Patent Grant
7429463
The present invention relates to a method for diagnosing a TSSE caused by a UTA strain, in a biological sample, by detecting PrP-res, and also to the use thereof in the context of differential diagnosis of the various UTA strains in a biological sample.
Transmissible spongiform subacute encephalopathies (TSSEs) are caused by unconventional transmissible agents (UTAs), also called prions, the precise nature of which to date remains disputed. TSSEs essentially comprise Creutzfeldt-Jakob disease in humans (CJD), scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) in bovines. Other encephalopathies have been demonstrated in the Felidae, in mink or certain wild animals, such as deer or elk.
These diseases are always fatal and, at the current time, there is no effective treatment.
In TSSEs, there is an accumulation of a host's protein, PrP (or prion protein), in an abnormal form (PrP-res), mainly in the central nervous system; PrP-res copurified with infectiousness and accumulation thereof precedes the appearance of histological lesions. In vitro, it is toxic for cultures of neurons.
The two isoforms of PrP have the same amino acid sequence (see
Two biochemical properties make it possible, in general, to distinguish these two isoforms.
The normal form of the prion protein (PrP-sen) is, in principle, completely degraded by proteases and is entirely soluble in the presence of nonionic detergents.
The peptide sequences of hamster, human, bovine and ovine PrP-sen are presented in
For detecting the presence of the infectious agent, the most recent methods are based on selective detection of the abnormal PrP (PrP-res) linked to the infectious agent, by taking advantage of its partial resistance to proteases.
It is possible to distinguish:
Among these various tests, which involve treating the tissue extracts with a protease, mention may be made of:
In general, these various tests have the drawback of lacking sensitivity and thus of causing false-negatives.
Other methods propose treating the sample with denaturing products (Oesch et al., 1994 and 1999; WO 00/22438, The Reagents of the University of California), a limited treatment with proteinase K (WO 00/29850, Wallac Oy et al.) or treatment with a metallopeptidase (WO 00/22438), which make hidden antigenic sites accessible, which sites can be detected with the monoclonal antibody 3F4 which recognizes region 109-112 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, due, in particular, to incomplete elimination of PrP-sen under the recommended treatment conditions, while the method described in WO 00/22438, The Regents of the University of California, lacks sensitivity due to the use of the detecting antibody 3F4 (WO 00/22438), which binds to only one motif on the protein.
The applicant has recently provided a test for the quantitative detection of PrP-res, which comprises a purification step which leads to significantly more sensitive detection and which represents a great advance for medical monitoring and assaying PrP-res in abattoirs. This method is, in particular, described in PCT international application WO 99/41280 and in a preliminary report of Directorate General XXIV of the European Commission (consumer policy and consumer health protection; http://europa.eu.int/comm/dg24/health/).
However, given the need for a particularly reliable test for diagnosing TSSEs, the applicant has continued its studies.
It has in particular established that, in order to produce a detection test:
(i) which is sensitive, i.e. which has the ability to correctly identify uninfected animals,
(ii) which is specific, i.e. which has the ability to correctly identify infected animals exhibiting. clinical symptoms,
(iii) which has as low a detection limit as possible, i.e. which can allow detection of small amounts of PrP-res (detection of PrP-res before the appearance of clinical symptoms), and
(iv) which is reproducible,
the biological sample to be analyzed must be treated under conditions which make it possible to conserve all or some of the octapeptide motifs exclusively in PrP-res.
It has been found in particular that, in order to effectively satisfy the four conditions (i)-(iv) stated above, it is necessary to define precise conditions for treating the sample to be analyzed, which completely eliminate PrP-sen from the sample, while at the same time allowing specific capture of PrP-res, under the defined conditions.
A subject of the present invention is a method for diagnosing (method A) a TSSE or prion disease caused by a UTA strain, by detecting PrP-res in a biological sample characterized in that it comprises:
(1) treating said sample with at least one proteinase K (PK) in such a way as to completely degrade PrP-sen while at the same time conserving all or some of the octapeptide motif repeats of PrP-res, whatever the UTA strain; preferably, said PK treatment is carried out at a concentration of between 30 μg/ml and 200 μg/ml for 10 minutes at 37° C. for a homogenate at 10% (biological sample in the form of a homogenate in a suitable buffer), or for a period and at a concentration which are equivalent to the concentration of between 30 μg/ml and 200 μg/ml for 10 minutes at 37° C. for a homogenate at 10%.
(2) bringing said treated sample into contact with a ligand for said octapeptide motifs, in particular antibodies directed against said. octapeptide motif repeats, and.
(3) detecting the possible presence of the octapeptide motif repeats/ligand complex.
According to an advantageous embodiment of the method, the treatment with said proteinase K is between 30 seconds and 2 hours, at a temperature of less than 80° C., preferably between 10 minutes and 30 minutes at 37° C., at the abovementioned concentrations, and even more preferably for 10 minutes at a concentration of between 30 μg/ml and 200 μg/ml for a homogenate at 10% (final concentration) or for 30 minutes at a concentration of between 10 μg/ml and 70 μg/ml for a homogenate at 10% (final concentration) or at a concentration of between 25 μg/ml and at 175 μg/ml for a homogenate at 25% (final concentration).
It should be noted a certain number of parameters are closely related to one another: the concentration of proteinase K depends directly on the duration of treatment (incubation time) of the sample; it may be considered that, at 37° C. for example, a 10 minute incubation with a PK at a concentration of between 30 and 200 μg/ml of homogenate at 10% is equivalent to a 30 minute incubation with a PK at a concentration of between 10 and 70 μg/ml of homogenate at 10%. For example, an incubation for 30 minutes with a PK at 25 μg/ml is equivalent to a 10 minute incubation with a PK at a concentration of 75 μg/ml.
Whatever the concentration of PK and the duration of incubation, the sample treated should no longer contain undegraded PrP-sen, whereas PrP-res, possibly present, has conserved all or some of the octapeptide motifs.
Other parameters may be involved, to a lesser degree, (i.e. in a nonessential way) in establishing the active concentration of PK: this involves, in particular, the buffer in which said PK is dissolved; when the PK is dissolved in the sample homogenization buffer, depending on the homogenization buffer used, the minimum concentration of PK may vary within the range of concentrations specified above:
Advantageously, the PK can be used in a buffer which is 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 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:
Such has test has the advantage of being particularly suitable for the differential diagnosis of the TSSEs in the same host, caused by various UTA strains, and in particular for the differential diagnosis of BSE strains compared to the conventional strains of scrapie in sheep.
It has, for example, been shown that the BSE strain has infected humans in Great Britain, by analyzing the electrophoretic profiles of the the PrP-res, which has shown differences in migration depending on the UTA strains; a characteristic profile (type 4) has been found in patients who develop a new variant of Creutzfeld-Jacob disease (vCJD), which appears to be linked to the passing of cattle infected with the BSE agent into human food (Collinge et al., Nature, 1996). A similar profile has been observed in a macaque infected with the BSE agent (Lasmezas et al., 1996) and a cat probably contaminated with this agent (Priola et al., Nature Med., 1996).
However, it is a long method which is tricky to implement if reproducible results are desired, which probably constitutes an element of the controversy which exists regarding the classification of the various types of PrP-res 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, although among ovines, there exists an endemic disease, scrapie, another TSSE, which has close and undifferentiatable clinical and histological characteristics. The only reference method for differential diagnosis between the BSE agent and the scrapie agent is inoculating mice and studying the lesional profile, which necessitates a wait of approximately two years, and which has been performed in Great Britain only on 9 sheep for a population of several tens of millions of heads of sheep.
First attempts have been made to try to distinguish the various UTA strains in a sample; Kuczius T. et al., 1999, had considered that the variations observed between the various TSSE strains (scrapie, BSE and CJD) using the glycotyping technique do not make it possible to distinguish each strain; they have selected other biochemical and biological markers for PrP-res which make it possible to distinguish the various prion strains from one another more clearly. The analytical parameters which they have used are as follows: long-term resistance to proteinase K, molecular mass of the PrP-res, topology and amount of the deposits of PrP-res. The results obtained show that the PrPs-res of various strains of BSE and of scrapie exhibit significant differences in their long-term resistance to proteinase K. The resistance to PK varies depending on the strain of scrapie: low resistance: Chandler strain; intermediate resistance: 22A strain; relative stability: 87V strain. Under the same conditions, the BSE strains exhibit intermediate resistance. Although glycotyping does not make it possible to distinguish between scrapie strain 87V and the BSE strains, these two types of strains are clearly distinguishable after long-term treatment with PK. However, these are not protocols which are sufficiently reliable and which can be used on a large scale and in the field.
In this context, it is important to have a reliable, sensitive method for detecting PrP-res which allows differential diagnosis, is relatively inexpensive and easy to carry out, using a biological sample, such as a tissue sample.
Consequently, a subject of the present invention is also a method for the differential diagnosis (method B) of TSSEs caused by UTA strains, in a biological sample, by detecting the PrPs-res associated with the various UTA strains, characterized in that it comprises:
(a) detecting PrP-res in a first fraction of said sample, in accordance with steps (1) to (3) of the method for diagnosing a UTA strain as defined above (method A), and then:
(b) for each sample for which the presence of an octapeptide motif repeats/ligand complex is detected in step (a):
A subject of the present invention is also a method for the differential diagnosis (variant of method B) of TSSEs caused by UTA strains, in a biological sample considered to contain PrP-res (detection tests carried out by any method), characterized in that it comprises, for each sample for which the presence of a PrP-res has been detected:
In the context of the differential diagnosis (method B), besides the concentration of PK, other conditions may possibly have an effect on the degradation of the octapeptide motifs, depending on the strain: specifically, when the PK is used in a buffer which is different from the homogenization buffer, such as 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 octapeptide motifs degraded may vary depending on the composition of said buffer and on the concentration of the various agents.
The term “strain of interest” is intended to mean the UTA strain(s), in particular the BSE strain, for which the intention is to eliminate the octapeptide motif repeats, for example.
One of the main characteristics of methods A and B is to exploit the conditions under which the protease treatment is carried out, so as to control the degradation of the octapeptide motif repeats. Depending on the use envisaged, the method will be carried out such that these motifs are conserved (method A) or destroyed (method B):
Surprisingly, under the conditions according to step (1) or step (a), the PK does not lead to the cleavage of all or some of the octapeptide motif repeats of the PrPs-res associated with all UTA strains or prions, whereas the conditions of step (b) lead to the cleavage of the octapeptide motif repeats of the PrP-res associated with the strain of interest, while all the PrPs-res associated with the other UTA strains conserve all or some of said octapeptide motif repeats.
Also surprisingly, such a test, in particular when the ligand is an antibody, has the following advantages:
Such tests therefore in particular find an application in the investigation of the contamination of sheep with the BSE strain, which is not usually possible to distinguish from the conventional strains of scrapie.
According to an advantageous embodiment of said methods, the PK is dissolved in a buffer which preferably comprises:
a. at least one surfactant, selected from the group consisting of:
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 the salts of metals which may or may not be alkali metals.
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 octapeptide motif repeats.
A subject of the present invention is also a diagnostic kit for carrying out 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 one salt and a protease, as defined above.
The octapeptide motif repeats/antibody complex (when the ligand is an antibody) is detected by standard immunological methods.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
Besides the above arrangements, the invention also comprises other arrangements which will emerge from the following description, which refers to examples of implementation of the method which is the subject of the present invention and also to the appended drawings, in which:
It should be clearly understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation.
Synthesis and labeling of the peptide
A peptide representative of the PrP octapeptide motif repeat, for example the motif G-G-W-G-Q-P-H-G-G-G-W-G-Q-G-(NH2) (SEQ ID NO:2), corresponding to sequence 79-92 of the human PrP, was synthesized using an automatic synthesizer (Milligen 9050, Waters, Milford, Mich.). The peptide was covalently coupled to acetylcholinesterase (AchE) via a heterobifunctional reagent, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxlate (SMCC, Calbiochem, France), as described previously for other peptides or proteins (McLaughlin et al., 1987, Grassi et al., 1989). This method involves reacting a thiol group introduced into the peptide with the maleimide function which was attached to the AchE by reaction with the 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 the AchE-SMCC with an excess of thiolated peptide.
Immunization and Preparation of Monoclonal Antibodies
A preparation of scrapie-associated fibrils (SAFs, PrP-res preparation) was obtained from infected hamster brains (263K scrapie strain) as described previously (Lasmezas et al., 1997). This preparation was inactivated by treatment with formic acid, before immunization of the mice. PrP knockout mice (in which the PrP gene has been eliminated) (PrP0/0 mice) were immunized with these SAF preparations and hybridoma cells were prepared as previously described (Grassi et al., 1988, 1989). The culture supernatants were screened as described above. It was possible to identify and stabilize 57 hybridomas; they were named SAF-1 to SAF-90. All these antibodies proved to recognize the SAFs immobilized on the microtitration plates, while a minority of them demonstrated an ability to recognize peptide-AchE conjugates. Among the latter, seven clearly recognize the octapeptide motif repeat (peptide 79-92); they are the antibodies SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35 and SAF-37. The list of antibodies obtained, and also the main characteristics thereof, are given in the table below. After cloning and expansion in the form of ascites 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 antibody was determined by radial immunodiffusuion according to the Ouchterlony technique.
Screening of Hybridoma Culture Supernatants
The presence of a PrP-specific antibody in the hybridoma culture supernatants was demonstrated in two ways, by testing their ability to bind either peptide-AchE conjugates or hamster SAFs. In the first case, the screening was performed in plates containing an anti-mouse IgG antibody immobilized as previously described (Créminon et al., 1993, Frobert et al., 1991). In summary, 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 the plates had been washed, 200 μl of Ellman reagent (Ellman et al., 1961) were added to the wells in order to detect the presence of AchE attached to the solid phase. In the second case, plates containing an immobilized SAF preparation were prepared by reacting 50 μl of a 2 μg/ml solution in a 0.05 M phosphate buffer, pH 7.4, overnight at room temperature. After washing, the plates were saturated with the EIA buffer (100 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, 0.1% of bovine serum albumin (BSA) and 0.01% sodium azide) overnight at +4° C. and were kept at this temperature until they were used. The binding of the monoclonal antibodies to the immobilized SAFs was revealed using goat anti-mouse IgG antibodies labeled with AchE as previously described (Negroni et. al., 1998).
I: Treatment of the Sample
(i) Preparation of a Tissue Homogenate from the Various biological samples
350 mg of bovine brain are taken: it is ground and homogenized at 20% (w/v) in a 5% glucose solution.
To perform the homogenization, the brain sample (350 mg) and 1.4 ml of glucose solution are introduced into tubes comprising ceramic beads, and vigorously agitated (Hybaid Ribolyser).
The positive samples were diluted in a homogenate originating from healthy brains of the corresponding species, as follows:
(ii) Conditions for Step (I)
A first fraction (500 μl) of homogenate at 20% obtained in (i) is incubated with 500 μl of a buffer comprising 10% sarkosyl (SK10), 10% Triton X100 (T10), 2M urea (U2) and proteinase K (PK) at 60 μg/ml of buffer (PK1), for 10 minutes at 37° C. (corresponding to a final concentration of 30 μg/ml for a homogenate at 10%).
In
(iii) 500 μl of a buffer consisting of 1-butanol (corresponding to buffers B, as described in international application WO 99/41280) are added; the mixture is centrifuged at 15 000 rpm for 5 min (approximately 17 000 g).
(iv) The centrifugation pellet, which contains the PrP-res, is taken up in 80-100 μl of a buffer C as described in international application WO 99/41280, preferably a Laemmli buffer containing 4% of SDS, and heated at 100° C. for 5 min, in order to carry out Western blotting, or taken up successively in a buffer C1 comprising 6M urea and 0.5% sarkosyl, followed by heating for 5 min at 100° C., and then in a buffer C2 comprising 2M guanidine, followed by heating for 5 min at 100° C., in order to perform an immunometric assay.
(v) Conditions for step (b)
A second fraction (500 μl) of homogenate at 20% obtained in (i) is incubated with 500 μl of a buffer comprising 5% sarkosyl (SK5), 5% SDS (SDS5), 1M urea (U1) and proteinase K (PK) at 180 μg/ml (PK6) for 10 min at 37° C., and then steps (iii) and (iv) above are carried out.
II. Western Blotting
The samples obtained are used to perform SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane under the conditions set out in example 1 and in example 3 of the abovementioned international application WO 99/41280.
Immunodetection of the PrP-res is carried out with the monoclonal antibodies SAF70 and SAF37 as described in example 1, above, and peroxidase-conjugated anti-rabbit goat Igs (1/2 500). The immunoreactivity is revealed by chemiluminescence (ECL, Amersham), quantified and visualized on autoradiographic films, as illustrated in
In this figure:
The results obtained show that:
To carry out a two-site immunometric assay, 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 ¼ or to ½), after heating, with a buffer containing albumin, producing a final albumin concentration of between 0.1 and 1% (w/v), or with a buffer containing 1% deoxycholate.
The two-site immunometric assay is performed in microtitration plates containing an antibody which has been immobilized under the conditions already described for other proteins (Grassi et al., 1989). The principle thereof is as follows: the PrP analyzed is recognized by an antibody attached to the solid support (capture antibody) and by a second antibody, which recognizes another part of the molecule, and which is labeled with an enzyme (in this case, acetylcholinesterase, tracer antibody), at 5 Ellman units/ml.
In the context of the invention, the capture antibody is directed against the octapeptide motif repeats and the tracer antibody recognizes a sequence included in region 94-230 of the PrP, for example region 142-160 of the PrP. After washing the solid phase, the enzymatic activity attached to the plate is proportional to the amount of PrP-res having the octapeptide motif repeats initially in the sample analyzed.
In practice, the assay is performed in the following way:
100 μl of the solution of PrP to be analyzed are deposited into the wells of the microtitration plate containing the antibody which recognizes the octapeptide motif repeats. After reaction for 3 hours at room temperature, the plate is washed before adding 100 μl of a solution of the tracer antibody (5 Ellman units ml). After overnight reaction at +4° C., the plates are washed again before adding 200 μl of a solution of substrate (Ellman reagent, Grassi et al., 1989) which will make it possible to measure the activity of the acetylcholinesterase attached to the solid phase. After 30 minutes of enzymatic reaction, the absorbence (N.D. at 414 nm) of each well is measured.
The samples are prepared as described in example 2.
The homogenates are prepared as in example 2.
In Humans
The amount of PrP-res having conserved the octapeptide motif repeat is measured using the two-site immunometric assay (absorbence or O.D. at 414 nm, see example 3; the appearance of a yellow coloration, Ellman reagent, is measured): negative control (□) sporadic CJD type 1 (□) and vCJD type 4 (▪)
In Ruminants
The amount of PrP-res having conserved the octapeptide motif repeat is measured using the two-site immunometric assay (absorbence or O.D. at 414 nm, see example 3); negative control (□), bovine UTA (□) and ovine UTA (▪).
under the same conditions as above.
Increasing only the dose of PK makes it possible to reveal a differential sensitivity of the region of octapeptide motif repeats 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 reveal a differential sensitivity of the region of octapeptide motif repeats in all cases.
by Western blotting (
by immunometric analysis (
The results obtained show that increasing only the dose of PK makes it possible to reveal a differential sensitivity of the region of octapeptide motif repeats between BSE and scrapie, in mice.
by Western blotting (
by immunometric analysis (
Detection
by Western blotting (
by immunometric analysis (
The results obtained show that increasing the dose of PK makes it possible to reveal a differential sensitivity of the region of octapeptide motif repeats in the various types of CJD. Under these conditions, no significant difference exists between type 1 and type 4; changing the composition of surfactant and of chaotropic agent, at the same dose of PK (E,
In addition,
Moreover,
Homogenates, obtained under the conditions according to example 2, are treated as follows:
20% homogenate (500 μl)+20% NaCl (500 μl)+[20% sarkosyl+2% SB314] (500 μl)+PK (20 μg/ml final concentration) for 1 hour.
b and d:
It is possible to entirely eliminate the BSE signal.
a and c: the results obtained confirm that there is a significant increase in the signal in the presence of antibodies directed against the octapeptide motif repeats.
e: this figure shows that it is possible to obtain a decrease in the signal with BSE, both in monkeys and in humans (lanes 4 and 7).
As emerges from the above, the invention is in no way limited to its methods of implementation, preparation and application which have just been described more explicitly; on the contrary, it encompasses all the variants thereof which may occur to a person skilled in the art, without departing from the context or scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 99 14242 | Nov 1999 | FR | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5733734 | Trojanowski et al. | Mar 1998 | A |
| 5773253 | Linsley et al. | Jun 1998 | A |
| 5876712 | Cheever et al. | Mar 1999 | A |
| 5877012 | Estruch et al. | Mar 1999 | A |
| Number | Date | Country |
|---|---|---|
| 0 861 900 | Sep 1998 | EP |
| 10 267928 | Oct 1998 | JP |
| 98 30909 | Jul 1998 | WO |
| 99 41280 | Aug 1999 | WO |
| WO 9941280 | Aug 1999 | WO |
| 99 66956 | Dec 1999 | WO |
| 00 22438 | Apr 2000 | WO |
| 00 26238 | May 2000 | WO |
| 00 29849 | May 2000 | WO |
| 00 29850 | May 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060084130 A1 | Apr 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10129111 | US | |
| Child | 11224951 | US |
