and humans (tracks 4 and 7)

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