Device and method for production and analysis of prions

Abstract

The invention provides a method for producing prion protein having an aggregated conformation by contacting native conformation prion protein with aggregated conformation prion protein in a liquid preparation and subjecting this to at least one cycle or to a number of cycles of application of shear-force for fragmenting aggregates of prion protein, wherein the shear-force applied is precisely controlled. In addition to this process for amplification of aggregated state prion protein from native conformation prion protein, the invention relates to the aggregated state prion protein obtained by the amplification process, which aggregated state prion protein has one conformation, which is e.g. identical within one batch and reproducible between batches, e.g. as detectable by proteinase resistance in a Western blot.

Claims

1. A device for use as a controlled intensity shear-force generator in a process for amplification of aggregated conformation prion protein from native conformation prion protein by contacting the native conformation prion protein with the aggregated conformation prion protein in a liquid composition and subjecting the liquid composition to at least one cycle comprising generation of shear-force and a resting phase, the device containing an array of two or more devices, arranged with their longitudinal axes vertical in a temperature-controlled housing, each device in the array comprising a shear-force generator arranged to exert the shear-force to each volume element of the liquid composition containing the native conformation prion protein and the aggregated conformation prion protein, wherein a control unit controls the shear-force generator to generate the shear-force acting on each volume element of the liquid composition only at one uniform shear force intensity which is limited to an intensity range of maximally 10% of a maximum shear-force, wherein the shear-force generator comprises a rotary element arranged coaxially within a vessel and an outer surface of the rotary element is parallel to an inner wall of the vessel.

2. The device according to claim 1, wherein the control unit comprises a computer that is connected to the devices of the array and controls a rotation rate of each rotary element individually.

3. The device according to claim 1, wherein the rotary element and a coaxial section of the inner surface of the vessel are spaced and cylindrical or conical.

4. The device according to claim 3, wherein a bearing of the rotary element comprises an axle, one end of which is arranged contacting a bottom section of the inner wall and the other end of which runs in a bearing attached next to a rim of the vessel.

5. The device according to claim 1, wherein the rotation of the rotary element is controlled to within 10% of one rate.

6. The device according to claim 1, wherein the shear-force generator is controlled precisely to a range of maximally 1% of one pre-set shear-force corresponding to a rotation rate of the rotary element between 10 and 10,000 Hz.

7. The device according to claim 6, wherein the shear-force generator is controlled such that the rotation rate of the rotary element is within a range of maximally +/2 Hz.

8. A device for use as a controlled intensity shear-force generator in a process for amplification of aggregated conformation prion protein from native conformation prion protein by contacting the native conformation prion protein with the aggregated conformation prion protein in a liquid composition and subjecting the liquid composition to at least one cycle comprising generation of shear force and a resting phase, the device containing an array of two or more devices, arranged with their longitudinal axes vertical in a temperature-controlled housing, each device in the array comprising a shear-force generator arranged to exert the shear-force to each volume element of the liquid composition containing the native conformation prion protein and the aggregated conformation prion protein, wherein a control unit controls the shear-force generator to generate the shear-force acting on each volume element of the liquid composition only at one uniform shear force intensity which is limited to an intensity range of maximally 10% of a maximum shear-force, wherein the shear-force generator comprises a tube and a coaxial rotary element arranged along the longitudinal axis of the tube at a spacing from the tube, the rotary element run on bearings, and at least one exit opening arranged between the tube and the bearings, the exit opening having a cross-section of at least a cross-section between the rotary element and the tube.

9. The device according to claim 8, wherein the rotary element is fixed to one end of a coaxial axle, which axle is arranged in a first bearing formed by a poly tetrafluoro ethylene (PTFE) polymer tube arranged around a section of the axle and arranged within a sleeve, wherein the low-friction polymer tube is arranged between a shoulder of the axle and an inner shoulder of the sleeve.

10. The device according to claim 8, wherein the rotation of the rotary element is controlled to within 10% of one rate.

11. A device for use as a controlled intensity shear-force generator in a process for amplification of aggregated conformation prion protein from native conformation prion protein by contacting the native conformation prion protein with the aggregated conformation prion protein in a liquid composition and subjecting the liquid composition to at least one cycle comprising generation of shear force and a resting phase, the device containing: an array of two or more devices, arranged with their longitudinal axes vertical in a temperature-controlled housing, each device in the array comprising a shear-force generator arranged to exert the shear-force to each volume element of the liquid composition containing the native conformation prion protein and the aggregated conformation prion protein, wherein a control unit controls the shear-force generator is controlled to generate the shear-force acting on each volume element of the liquid composition only at one uniform shear force intensity which is limited to an intensity range of maximally 10% of a maximum shear-force, wherein the shear-force generator comprises a rotary element arranged coaxially within a vessel and an outer surface of the rotary element is parallel to an inner wall of the vessel; and a controlled positioning apparatus having a clamping means for holding the vessel, which positioning apparatus is adapted for positioning the vessel at a pre-determined position in relation to the shear-force generator.

12. The device according to claim 11, wherein the controlled positioning apparatus is adapted for repeatedly positioning the vessel at a pre-determined first position in relation to the shear-force generator during the generation of the shear-force and for subsequently removing the vessel from the first position and positioning the vessel at a spaced second position during a resting phase.

13. A device for use as a controlled intensity shear-force generator in a process or amplification of aggregated conformation prion protein from native conformation prion protein by contacting the native conformation prion protein with the aggregated conformation prion protein in a liquid composition and subjecting the liquid composition to at least one cycle comprising generation of shear force and a resting phase, the device containing an array of two or more devices, arranged with their longitudinal axes vertical in a temperature-controlled housing, each device in the array comprising a shear-force generator arranged to exert the shear-force to each volume element of the liquid composition containing the native conformation prion protein and the aggregated conformation prion protein, wherein the shear-force generator has a rotary element coaxially arranged in a radially spaced tube section, the radial spacing of the rotary element and the tube and the axial section in which both the rotary element and the tube extend defining a space, in which space upon rotation of the rotary element the shear-force is generated, wherein a control unit controls the shear-force generator to generate the shear-force acting on each volume element of the liquid composition only at one uniform shear force intensity which is limited to an intensity range of maximally 10% of a maximum shear-force.

14. The device according to claim 13, wherein the rotary element along its longitudinal and rotary axis has a constant outer diameter and is arranged at a constant spacing from the encircling tube section.

15. The device according to claim 13, wherein the rotation of the rotary element is controlled to within 10% of one rate.

16. The device according to claim 13, wherein the shear-force generator is controlled precisely to a range of maximally 1% of one pre-set shear-force corresponding to a rotation rate of the rotary element between 10 and 10,000 Hz.

17. The device according to claim 16, wherein the shear-force generator is controlled such that a rotation rate of the rotary element is within a range of maximally +/2 Hz.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention is now described in greater detail with reference to the figures, wherein

(2) FIG. 1 schematically shows a shear force generator of the invention,

(3) FIG. 2 schematically shows another shear force generator of the invention,

(4) FIG. 3A schematically shows a sonotrode as a shear-force generator, with the areas between vibration nodes (hatched) and shear-force intensities (white boxes) represented in the lower side view,

(5) FIG. 3B shows a top-view onto a sonotrode as a shear-force generator of the invention with vibration amplitudes given in grey shading according to the right-hand scales,

(6) FIG. 3C shows a top-view onto a sonotrode with vibration amplitudes at a distance of 0.5 mm above the sonotrode surface,

(7) FIG. 3D shows the shear-force intensity at a distance of 0.5 mm above the sonotrode surface in a partial cross-section of FIG. 3B as indicated by the dashed line in grey shading according to the right-hand scale,

(8) FIG. 4 shows Western blots of Proteinase K digested aggregated conformation prion protein produced according to the invention with different shear forces applied,

(9) FIG. 5 shows a graph of the normalized concentrations of aggregated conformation prion from the Western blots of FIG. 4,

(10) FIGS. 6A and 6B show a Western blot analysis of Proteinase K digested aggregated conformation prion protein produced according to the invention with different shear forces applied,

(11) FIG. 7 shows sections of Western Blots of proteinase K-resistant prion protein produced at different controlled shear-forces,

(12) FIG. 8 shows a graphic analysis of the relative amount of aggregated conformation prion protein of FIG. 7,

(13) FIG. 9 shows the kinetics of the amplification at controlled shear-forces (Hz),

(14) FIG. 10 shows shows an SDS-PAGE (silver stained) of the initial admixture of Sc237 with the native conformation prion protein (left lane) and of the produced aggregated conformation product,

(15) FIG. 11 shows the CP-MAS NMR: .sup.13C, .sup.13C correlation of a comparative spontaneous amplification product,

(16) FIG. 12 shows the CP-MAS NMR: .sup.13C, .sup.13C correlation of an amplification product produced according to the invention,

(17) FIG. 13 schematically shows an embodiment of a shear-force generator in cross-sectional view, and

(18) FIG. 14 shows an embodiment of a holder for holding a reaction vessel.

(19) In the examples, same reference numerals refer to functionally identical elements.

(20) FIG. 1 depicts a device containing a rotary element 1, which is arranged on an axle 2 that is coaxial to the vessel and runs on a first bearing 3 and a second bearing 4. The first bearing 3 is formed by a first end of axle 2 contacting the bottom of vessel 5, in which the rotary element 1 is arranged, and the second bearing 4 is formed by the second end of the axle 2 opposite its first end. As preferred, the second bearing is formed by a boring 6 of a holder 7, which is e.g. held by frictional connection within a recess formed within the lid 8. The rotary element 1 comprises a first coupling element 10 of a coupling, e.g. a permanent magnet having at least two poles, which coupling is connected to a drive 11. For driving the rotary element 1, the device is arranged in a coupling position to the second coupling element 12, e.g. between coils, and the drive 11 is an electric field generator connected to the coils. The drive 11 is closely controlled by a control unit 13, which preferably is also connected to a sensor 14 monitoring the drive 11. The control unit 13 is equipped to control the drive 11, e.g. in a closed loop circuit, to high precision for generating a shear force having an intensity range of 2%, preferably of 1% or narrower about one intensity value, e.g. by controlling the rotation of the rotary element 1 within vessel 5 to one frequency with a variation of 2%, preferably 1% maximally.

(21) The surface of the rotary element 1 can be parallel to the wall of the vessel 5, forming a homogenous shear force for a cylindrical rotary element in a cylindrical vessel 5. In the case of a conical vessel 5, a rotary element that is conical for being in parallel to the vessel 5 will generate a shear force gradient in accordance with the change in radius. Generally, if the shear force gradient caused by the angle of the conical shape of the vessel and the parallel conical shape of the rotary element exceeds the narrow range of one shear force intensity, it is preferred that the angle of the cone of the rotary element is smaller to the axle than the cone of the inner vessel wall to its longitudinal axis, preferably such that the spacing between the vessel and the rotary element increases with increasing radius of the rotary element and vessel, respectively.

(22) This embodiment has the advantage that it can be realized using a commercial vessel 5, e.g. an Eppendorf vial, into which the rotary element 1 is positioned and held by a second bearing 4 which is provided by a plate serving as a holder 7 arranged in a recess of a lid 8.

(23) Rotation of the rotary element 1 within vessel 5 at a closely controlled rotation frequency, i.e. at a narrow range about one pre-set rotation frequency generates a shear force of a narrow range about one pre-set shear force between the rotary element 1 and the vessel 5.

(24) FIG. 2 shows a preferred embodiment of the device of the invention, wherein a rotary element 1 is encircled by a spaced tube 20 of cylindrical inner diameter, the inner wall of the tube 20 forming a space of annular cross-section around the rotary element 1. In the alternative, a section of the rotary element 1 that is encircled by tube 20 can have a flat form, e.g. of rectangular cross-section not exceeding, preferably having the radius of the rotary element 1. As preferred, the tube 20 exceeds the first (lower) end 1a of the rotary element 1, and the tube 20 provides for an exit opening 21 having or exceeding the diameter of the cross-section between the rotary element 1 and tube 20. The rotary element 1 runs on a first bearing 3 which is formed by a low-friction polymer tube 22 arranged between the axle 2 of the rotary element 1 and a sleeve 24 that arranged around axle 2, wherein the sleeve 24 has an inner shoulder 25 against which the low-friction polymer tube 22 abuts, and a second bearing 4 which is formed by shoulder 23 and the low-friction polymer tube 22 abutting against the shoulder. The sleeve 24 and the tube 20 are coaxial and can optionally be formed of one tube having the exit opening 21, e.g. at least one boring and preferably 2 opposite borings, arranged between the sleeve 24 and the tube 20. Preferably, the sleeve 24 and the tube 20 are connected to one another, e.g. by at least one connector, preferably by at least 1, preferably two sections of a tube, which sections separate the exit openings 21.

(25) A first element of a coupling 10, e.g. a permanent magnet, is fixed to the axle 2. Preferably, the device is encased within a sealable housing 30, and the second element of the coupling 12 is arranged outside the housing 30, allowing the drive 11 coupled to the second element of the coupling 12 to drive the first element of a coupling 10, and hence the axle 2 which bears the rotary element 1.

(26) The embodiment of the device depicted in FIG. 2 is for arrangement of the tube 20 within a vessel, which can e.g. be the housing 30, or which vessel 31 can be arranged with in a housing 30. This embodiment has the advantage that the shear force is generated without interaction with the vessel 31.

(27) FIG. 3A shows a sonotrode surface in top view, wherein the hatched dark area indicates one of several vibration nodes, and adjacent surface areas which significantly vibrate. The lower side view shows that the one exemplified vibrating area generates shear-force which is limited both to a surface section of the sonotrode and to a certain spacing from the sonotrode surface, as indicated by the curve that represents both the spatial extent of the vibration, i.e. shear-force maximum in parallel to the plane of the sonotrode surface, and in the distance form the sonotrode surface.

(28) FIG. 3B shows a sonotrode surface in top view, wherein the dark area schematically shows one of several node forming areas and adjacent surface areas which significantly vibrate. The dark lines represent vibration nodes, and increasing brightness shows increasing shear-force. FIG. 3C shows the spatial distribution of shear-force at a height of 0.5 mm above the sonotrode metal surface as determined e.g. in water. The side view given in FIG. 3D shows that the vibrating areas are not only limited to a surface section of the sonotrode, but are also limited to a certain spacing from the sonotrode surface, as indicated by the intensity curve of the shear-force between vibration nodes, which curve represents both the spatial extent of the vibration energy maximum in the plane of the sonotrode and the distance from the sonotrode surface, to which the vibration energy maximum extends. The shear force of one intensity that is used in the invention is exerted only onto the volume elements of a liquid composition which are located in the volume above the sonotrode in which maximum vibration energy is generated. Accordingly, in devices and processes using a sonotrode as the shear force generator, all the volume elements of the liquid composition are arranged in a distance from the sonotrode surface and in parallel to a surface fraction of the sonotrode surface between vibration nodes, in which the vibration energy maximum is generated, preferably at a resonant frequency of the sonotrode, e.g. allowing a specific maximal ultrasonic shear force of a limited intensity range only to be transmitted to the vessel's inner volume consisting of the volume elements of the liquid composition, e.g. to a precision or limitation of 10%, preferably 2% or 1% of the shear-force maximum at the one frequency applied. It has been found that additional agitation for mixing the liquid preparation is not necessary, as the shear-force applied to each volume element of the liquid composition consists of the controlled intensity range of shear-force, which results in the homogeneity of aggregated-state prion protein generated from the native conformation prion protein in admixture with aggregated state prion protein of the liquid composition.

EXAMPLE 1

Device for Generating Controlled Shear-force of a Limited Intensity Range

(29) As an example, a device as schematically shown in FIG. 2 was used with the following dimensions: a rotary element with a square cross-section of 1.95 mm on an axle of 1.95 mm diameter was arranged at a spacing of 0.3 mm coaxially in a cylindrical tube. The sleeve, the tube and the rotary element including the axle arranged in the sleeve and the permanent magnet at the end of the axle opposite the square rotary element were obtained from Heidolph, Germany, and the original exit borings were drilled to diameter of 3 mm to form two opposing exit borings of 3 mm diameter each arranged adjacent the tube.

(30) The bearing was as shown in FIG. 2, using a Teflon tube cut to size between the shoulder of the axle and the inner shoulder of the sleeve. For reduced friction, the Teflon tube was folded lengthwise by flattening two times, generating 4 lengthwise folds separating 4 lengthwise convex sections, as is generally preferred for this tube. The sleeve was formed by a tube section that was connected to the tube encircling the rotary element by the tube wall sections forming the boundaries to the exit openings. For the coupling, the set of stator coils available from Heidolph were used, including part of their individual control electronics.

(31) For forming an array of the devices, two or more devices, preferably 14 or 21 were arranged with their longitudinal axes vertically in a temperature-controlled housing. All devices of the array were controlled by one computer.

(32) The shear rate was controlled by a potentiometer and multimeter, regulating the rates of rotation between 20 to 1300 Hz with a variation of 5 Hz, preferably 2 Hz, and more preferably of 1 Hz.

(33) It was found that these devices could be run for 136 cycles of 60 s rotation at 20 to 1300 Hz and 540 s resting phase without rotation with an accuracy of rotation of 1 Hz for treatment of aqueous liquid compositions at 5 C. to 40 C. in one experiment, and these devices could be used for up to 10 experiments. The bearing was essentially stable, maintained a low-resistance movement of the rotary element which is essential for reproducibility of rotation rates, and did not show excessive wear.

(34) As a comparative bearing, a rolled-up sheet of PTFE foil was arranged between the rotary element and the tube. After rotating for 10 cycles of 60 s with 540 s resting phase, the rolled-up sheet forming the bearing had run hot and was partially destroyed, whereas a bearing consisting of a PTFE tube arranged between the rotary element and a tube section could run for at least 5 min at the same speed, and could be used to a total service life of at least 60 min up to 24 h.

(35) For comparison to a device of the invention having two exit openings with a total cross-section of the cross-section that is limited by the rotary element and the tube section encircling it, a device was used, wherein the exit opening was one boring of 1.5 mm diameter, i.e. a cross-section of 1.767 mm.sup.2. It was found that this smaller exit opening resulted in irreproducible products from liquid compositions containing native conformation prion protein with aggregated conformation prion protein. Currently, it is assumed that the smaller exit opening results in shear forces which are generated in addition to the shear forces generated by the rotary element. Further, it was observed that in these devices, liquid composition was drawn into the bearing, allowing the bearing to exert additional shear forces onto the liquid.

EXAMPLE 2

Production of Aggregated Conformation Prion Protein

(36) As a first experiment, the starting liquid composition contained 5% vol/vol of the Protease K-resistant prion protein Sc237 BH, representing an aggregated conformation prion protein which is known to induce amplification of the aggregated conformation in the native conformation shNBH, was admixed with a 10% wt/vol preparation of hamster prion protein shNBH (Syrian hamster normal brain homogenate) having the native conformation, which was produced by homogenization of brain tissue detergent containing aqueous buffer solutions. From this stock, aliquots were each subjected to different shear force intensities using an array of the devices of Example 1 at cycles of 60 s shear force and 9 min resting phase without agitation for a total of 46 h in a thermostat at 37 C. to a shear force by rotation rates as indicated in FIG. 4 with an accuracy of 1 Hz. The rotary elements of the devices were introduced into 1.5 mL Eppendorf vials containing the liquid composition aliquots.

(37) No deterioration of the precision of the shear force, i.e. no deviation of the control of rate of rotation was observed over the number cycles, indicating the reliability of the device.

(38) For analysis of the amplification reactions, aliquots from each reaction were taken and digested with Proteinase K added to 50 g/ml or 10 g/ml, respectively. Samples were separated by SDS PAGE, detection was in a Western blot using anti-PrP antibody and Western Pico ECL solution (Pierce) for signal generation. The results are shown in FIG. 4, wherein the number of the tool identifies the individual device of the array, Hz indicates the rate of rotation used by the individual device, neg. denotes a 5% vol/vol dilution of 10% wt/vol Sc237 BH prion protein in 10% wt/vol shNBH, corresponding to the starting liquid composition, and ScBH, separated from neg. by an empty lane, denotes 10% wt/vol Sc237 BH prion protein digested at 50 g/ml Proteinase K in each blot as a common positive control.

(39) The Western blots show that the amount of Proteinase K resistant prion protein generated by amplification differs in dependence on shear force intensity, as indicated by the rates of rotation. Further, the comparison of samples generated at one shear force intensity but digested with different concentrations of Proteinase K indicates that the change in signal intensity differs between samples according to the shear force intensity. A quantitative analysis is shown in FIG. 5, wherein the signal intensities are shown after normalization to the positive control signal. In the graph, the shear force intensity is given as the speed of rotation (.box-tangle-solidup.), each pair of columns gives Proteinase K resistant prion protein of the same rotation rate as indicated, with the left column indicating the signal intensity at 10 g/ml Proteinase K, the right column indicating the signal intensity at 50 g/ml Proteinase K. This analysis shows that in aliquots of one starting liquid composition of native conformation prion protein, amplification depends on the shear force intensity applied, e.g. different total concentrations of aggregated conformation prion protein is produced when using a different shear force intensity, e.g. generated by a speed of rotation differing by e.g. 10 or by 100 Hz.

(40) This indicates that there is an optimum shear force intensity for amplification of the aggregated conformation of a prion protein.

(41) A further effect of the different shear force intensities applied during amplification can be seen for the different Proteinase K concentrations, which show that the relative resistance against proteolysis differs between shear force intensities. This result indicates that the different shear force intensities result in different aggregated conformations of one prion protein during amplification.

(42) As a second experiment, 0.001 vol/vol of 10% wt/vol aggregated conformation Sc237 (hamster) prion protein was added to native conformation prion protein hamster PrP (amino acids 23-230) at 100 g/ml to form a liquid composition for amplification using a total volume of 10 ml per reaction. For the process, 144 cycles of 60 s shear force and 9 min resting phase without agitation were performed at 37 C. An array of 7 devices as above was used, with the rotation rates controlled to 109 rpm to 406 Hz, controlled to a range of 1 Hz. Aliquots of samples were digested with 0.25 g/ml Proteinase K at 37 C. for 30 min and analysed by SDS-PAGE and Western blotting. The result is shown in FIG. 6A, showing the Western blot for samples including Sc237 hamster prions, demonstrating amplification in a shear-force dependent manner. In FIGS. 6A and 6B, the individual rates of rotation generating the shear force intensity are indicated above lanes; markings to the right of the Western blots indicate expected positions of bands for the indicated prions. As shown in the Western blot of FIG. 6B, in the absence of Sc237 hamster prions, no proteinase K resistant band is observed, indicating that no amplification occurred in this aliquot.

(43) FIG. 6 demonstrates that the amplification is strongly dependent on the shear rate, in the present example showing an optimum of 169 Hz, with the aggregated conformation prion protein PrP.sup.C at 0.25 g/ml as a positive control.

EXAMPLE 3

Analysis of Interaction of Test Compound on Amplification

(44) Analysis of interaction of test compounds with prion protein that influences the amplification of aggregated conformation prion protein from native conformation prion protein was performed using aggregated prion protein produced according to Example 2 at one shear force intensity. A test compound was added at 6 g/ml to 600 g/ml to a liquid composition of 5% vol/vol Sc237 BH in 10% wt/vol native conformation hamster prion protein shNBH. Aliquots of the composition were subjected to 144 cycles of 60 s shear force and 9 min resting phase without agitation at 37 C. An array of 12 devices as above was used, with the rotation rates controlled to 1000 rpm to 16000 rpm, controlled to a range of 1 Hz. Aliquots of samples were digested with 5 to 50 g/ml Proteinase K at 37 C. for 30 min and analysed by SDS-PAGE and Western blotting.

EXAMPLE 4

Production of Distinct Aggregated Conformation Prion Protein Using Different Shear-force Intensities

(45) Using an admixture of aggregated conformation Sc237 prion protein and a 10% preparation of normal hamster brain extract, aliquots were subjected to different shear-forces using the array of devices of Example 1. Shear-forces were generated at the rotation speeds indicated in Hz, samples were taken at 0, 1, 3, 6, 12, and 22 h during amplification (cycles of 5 s rotation, 5 min resting phase) as indicated.

(46) FIG. 7 shows sections of the Western Blots of the immunologically detected amounts of proteinase K-resistant, i.e. aggregated conformation prion protein. Proteinase K digestion was at 50 g/mL for 1 h at 37 C., followed by SDS-PAGE and Western Blotting. The progress of the amplification can be seen as the increase in intensities of aggregated conformation prion protein for each shear-force (Hz). The graphic analysis of the relative amount of aggregated conformation prion protein as derived from the Western Blot analysis is shown in FIG. 8 for each of the sampling periods. This analysis shows that amplification efficacy is dependent on the shear-force applied, in this experiment generating three distinct maximal amplification rates. FIG. 9 shows a graphic analysis of the kinetics of the amplification for those shear-forces (Hz) yielding maximum amplification.

(47) These results show that the method produces a homogenous aggregated conformation prion protein at specific distinct shear-forces, because the efficacy and rate of the amplification are dependent on the intensity of the shear-force applied.

EXAMPLE 5

Production of Homogenous Aggregated Conformation Prion Protein Using One Shear-force Intensity

(48) For producing a preparation of aggregated conformation prion protein, Sc237 was admixed at 1/2500 with 50 g/mL recombinantly produced and purified native conformation prion protein in a total reaction volume of 10.0 mL. For generating the shear-force, a rotating shear-force generator as described in Example 1 was rotated at 756 Hz at +/1 Hz for 60 s with 540 s resting phase for 136 cycles. The comparative spontaneous amplification product was generated under the same processing conditions but with no aggregated prion protein added to the initial reaction, i.e. without Sc237 as a seeding agglomerated conformation prion protein.

(49) FIG. 10 shows an SDS-PAGE (silver stained) of the initial admixture of Sc237 with the native conformation prion protein (left lane) and of the produced aggregated conformation product. To the left, marker protein sizes are given in kDa, on the right, the migration patterns of three different PrP.sup.c (aggregated conformation prion protein) fragments are indicated. The increase in homogeneity of the protein in the reaction volume due to the shear-force applied is evident.

(50) The produced aggregated conformation product was pelleted by centrifugation, yielding a total of 6 mg proteinase K-resistant protein when isolated. This protein, which following the production process is unperturbed, was used for solid-state .sup.13C-NMR.

(51) As a comparative sample, the product of spontaneous amplification (without initial aggregated prion protein) was used similarly for .sup.13C-NMR.

(52) The CP-MAS NMR: .sup.13C, .sup.13C correlation is shown in FIG. 11 for the comparative spontaneous amplification product, and in FIG. 12 for the aggregated conformation product obtained by the process of the invention using controlled shear-force. The enlarged sections of the correlation graph show the upper right hand sections, namely the spectral region in which characteristic cross-peaks for isoleucine can be observed. In comparison to the spontaneous amplification product, the aggregated conformation product obtained by the process of the invention shows distinct signals for four of four isoleucine residues, indicating that this product predominantly consists of one conformation of aggregated prion protein, i.e. the distinct signals show a high homogeneity of the conformation of the product of the process of the invention. Further, the comparison of the enlarged section of the NMR spectrum of FIG. 12 shows enlarged peaks that correspond to .sup.13C.sup./.sup.13C.sup.1 and .sup.13C.sup./.sup.13C.sup.2 atom pairs of individual amino acids. These results show that the chemical shifts depend on the shear force, as the product obtained at 756 Hz (Sc237-756 Hz) shows four paired isoleucine peaks, which differs from the product obtained at 189 Hz (Sc237-189 Hz) showing three paired isoleucine peaks and from the product of the comparative spontaneous agglomeration (Spont. 189 Hz) which shows at least eleven paired isoleucine peaks, indicating structural inhomogeneity.

EXAMPLE 6

Analysis of a Mammalian Sample for Presence of Aggregated Conformation Prion Protein

(53) Analysis of the presence of aggregated conformation, i.e. disease-associated prion protein in a mammal was done using serial dilutions of 5% vol/vol Sc237 BH . The sample dilution was added to 10% wt/vol native conformation hamster prion protein, e.g. shNBH. Aliquots of the composition were subjected to 144 cycles of 60 s shear force and 9 min resting phase without agitation at 37 C. An array of 12 devices as above was used, with the rotation rates controlled to 1000 rpm to 16000 rpm, controlled to a range of 1 Hz. Aliquots of samples were digested with 5 to 50 g/ml Proteinase K at 37 C. for 30 min and analysed by SDS-PAGE and Western blotting.

EXAMPLE 6

Device for Controlled Shear-force Generation Using Sonotrode

(54) This Example shows a currently preferred embodiment of a device for application of controlled shear-force to the inner volume of reaction vessels using ultrasound.

(55) As shown in FIG. 13, the surface 40 of a sonotrode 40 forms the bottom wall of a transfer liquid bath. Side walls 42 of the transfer liquid bath are arranged in a fluid-proof manner to the sonotrode 41, e.g. by sealing spacers 43. The transfer liquid bath is adapted to provide a thickness of the transfer liquid corresponding to the wave-length of the resonance frequency of the sonotrode 41 by the side walls 42 extending to the wave-length of the resonance frequency of the sonotrode 41 in perpendicular to the sonotrode surface 40. Accordingly, the transfer liquid level is pre-set by the side-walls 42 when filling the bath with transfer liquid, e.g. using filling pipe 44. Preferably, the device has an exit 45 for transfer liquid passing over the rim of side walls 42. The sonotrode 41 is coupled to a drive and converter unit 46. Reaction vessels 31 are each positioned at a distance to the sonotrode surface 40. The reaction vessels are each held in a clamping device 50 of a holder 51 that preferably is part of a controlled positioning device, which preferably is computer-controlled, e.g. having servo-drives for accurately positioning vessels 31. As depicted, in a simple embodiment for positioning vessels 31, the holders 51 are guided, e.g. in a threaded bore 53 provided at a distance over the transfer liquid and at a spacing from the sonotrode 41. The threaded bore can e.g. be arranged in a cover 54 over the transfer liquid bath. Cover 54 preferably is vibrationally decoupled from the sonotrode 41 and side walls 42, e.g. by arranging cover 54 on a support which is spaced from the sonotrode 41 and from the side walls 42.

(56) The arrangement of holder 51 in a guiding bore 53 allows to accurately and repeatedly position the vessel 31 in relation to the shear-force generator, in this embodiment represented by the sonotrode 41.

(57) FIG. 14 in greater detail shows that each reaction vessel 31 is held in a clamping means 50 that is mounted on a holder 51. The reaction vessel 31 contains an inner abutment face 52, against which the reaction vessel 31 is located for providing a reproducible position of the reaction vessel 31 at each holder 50. Preferably, the holder 51 is part of a computer-controlled positioning device, e.g. a robotic arm.

REFERENCE NUMERAL LIST

(58) 1 rotary element 2 axle 3 first bearing 4 second bearing 5 vessel 6 boring 7 holder 8 lid 10 first element of coupling 11 drive 12 second element of coupling 13 control unit 14 sensor 20 tube 21 exit opening 22 polymer tube 23 shoulder 24 sleeve 25 inner shoulder 30 housing 31 vessel 40 sonotrode surface, sonicator surface 41 sonotrode, sonicator 42 side wall 43 spacer 44 filling pipe 45 exit for transfer liquid 46 drive and converter unit 50 clamping means 51 holder 52 abutment face 53 threaded bore 54 cover