Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2828—Prion diseases

Definitions

• the present invention relates to a method for in vitro determination of infectivity related to unconventional transmissible agents (NCTA), its application, in particular to a method of evaluation and / or in vitro control of the efficacy of a method of obtaining or treating a biological product or method for evaluating and / or controlling in vitro a decontamination procedure or an in vitro evaluation method of the ability of a compound to modulate the infectivity associated with NCTA.
• NCTA unconventional transmissible agents
• TSEs Transmissible spongiform encephalopathies
• CNS central nervous system
• PrP prion protein
• PrPsc is co-purified with infectivity and its accumulation precedes the appearance of histological lesions. It results from a modification of the conformation of the prion protein PrP. There has been no evidence of altering the expression of the gene encoding PrP or altering its translation (Prusiner, Biochemistry 1992; 31: 12277-88).
• NCTAs prevents the use of inactivation processes conventionally used, such as the Tween-TNBP solvent / detergent treatment, which have proved their effectiveness in reducing the viral load of blood derivatives.
• inactivation processes conventionally used such as the Tween-TNBP solvent / detergent treatment, which have proved their effectiveness in reducing the viral load of blood derivatives.
• cryoprecipitable plasma proteins factor VIII, von
• the method of titration of the infectivity associated with NCTAs conventionally used uses an in vivo titration method (for example the golden hamster to titrate infectivity related to the strain 263k), by intracerebral injection of different dilutions of a test product loaded with ATNC.
• an in vivo titration method for example the golden hamster to titrate infectivity related to the strain 263k
• intracerebral injection of different dilutions of a test product loaded with ATNC Depending on the number of affected animals in the different groups corresponding to the dilutions made, one can calculate an infectious titer and establish the reduction factor of a given method from an untreated reference.
• this method has the disadvantage of being long (about one year), expensive and not compatible with a development on an industrial scale which requires a result on the effectiveness of the elimination of the prion, as quickly as possible.
• PrPsc by Western-Blot (MacGregor, Transfusion J. Medicine 2001; 11: 3-14) or by ELISA generally require prior digestion. of the sample to be analyzed by Proteinase K, or denaturation by chaotropic agents to distinguish the pathological protein (PrPsc) from the normal protein (PrP).
• WO 2005/022148 discloses an in vitro titration method, called "tissue culture infectivity assay” (TCIA), of an ATNC in a biological product, by means of bringing into contact stable transgenic cells supporting replication said ATNC with the biological product, and then culturing these cells during one or more passages to amplify the amount of ATNC present in the biological product by replicating the ATNC.
• TCIA tissue culture infectivity assay
• the TCIA consists of bringing together successive dilutions of an infectious homogenate (127-S scrapie strain) with cells in multiwell plate culture, at the rate of 5 wells per dilution. The number of positive wells is then evaluated by detection of PrPsc (indissociable marker of infectivity) after 8 to 10 passages, and the titre determined by the Spearman - Karber method.
• PMCA Protein misfolding cyclic amplification
• TCIA and PMCA processes nevertheless have disadvantages.
• the in vitro TCIA titration method is still long (10 weeks) for routine use.
• it is currently slightly less sensitive than the bioassay method involving infection of a laboratory animal.
• the PMCA method although still in development, is at least as sensitive as the bioassay. However, it provides no evidence on the infectivity associated with the detected PrPsc and proves difficult to implement with plasma matrices.
• the invention now provides an improved in vitro method for detecting and / or in vitro titrating an unconventional transmissible agent (ATNC) or a pathological conformation protein, a marker of ATNC-related infectivity, in a sample.
• the method of the invention comprises, in a cell system in vitro culture, the replication or propagation, in cells in culture or on their surface, of the ATNC present in the sample, then the repeated incubation with a substrate allowing the amplification of the ATNC or the pathological conformation protein, prior to determining the presence and / or amount of the ATNC or pathological conformation protein in the sample.
• the inventors have indeed demonstrated that it is possible, by associating the product resulting from the culturing of the cells with a substrate source for the pathological conformation protein (the substrate may contain, for example PrP) and / or the ATNC, to convert this substrate to pathological conformational protein (PrPsc) and / or detectable and quantifiable ATNC.
• the substrate may contain, for example PrP
• PrPsc pathological conformational protein
• the invention relates to an in vitro method for detecting and / or titrating an unconventional transmissible agent (ATNC) or a pathological marker protein of the ATNC infectivity in a sample, comprising the steps of: i) contacting said sample with cells supporting replication or propagation of the ATNC, ii) culturing said cells to replicate or propagate ATNC present in said sample, iii) contacting and incubating the ATNC; ATNC with a substrate source for the pathological conformational protein and / or the ATNC, whereby the amount of pathological conformational protein and / or ATNC is amplified, iv) disaggregating the eventually formed aggregates, v) determining the presence and / or amount of pathological conformational protein and / or ATNC in the sample, wherein steps (iii) and (iv) constitute a cycle of operations that is repeated at least two times before step (v).
• ATNC unconventional transmissible agent
• the sample is selected from the group consisting of blood products and their derivatives, foods, and cosmetics
• the invention also relates to an in vitro method of evaluating and / or controlling a process for obtaining or treating a biological product or a material likely to be contaminated by an NCTA, a method in which applies to said biological or material product, (A) upstream and (B) downstream of said process, a titration method as defined above, and in that the two values (A) and (B) are compared of title obtained.
• Another subject of the invention concerns an in vitro method for evaluating and / or controlling a procedure for decontaminating a biological product or a material, in which method is applied to said biological or material product, (A) ) upstream and (B) downstream of said procedure, a titration method as defined above, and in that the two obtained values (A) and (B) of title are compared.
• Yet another subject of the invention is an in vitro method for diagnosing a transmissible spongiform encephalopathy in a human or non-human animal, comprising detecting the presence in a biological sample of said subject of a non-transmissible transmissible agent. conventional (ATNC) or a pathological conformation protein, marker of the infectivity of the NCTA, by the method as defined above.
• Another subject of the invention is an in vitro method for evaluating the capacity of a compound to modulate (ie inhibit or increase) the infectivity or replication or propagation of an ATNC.
• the detection method of the invention makes it possible to detect amounts of ATNC at least equivalent to those detected by the methods known from the prior art, including the bioassay method, considered as the reference method. Moreover, it makes it possible to obtain results more quickly than the known methods of the prior art.
• the Figure is an autoradiograph of a gel showing the detection of PrP sc by the method of the invention.
• the tracks are as follows:
• the term "ATNC" represents any unconventional transmissible agent, such as those responsible in humans for familial or sporadic CJD, Kuru disease or variant CJD, or those officials in natural TSE animals, such as ovine scrapie, bovine or feline spongiform encephalopathy, chronic wasting of deer or spongiform encephalopathy of mink, or EST strains experimentally adapted to laboratory animals .
• PrPsc is here used to denote the prion protein of pathological conformation (or pathological conformation).
• pathological form of PrP is the form of the protein whose conformation is correlated with the appearance of an EST in the infected human or non-human animal.
• the ATNC is also referred to as "infectious agent".
• the NCTA titrated by the method according to the invention is preferably of ovine, bovine, murine, cricetid, cervid, primate or human origin.
• the ATNC may be the pathological conformation protein itself.
• sample means any source of material that may be contaminated by a NCTA.
• a source of material may for example be a liquid, a food product, a beverage, a cosmetic product or a product resulting from genetic engineering, a molecule capable of inhibiting the infectivity of an ATNC, this list not being limiting.
• a biological sample for example a biological fluid or tissue or tissue extract.
• the method of the invention can be carried out on homogenates or directly on samples ex vivo.
• a tissue may be a tissue of the brain, vertebral column, or tonscillary tissue, this list not being limiting.
• the sample may also be a composition derived from a human or animal source, such as growth hormones or cell extracts, such as pituitary extracts.
• a composition can indeed be contaminated by an NCTA.
• a biological fluid it may be blood, lymph, urine or milk, this list not being limiting.
• the sample is a blood product or a derivative, for example a plasma derivative or a plasma protein concentrate.
• cells supporting the replication or propagation of the ATNC is meant any cell capable of being infected by an ATNC, of replicating or propagating this ATNC and of producing PrPsc and / or infectivity. It is a preference cells in which the gene coding for the non-pathological conformation of the prion protein PrP has been integrated and / or overexpressed. More preferably, these cells are stable transgenic cells, which are capable of expressing PrP.
• a "passage” is generally the transplanting of a portion of the cells of one culture dish into another box containing new culture medium. Each passage therefore dilutes the cells that no longer have room to divide into another box and the time of culture in a box allows the NCTA to replicate.
• substrate source for the NCTA any non-infectious matrix capable of supporting replication of NCTAs by in vitro amplification cycles.
• substrate source for the pathological conformation protein is meant any source of non-pathological conformational protein that can not modify the conformation of another protein to render it insoluble.
• the substrate source for the ATNC and / or the non-pathological normal form of the protein may belong to the same species, or to a species different from the tested biological product.
• This source may be, for example, derived from a non-pathological normal form of the protein of the same product as that contained in the sample to be tested.
• the source of non-pathological normal form can be produced recombinantly or synthetically, using means known to those skilled in the art.
• the cells used in step (i) of the method of the invention advantageously have the following criteria:
• nerve cells nerve cells (neurons, glial cells) are not necessarily the best substrates for in vitro assay purposes, because of their poor adaptation in culture, it is preferred to establish transgenic cell lines by combining a type of specific cells and a particular transgene, by known techniques, necessary to provide an ideal environment for replication of the ATNC.
• the cells of step (i) are rabbit epithelial cells, in particular cells of the Rov9 line (Vilette et al., PNAS, March 2001; 98, 4055-9).
• transgenic and stable rabbit epithelial cells expressing ovine PrP are able to replicate a scrapie strain and produce ovine PrPsc after infection with an infectious brain homogenate containing ovine PrPsc.
• the cell model constructed, the Rov9 line inducibly expresses exogenous PrP, which guarantees its maintenance during the incubation period necessary for the accumulation of PrPsc in these cells placed in contact with an infectious material.
• the cells of step (i) are murine glial cells, particularly cells of the MovS2 or MovS6 line (Archer et al., Journal of Virology, 2004; 78 pp. 482-90). These lines consist of murine glial cells expressing ovine PrP and supporting the replication of ATNC of ovine origin. Contacting the sample to be tested:
• Cells supporting the replication or propagation of the ATNC are contacted with the test sample likely to be infected by this ATNC, or with infectious material containing the NCTA as reference material, by examples of brain extracts from animals infected with NCTA, such as a sheep prion.
• the contacting is carried out according to methods known to those skilled in the art (see, for example, Archer et al., Journal of Virology, Jan. 2000, pp. 4 82 30 49 0).
• These cells are then cultured during one or more passages to allow the ATNC to replicate or propagate.
• the transgenic cells may be brought into contact with at least one dilution of the sample that may be infected with the ATNC in a biologically acceptable aqueous solution, in particular with several dilutions, more particularly with serial dilutions or successive. These dilutions make it possible to refine the quantification of the infectivity in the sample tested.
• the step of culturing the cells potentially infected with the biological product is required for the replication or the propagation of the ATNC, therefore to amplify an insufficient amount of ATNC, initially, to be detected.
• the stable transgenic cell culture step ii) performed to amplify the amount of ATNC present in said biological sample by replication of the ATNC is carried out in DMEM + Ham-F12 medium (4: 1 ) supplemented with glutamine and fetal calf serum (5% final).
• the cells are incubated at 37 ° C. under 5% CO 2 .
• a passage of the cells (split ratio of: 1 for 10) is carried out every week.
• the product resulting from the culturing of the cells contains the marker protein (in particular PrP) in its pathological form, the quantity of which is greater than the quantity initially present in the biological sample, if it contains therein.
• the marker protein (in particular PrP) in its pathological form and the ATNC accumulate within the cell culture (at the level of the infected cells and in the culture medium).
• Step ii) of the method of the invention is followed by contacting and incubating the product resulting from culturing the cells with a substrate source for the pathological conformer and / or ATNC. .
• This source of substrate may be provided for example in the form of a product of animal origin, for example a healthy brain homogenate, or material derived from in vitro culture.
• This material may for example be derived from cells, such as MovS, Rov N2A (Weissmann et al., (2003), PNAS vol.100 No. 20, 666-11671), or even yeasts, fungi, or bacteria, this list is not limiting.
• This material derived from in vitro culture can be expressed in various cellular compartments, such as the extracellular compartment (for example the supernatant, the exosomes, this list not being limiting) and / or membrane and / or cytosolic compartments (cell lysate). for example).
• This step serves to allow the in vitro amplification of the pathological conformation protein (in particular PrPsc) and / or the ATNC harvested during step ii) by the conversion of the non-pathological form of the protein a marker (in particular PrP) (contained in the substrate) in pathological form, a non-pathological autoconversion of the form being theoretically impossible.
• the protein in its pathological form would initiate the transformation of the non-pathological form into a pathological form.
• step (ii) Incubation of the cell culture product of step (ii) with a substrate source for the pathological conformation protein and / or the ATNC is performed for a time sufficient to allow at least a portion of the proteins having non-pathological form, to be transformed into a pathological form of the protein, or to the NCTA to amplify.
• each incubation step is carried out for a period of between 10 seconds and 4 hours, preferably between 20 minutes and 1 hour, and particularly preferably for 30 minutes.
• the amplification medium may advantageously consist of a buffer typically composed of: (Ix PBS), 15mM NaCl and 1% Triton. and at least one substrate containing the marker protein (in particular PrP) in a non-pathological conformation (for example of a volume 10 times greater than the volume of the sample to be amplified).
• a buffer typically composed of: (Ix PBS), 15mM NaCl and 1% Triton.
• at least one substrate containing the marker protein (in particular PrP) in a non-pathological conformation for example of a volume 10 times greater than the volume of the sample to be amplified.
• step iv) of the method of the invention consists of the disaggregation of the aggregates possibly formed, so as to release the particles of pathological conformation marker proteins and / or ATNC so that they can convert from other non-pathological proteins.
• a solvent such as sodium dodecyl sulphate, dimethyl sulphoxide, acetonitrile, guanidine, urea, trifluoroethanol, diluted trifuroacetic acid, diluted formic acid, this list not being limiting
• the modification of the physicochemical characteristics of the solution such as the pH, the temperature, the ionic strength, the dielectric constant
• physical methods such as sonication, laser irradiation, freezing / thawing, autoclave incubation, high pressure, gentle homogenization, or other sources of irradiation, this list not being not limiting.
• sonication is used. Sonication is a method known to those skilled in the art, and often used in PrP sc purification methods, by making it possible to increase the solubility of the aggregates.
• the duration of the disintegration step is readily determinable by those skilled in the art, and may depend on the method of disintegration chosen. Preferably, the duration of the disintegration step is between 1 second and 60 minutes, more preferably between 5 seconds and 30 minutes, and more particularly between 5 seconds and 30 seconds.
• the succession of steps iii) and iv), called the cycle is repeated at least twice, preferably between 5 and 100 times, preferably between 20 and 60 times.
• This repeated cycle between 2 and 100 times constitutes a series of amplification cycles.
• a series of cycles can also be repeated several times. In this case, the new series will be initiated from a volume of the previous series in place of the original NCTA sample.
• Step (v) for detecting pathological proteins can be carried out by any method known to those skilled in the art.
• the specific detection of PrPsc can be carried out by a first step of separation of the two isoforms PrPc and PrPsc.
• This separation is carried out on the basis of the biochemical properties of PrPsc which make it possible to distinguish it from non-pathological proteins, in particular the fact that PrPsc is reported to be more resistant to protease-based treatments and less soluble even in the presence of detergents.
• the first step after amplification is preferably the separation of the PrPc (non-pathological soluble normal form of PrP) from the sample, which can be effected for example by means of protease treatment, for example proteinase K or by centrifugation to separate soluble forms (PrPc) from insoluble forms (PrPsc).
• digestion with proteinase K is a step prior to Western Blot, mainly digests PrPc and not PrPsc. It is indeed a property of PrPsc that it is relatively resistant to PK compared to PrPc. The subsequent detection step therefore no longer detects the non-pathological form of the protein because it has been digested by the protease.
• the detection step can be carried out using the following methods: immunocytochemistry (for example by cell labeling and Facscan analysis) or immunochemistry (such as the Western blot or an ELISA test), or radioactivity test, test of fluorescence, electron microscopy, turbidimetry to detect aggregates, as well as structural tests including NMR (nuclear magnetic resonance), circular dichroism, Raman spectroscopy, UV absorption, a monoclonal antibody recognizing the pathological form of protein, this list not being limiting.
• immunocytochemistry for example by cell labeling and Facscan analysis
• immunochemistry such as the Western blot or an ELISA test
• radioactivity test test of fluorescence, electron microscopy, turbidimetry to detect aggregates, as well as structural tests including NMR (nuclear magnetic resonance), circular dichroism, Raman spectroscopy, UV absorption, a monoclonal antibody recognizing the pathological form of protein, this list not being limiting.
• an antibody specifically recognizing the pathological form and not the non-pathological form may itself be labeled.
• an antibody may be for example the 15B3 antibody (Korth et al., 1997, Nature 1997 Nov6, 390 (6655): 74-7).
• Titration of TNC A The detection of the pathological forms of the marker proteins or the NCTA may be associated with a determination of the amount of these proteins or the ATNC present in the sample.
• the titration can be carried out using any titration method known to those skilled in the art. In particular, it can be performed on the model of the methods described in the documents WO2005022148 and WO2006117483 (in particular reference examples A and B).
• the set of Western-blot results for the different dilutions and the different replicates can be analyzed by a statistical method known to those skilled in the art for establishing an infectious titer, such as the method of Spearman Karber (Schmidt NJ , Emmous RW, Diagnosis Procedures for Viral, Rickettsial and Chlaveydial Infection, 1989, 6th Edition).
• the method of calculating the title is the Spearman - Karber method. This method assumes the dilution of the sample to be tested according to a geometric progression, that is to say of constant reason between successive dilutions, and a constant volume inoculation (generally 0.150 ml) of each dilution in five well at least. The most commonly used dilution factor is the decimal factor.
• Logio median dose (Xo) - (d / 2) + dS (T 1 Ai 1 )
• Xo logio of the reciprocal value of the lowest dilution at which all test inocula are positive.
• d log10 of the dilution factor, also referred to as "no dilution” (i.e. the difference between log dilution intervals).
• U 1 number of test inocula used at each dilution.
• R 1 number of positive inocula of test (on ni).
• the estimated standard deviation is calculated using the following formula:
• V 1 Zn 1 proportion of positive assays (i.e., inoculum cups with reaction) at each dilution.
• the method of the invention makes it possible to quantify the infectivity associated with NCTAs over a range of about 4 log, that is, 10,000 infectious units in vitro. This may, for example, make it possible to satisfy the criteria for validating the efficiency of the processes for obtaining biological products with respect to the elimination of NCTAs.
• Applications of the NTCA infectivity determination method :
• the invention also relates to the application of the titration method according to the invention to a method of evaluation and / or in vitro control of a process for obtaining or treating a biological product likely to be contaminated. by an NCTA.
• This evaluation and / or control method is characterized in that a titration method according to the invention, as described above, is applied to the biological product, upstream and downstream of said process, and in that the we compare the two title values obtained. By comparison between the two measurements, the degree of elimination or the reduction factor of the NCTA is determined.
• the titration method according to the invention has the capacity to be easily applicable to any type of process for obtaining or purifying biological products, in particular blood products, such as blood plasma derivatives, using for example, chromatography or nanofiltration, in particular the chromatographies described in documents EP 0 359 593 and WO 02/092632, or the nanofiltration described in document WO / 2005/022148.
• the implementation of the method of the invention makes it possible to evaluate and / or control the efficiency of a process (or a part of a process) for obtaining or treating, or even purifying of any biological product likely to be contaminated by an NCTA, in the elimination of this NCTA, by means of a titration using specific transgenic cell lines, promoting the replication of the ATNC, brought into contact with an infectious material or potentially infectious containing the ATNC to be tested.
• the amounts of NCTA are measured upstream and downstream of the process (or part of the process) whose performance with respect to the NCTA is to be assessed. By comparing the two measurements, the degree of elimination of the pathogen is determined.
• the implementation of the present method can be carried out during a process for obtaining a biological product or in the context of an elimination treatment of the NCTA according to obtaining the biological product.
• the invention also relates to the application of the titration method according to the invention to a method of evaluation and / or in vitro control of a decontamination procedure.
• a material In this case, the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention. This infected biological product is then contacted with the material to be decontaminated and the decontamination procedure is applied to this material. Finally, the title of the biological product having undergone the decontamination procedure is again determined. The two titration measurements performed upstream and downstream of the decontamination procedure are compared to evaluate the effectiveness of the decontamination procedure.
• the material may, for example, be purification equipment, in particular a chromatography column, or be the sanitization of a chromatography column using sodium hydroxide.
• the invention also relates to the application of the titration method according to the invention to a selection procedure and / or method for evaluating the ability of a candidate compound to modulate (reduce or increase) the titre of the infectious material.
• the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention.
• This infected biological product is then brought into contact with the test compound and the title of the biological product having undergone the decontamination procedure is again determined.
• the two titration measurements made upstream and downstream of the contacting of the sample with the test compound are compared.
• the invention also relates to the application of the titration method according to the invention to a selection procedure and / or evaluation method of a compound capable of inhibiting the infectivity of an ATNC.
• the ATNC titre of a biological product containing an NCTA is determined, in the presence and then in the absence of the compound to be evaluated, by means of the titration method according to the invention.
• the methods for bringing the compound into contact are determined according to whether the action of the compound prevents the initiation of an infectious cycle or blocks an infectious cycle already initiated. In all cases, the title of the biological product is determined with and without treatment with the product to be tested.
• the two titration measurements performed are compared to evaluate the inhibitory activity of the ATNC infectivity of the compound.
• the invention also relates to the application of the titration method according to the invention to a method for identifying a compound making it possible to modulate the transformation of the non-pathological form in the pathological form of a marker protein or the ATNC, for example the transformation of PrP into PrP sc .
• the ATNC titre of a biological product containing an NCTA is determined using the titration method of the invention.
• This infected biological product is then contacted with the test compound and the titration method of the invention is applied to again determine the title of the biological product.
• the two titration measurements made upstream and downstream of the contact with the compound are compared to evaluate the effectiveness of the test compound.
• MovS6 cells (Archer F. et al., 2004 supra) were selected as replication-supporting cells of the ATNCs, and the 127-S scrapie strain adapted to the Tg301 transgenic mice was used as a source of natural infectivity (Vilette JL and 2001, supra).
• Tg301 transgenic mice carry a large DNA fragment isolated from an ovine artificial chromosome library, and ovine PrP expression levels are approximately 8-fold higher than those observed in sheep brains.
• the source of initial infectivity was a brain homogenate (lot LN-3326) from infected mice at 200 mg / ml. The title of this sample is 5.7 logio uWB / ml and 6.35 TCIDso / ml.
• MovS6 cells were cultured in DMEM + Ham-F12 medium (4: 1) supplemented with glutamine and fetal calf serum (final 5%) and incubated at 37 ° C. under 5% CO 2 . Each week 10% of the cells were re-seeded in each well. The remaining 90% of cells were either stored as a dry cell pellet previously washed in PBS, or removed. Assay plates were prepared 24 hours before inoculation. The cells were seeded in wells of approximately 2 cm 2 (24-well plate format), at the rate of 100,000 cells per well, ie approximately 50,000 cells / cm 2 .
• the inoculum consisted of either the LN-3326 10-fold diluted sample or a healthy brain homogenate sample that served as a "Negative Control" (lot: LN-3777). Inoculations were performed in 5 replicates.
• the cells were contacted for 24 hours with 150 ⁇ l of diluted inoculum, and then 1 ml of new culture medium was added. The cells were maintained in culture for an additional 72 hours, ie until the first passage during which, for each well, the entire cell layer was transferred to a well of about 9.6 cm 2 (6-well plate format).
• the cell culture was then maintained for 10 weeks. Each week, for each well, the culture medium was changed and 10% of the cells were re-seeded.
• the cells inoculated with the infected inoculum (LN-3326) not re-seeded were for 2 replicates on the 5, washed once in PBS and then stored at -80 ° C. in the form of a dry pellet (samples intended for amplification according to the method of the invention) and for the other 3 replicates preserved without prior dry-pellet washing (samples intended for detection by WB).
• the cells inoculated with the healthy sample and not re-seeded were, for all the replicates, preserved without prior washing in the form of a dry pellet.
• the cell pellets obtained made it possible to carry out the analyzes described below. Detection of PrPsc in cells:
• PrPsc present on the membranes was detected by incubation with the antibody 6H4 (Prionics) then a secondary antibody labeled with alkaline phosphatase (goat antibody "anti-mouse antibody”).
• the labeled membranes were revealed by chemiluminescence.
• One sample was considered positive if the electrophoretic profile of the three forms of glycosylated PrPsc was visible on autoradiograms.
• Table 1 Number of positive wells after detection of PrPsc in MovS6 cell lysates.
• PrPsc signal was observed in the lysates of MovS6 cells inoculated with the brain homogenate infected with strain 127-S. This signal was observed on the first pass (figure, wells 3 to 12).
• PrPsc associated with the 127-S strain in the sample of brain homogenate LN-3326 previously diluted 10 times can be amplified by the method of the invention. Detection of PrPsc in this diluted sample indicates that the amplification rate is at least 2.5 to 3 log 10. Moreover, this amplification is specific for PrPsc since no signal is observed with the undiluted healthy homogenate sample.
• Amplification of PrPSc associated with the 127-S strain in cell lysates A dilution studied (10 "), the homogenate LN-3326 induced the production of detectable PrP Sc by Western blot from the 4 th passage. This time corresponds to the time required for PrPsc propagation in the cell culture to reach the level of minimum concentration detectable by Western-blot. This delay also proves a de novo production of PrPsc by the cells.
• the PrPsc present in the infected cells is detected at the first pass and for all subsequent passages. This amplification seems specific since PrPsc was not detected in uninfected cells.

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  • 2009-03-25 EP EP09730577A patent/EP2269075A1/de not_active Withdrawn
  • 2009-03-25 CA CA2719152A patent/CA2719152A1/fr not_active Abandoned
  • 2009-03-25 CN CN2009801111193A patent/CN101981453A/zh active Pending
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  • 2009-03-25 WO PCT/FR2009/050520 patent/WO2009125139A1/fr not_active Ceased
  • 2009-03-25 JP JP2011501280A patent/JP2011517772A/ja active Pending
  • 2009-03-25 US US12/933,492 patent/US20110151476A1/en not_active Abandoned
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