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WO2015160007A1 - Procédé et dispositif de détection de protéine prion pathogène infinitésimale - Google Patents

Procédé et dispositif de détection de protéine prion pathogène infinitésimale Download PDF

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Publication number
WO2015160007A1
WO2015160007A1 PCT/KR2014/003340 KR2014003340W WO2015160007A1 WO 2015160007 A1 WO2015160007 A1 WO 2015160007A1 KR 2014003340 W KR2014003340 W KR 2014003340W WO 2015160007 A1 WO2015160007 A1 WO 2015160007A1
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sample
pathogenic
pmca
horn
prp
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PCT/KR2014/003340
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English (en)
Korean (ko)
Inventor
박영재
박정호
최은경
김용선
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한림대학교 산학협력단
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Publication of WO2015160007A1 publication Critical patent/WO2015160007A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B06—GENERATING OR TRANSMITTING MECHANICAL VIBRATIONS IN GENERAL
    • B06B—METHODS OR APPARATUS FOR GENERATING OR TRANSMITTING MECHANICAL VIBRATIONS OF INFRASONIC, SONIC, OR ULTRASONIC FREQUENCY, e.g. FOR PERFORMING MECHANICAL WORK IN GENERAL
    • B06B3/00—Methods or apparatus specially adapted for transmitting mechanical vibrations of infrasonic, sonic, or ultrasonic frequency
    • 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 microscopic diagnostic method of infectious spongiform encephalopathy (prion disease) using an improved ultrasound apparatus, and more particularly, to a method for amplifying and detecting a trace amount of pathogenic prion protein through an improved ultrasound apparatus.
  • CJD Creutzfeldt-Jakob disease
  • electroencephalography / cerebrospinal fluid / radiography and clinical course, with characteristic clinical symptoms, and confirmed by laboratory tests.
  • Laboratory diagnosis is made the sequence analysis and histopathological examination of the 14-3-3 protein detection, PrP Sc detected prion gene in the brain tissues of CJD patients and the way in cerebrospinal fluid.
  • PrP Sc detected prion gene in the brain tissues of CJD patients and the way in cerebrospinal fluid.
  • all of these diagnostic methods are tests for diagnosis and confirmation after the onset of the disease, and the early diagnosis for the prevention is impossible.
  • CJD infection caused by blood transfusions is urgent for early diagnosis of prion disease.
  • Another object of the present invention is to select a glass as a sample tube material in order to increase the cavitation during the sonication process, and to amplify the pathogenic prion protein more effectively than the conventional PMCA by producing a horn cross section.
  • the present inventors applied electric energy to the cathode and the anode during the incubation process of the PMCA culture and the sonication cycle to improve the amplification of the pathogenic prion protein.
  • the technique of applying energy is mixed with "ePMCA").
  • ePMCA electrosonication
  • the vial and a horn section of a glass material with a curved surface buffers and trace amounts to an enhanced amplification technique than conventional PMCA conditions in an appropriate combination of the surface active agent additives pathogenic PrP Sc detection of It was confirmed that this is possible.
  • the present invention as a result of trying to detect the trace amount of the pathogenic prion protein using an automated ePMCA device, generates an ultrasonic wave such as a piezoelectric element, a horn, a booster, a converter (converter) in the existing PMCA device
  • an ultrasonic wave such as a piezoelectric element, a horn, a booster, a converter (converter) in the existing PMCA device
  • the device was improved, and the glass tube and horn cross section were modified to increase the cavitation, and the water-cooled heat retainer (constant circulation system) was installed to facilitate temperature control, thereby improving the limit of prion protein detection.
  • the present invention used at least one of MES, PIPES, ACES, MOPSO, TES, HEPES and HEPPS as a buffer and the concentration of 50 ⁇ 500mM further improved the detection limit of the prion protein.
  • the present invention was tested by the addition of non-ionic surfactants such as Tween 80, Triton X-100, Brij, Lubrol, PEG in the content of 0.5 ⁇ 2%, it was possible to detect even more trace amounts of pathogenic prion protein.
  • non-ionic surfactants such as Tween 80, Triton X-100, Brij, Lubrol, PEG in the content of 0.5 ⁇ 2%, it was possible to detect even more trace amounts of pathogenic prion protein.
  • Figure 1 schematically shows the protein misfolding cycle amplification apparatus (mixed with ePMCA) of the present invention.
  • Figure 2 shows the ultrasonic converter system of the protein misfolding cycle amplification apparatus according to the present inventors Patent No. 1098185, the right side shows that the horn upper cross section is formed in a concave curved center.
  • the left side of Figure 3 shows the ultrasonic converter system of the protein misfolding cycle amplification apparatus according to the present inventors Patent No. 1098185, the right side forms the upper surface of the horn to the concave curved surface of the center, and continuously applying electricity in the cup horn tank It shows a state in which the negative electrode and the positive electrode is installed so that.
  • Figure 4 shows a schematic diagram of the constant temperature cycling system of the protein misfolding cycle amplification apparatus (ePMCA) according to the present invention.
  • PrP C and PrP Sc were treated with concentration-dependent protease (PK) followed by Western blot.
  • PK protease
  • + treated
  • + not treated.
  • Lane 2 treated with 10% PrP C , 50 ⁇ g / ml PK;
  • Lane 3 treated with 10% PrP C , PK 100 ⁇ g / ml;
  • Lane 6 treated with 0.1% PrP Sc , PK 50 ⁇ g / ml;
  • PrP C used recHamPrP (recombinant Hamster Prion Protein).
  • PrP Sc homogenized hamster brain tissue infected with 263K, and experimented to make each concentration (10 -6 ⁇ 10 -30 ). Use 100 ⁇ g / ml recHamPrP to PrP C and PrP Sc was added to dilute the still at a concentration of 1/100 to PrP C was performed ePMCA, ilsong-PMCA and Misonix-PMCA.
  • Figure 6a is an experiment using the ePMCA device
  • Lane 1 was carried out 1.5 rounds with 0.001% (10 -5 ) PrP Sc in 100 ⁇ g / ml recHamPrP.
  • Lanes 2 to 15 show the result of performing ePMCA by continuously diluting 0.0001% (10 ⁇ 6 ) PrP Sc by 1/100 to 100 ⁇ g / ml recHamPrP.
  • ePMCA is confirmed that the detected at a concentration of 10 -30. This is the result of confirming that the ePMCA device is the most amplified.
  • Figure 6b is an experiment using the ilsong-PMCA apparatus, lane 1 was put in 0.001% (10 -5 ) PrP Sc in 100 ⁇ g / ml recHamPrP was carried out 1.5 rounds. Lanes 2 to 8 show the result of performing ePMCA by continuously diluting 0.0001% (10 ⁇ 6 ) PrP Sc by 1/100 to 100 ⁇ g / ml recHamPrP. It was confirmed that ilsong-PMCA was detected at a concentration of 10-18 .
  • Figure 6c is an experiment using the Misonix-PMCA apparatus, lane 1 was carried out 1.5 rounds with 0.001% (10 -5 ) PrP Sc in 100 ⁇ g / ml recHamPrP.
  • Lanes 2 to 8 show the result of performing ePMCA by continuously diluting 0.0001% (10 ⁇ 6 ) PrP Sc by 1/100 to 100 ⁇ g / ml recHamPrP. Misonix-PMCA was confirmed to be detected at a concentration of 10 -12 .
  • PK protease, +: treated,-: not treated.
  • FIG. 7 is a prototype photograph of the ePMCA device of the present invention.
  • the upper right and lower two photographs and the lower left photograph are both enlarged photographs of the upper side of the tube rack into which the sample tubes are inserted. It can be seen that the cathode and anode are installed.
  • the present invention is a.
  • steps (b) and (c) are repeated twice or more times prior to step (d).
  • the protein misfolding cycle amplification apparatus comprising an ultrasonic generator, a converter, a booster and a horn to perform protein misfolding cyclic amplification
  • An ultrasonic generator for providing electrical energy to generate ultrasonic waves to perform the step of decomposing the aggregates of (c),
  • a converter including a piezoelectric element for converting electrical energy provided by the ultrasonic generator into ultrasonic vibration
  • a booster for amplifying the ultrasonic vibrations converted by the converter is a booster for amplifying the ultrasonic vibrations converted by the converter
  • Cup horn tank coupled to the top of the horn and a constant temperature bath is formed therein to incubate the sample
  • a tube rack for placing a sample tube containing a sample on the cup horn tank
  • a constant temperature circulation system for supplying and discharging water to the cup horn tank
  • a direct current electricity supply member for continuously applying electric energy (electric energy) in order to supply the direct current electricity in the culturing step (b),
  • a protein misfolding cyclic amplification apparatus comprising a cathode and an anode respectively connected to the DC electricity supply member in a cup horn tank.
  • the constant temperature circulation system in the present invention is characterized in that it comprises a temperature sensor for measuring the temperature of the water or the sample of the cup horn tank.
  • the sample tube is characterized in that the glass material.
  • the thermal conductivity is higher than that of the synthetic resin such as polypropylene, so that the amplification efficiency is increased.
  • the upper cross section of the horn in the present invention is characterized in that the center portion is formed in a curved surface. If the upper cross section of the horn is flat rather than curved, when the tube rack contains multiple samples in the tube rack, the ultrasonic waves delivered to the sample tube through the horn are not uniform, and strong ultrasonic waves are transmitted only to the center sample tube. In order to solve the problem, the upper cross section of the horn was manufactured in a concave shape at the center. The upper cross section of the horn with a concave center portion actively causes cavitation to transmit stronger ultrasonic waves to the sample, thereby increasing the amplification efficiency.
  • step (d) measuring the presence or content of pathogenic isoforms in the sample, wherein steps (b) and (c) are repeated twice or more times prior to step (d). It provides a method for detecting pathogenic prion protein by protein misfolding cyclic amplification.
  • the DC electricity is preferably in the range of DC 5-50V and 10-100W, most preferably 24V / 30W. If the direct current energy is too strong, the equipment tends to corrode, and if too weak, the amplification efficiency is reduced.
  • the present invention also provides a method for detecting a pathogenic prion protein, characterized in that the sulfonic acid-containing buffer of pH 7.0 ⁇ 8.0 is added as a buffer in the above method.
  • the present invention also provides the sulfonic acid-containing buffer containing 2- ( N -morpholino) ethanesulfonic acid (MES), piperazine- N, N' -bis (2-ethanesulfonic acid) (PIPES), N -2-acetami Fig. 2-Aminoethanesulfonic acid (ACES), 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and N-2-hydroxyethylpiperazine-N'-2-propanesulfonic acid (HEPPS) is characterized in that at least one member selected from the group consisting of.
  • MES 2- ( N -morpholino) ethanesulfonic acid
  • PPES piperazine- N, N' -bis (2-ethanesulfonic acid)
  • MOPSO 3- (N-morpholino) -2-hydroxyprop
  • the present invention also provides a method for detecting a pathogenic prion protein, characterized in that the non-ionic surfactant is added to or below the critical micelle concentration in the buffer.
  • the present invention is characterized in that the nonionic surfactant is at least one selected from the group consisting of Tween 80, Triton X-100, Brij, Lubrol and polyethylene glycol.
  • the present invention is characterized in that the nonionic surfactant is in the range of 0.5 to 2% (w / w).
  • step (d) measuring the presence or content of pathogenic isoforms in the sample, wherein steps (b) and (c) are typically repeated two or more times prior to step (d).
  • DC electricity was continuously applied in the incubation process.
  • the aggregate decomposition step is generally performed using ultrasonic waves.
  • the PMCA apparatus also adopts a method of decomposing aggregates using ultrasonic waves.
  • An improved PMCA device of the present invention is an ultrasonic generator for providing electrical energy to generate ultrasonic waves for carrying out the step of decomposing the aggregates of (c), and converting the electrical energy provided by the ultrasonic generator into ultrasonic vibrations.
  • Converter a booster for amplifying the ultrasonic vibration converted by the converter, a horn for transmitting the ultrasonic vibration amplified by the booster to the sample, coupled to the upper portion of the horn and a constant temperature bath is formed therein to incubate the sample.
  • Cup horn tank, a constant temperature circulation system for supplying and discharging water to the cup horn tank, and comprises a tube rack for fixing the sample tube is located above the cup horn tank.
  • the present inventors selected a piezoelectric element capable of producing high output and excellent durability due to large electromechanical coupling coefficient and mechanical quality coefficient value as a piezoelectric element to be used in a converter.
  • the piezoelectric element composite piezoelectric element materials such as PbO, TiO 2 , ZrO 2 , Sb 2 O 3 , Nb 2 O 5, and MnO 2 were selected among the elements for converting electrical energy into mechanical vibrations. Bonding the ultrasonic radiating surface made of metal such as aluminum or stainless steel to the composite piezoelectric element, and applying an electric signal of 60Hz, vibration of 20KHz frequency is generated and output power can be controlled up to 0 ⁇ 2000W. A converter was configured.
  • Boosters and horns are made of titanium and are strong converters. It was used as a material to amplify the output of. Ultrasonically amplified by the booster, it is designed to spread out into the cup horn solution. Detailed drawings are shown in FIG. 1.
  • a 40-60% potency 40 second ultrasonic wave is applied for 40 seconds in a state of being maintained at 37 ° C. for 30 minutes. Normally, 96 cycles are carried out for two days with one round. Misonix's PMCA units are all placed in an incubator maintained at 37 ° C. Experiment with rounds, sometimes cycles from 15 to 30 days. Misonix's PMCA equipment will maintain temperature in an air-cooled heat-maintaining manner.
  • Supattapone and his colleagues performed PMCA experiments at 4, 25, and 37 ° C to obtain the best amplification results at 37 ° C (Lucassen R. et al., Biochemistry , 2003, 42, 4127-4135).
  • the present inventors have tried to improve the experimental error due to the temperature change that may occur when the Misonix PMCA device is put into the incubator and horn and / / water-cooled heat maintenance system that can provide a uniform temperature conditions Or it was invented to attach to a cup horn.
  • water inlet and outlet pipes were formed in the constant temperature water tank inside the cup horn to form the outside of the cup horn tank. It is designed to not affect the sample during the generation of ultrasonic waves and to reduce the experimental error due to temperature.
  • a temperature sensor is attached to the cup horn tank to check and control the temperature of the water in the cup horn tank in real time, and to prevent protein denaturation due to high temperature, and a protection box for maintaining the temperature and suppressing ultrasonic noise. Also, it can be added to be integrally movable. In addition, in the present invention, the angle of the plane horn shape was adjusted to a sphere so that ultrasonic waves are uniformly transmitted.
  • the ilsong-PMCA apparatus was detected more than the conventional Misonix apparatus. It was confirmed that the limit was improved (see Patent No. 1098185). This is because the improved PMCA device maintains a uniform temperature and immediately changes and maintains a high temperature generated after application of ultrasonic waves to a temperature set by a user, thereby suppressing protein denaturation due to high temperature.
  • the ultrasonic strength in the piezoelectric element of the converter can be set in a variety of 0 ⁇ 2000W, it is possible to apply ultrasonic waves stronger than the conventional PMCA device (maximum 600W power output).
  • Patent Publication No. 1098185 the same addition of CB (conversion buffer) (PBS, 0.15M NaCl, 1% Triton X-100, Complete Protease Inhibitor Cocktail, pH 7.0-7.3) used in the conventional PMCA method
  • CB conversion buffer
  • surfactants are substances that adsorb on the interface and significantly lower the interfacial tension, and are called emulsifiers, solubilizers, wetting agents, and detergents depending on the application. If structurally increases the configuration of ⁇ - sheet solubility (solubility) is applying a surfactant to the low PrP Sc of PrP Sc.
  • solubility solubility
  • the present inventors obtained an experimental result that the conversion of PrP C to PrP Sc is suppressed when a surfactant having a CMC concentration or higher is added. This seems to inhibit contact of normal prion protein with modified prion protein because surfactants added in excess of CMC form micelles.
  • Nonionic surfactants such as Tween 80, Triton X-100, Brij, Lubrol, PEG, etc. were found to increase the detection limit of prion protein when added at a concentration of 1-2% (see Patent No. 1098185). .
  • the present inventors selected composite piezoelectric element materials such as PbO, TiO 2 , ZrO 2 , Sb 2 O 3 , Nb 2 O 5, and MnO 2 from among ceramic piezoelectric elements in order to use them as ultrasonic transducers.
  • the frequency of 20KHz comes out when 60Hz electric signal is given, and the converter is configured to control the output power from 0 ⁇ 2000W.
  • Boosters and horns are made of titanium.
  • the incubation process is carried out, and a constant temperature circulation system is applied so that the desired temperature is uniformly applied in the cup horn tank. Was produced.
  • the water supply and drain pipes were connected to the cup horn tank and the flow rate was adjusted.
  • the temperature sensor was mounted on the cup horn tank to minimize the temperature change and to measure the water temperature of the tank in real time. Since the sample is contained in the water tank, the water temperature of the water tank and the sample temperature are the same. In fact, after the ultrasonic wave is applied, the temperature of the sample shows a high temperature (50 to 60 ° C). Therefore, we tried to solve problems such as protein denaturation and non-uniform amplification due to evaporation of moisture at high temperature.
  • the present inventors further improved the ilsong-PMCA device so that the cathode and the anode are positioned in the cup horn as shown on the right side of FIG. 3, and the DC electricity supply member is connected with the cathode and the anode to continuously connect the DC electricity to the cathode and the anode during the culturing process. Supplied.
  • the inventors of the present invention to form a curved upper surface of the upper cross section of the horn in order to evenly transmit the ultrasonic wave to a plurality of sample tubes to be inserted into the tube rack was formed in the center concave.
  • the inventors replaced the sample tube with a glass tube instead of the existing polypropylene tube.
  • the experiment was divided into a control group consisting of normal animals of the same age as the experimental group infected with scrapie.
  • ME7 and 139A scrapie strains were infected with SJL, ME7, ICR mice, and 22L scrapie strains were infected with C57BL mice.
  • 87V scrapie strains were infected in MB mice, 263K and 139H scrapie strains in hamsters.
  • Infection method was injected with 0.01M phosphate buffer (PBS, pH 7.4) containing 1% (w / v) brain homogenate infected with scrapie strain, 30 ⁇ l in mice and 50 ⁇ l in hamsters. I was.
  • PBS 0.01M phosphate buffer
  • Hamsters infected with scrapie strain 263K apparently develop 70 days after infection, 152 days for mice infected with scrapie strain 22L, 158 days for experimental animals infected with scrapie strain ME7, 139A, and 139H, and 287 days for experimental animals infected with scrapie strain 87V. Brain tissues were harvested at the expense of clinical symptoms. The brain tissues of each scrapie-infected brain and uninfected controls used for extraction were stored at -70 ° C.
  • the extracted brain tissues were placed in CB buffer selected for each experiment and homogenized using a homogenizer to make a concentration of 10% (w / v). Thereafter, centrifugation was performed at 1,500 rpm for 30 seconds using a centrifuge, and the mixture was separated into a supernatant and a precipitate layer (pellet). At this time, the supernatant was carefully taken and used in the experiment. Infected experimental animal brain tissue should be used immediately after the brain tissue was extracted and amplified.
  • the PMCA technique is a method consisting of repeated incubation ⁇ ultrasonic sonication as one cycle.
  • DC 24V / 30W electricity was applied while incubating for 29 minutes and 20 seconds.
  • Ultrasonic grinding was performed for seconds.
  • Ultrasonic grinding power was optimized for experiments of 40-60%.
  • the temperature was best maintained at 37 ° C. during the PMCA. Above 37 ° C, the moisture increased a lot and evaporated to the tube cap. Therefore, the amplification efficiency was lowered because the state in the tube containing the test sample was not uniform, and the concentration of the lower part where the ultrasonic wave was applied was increased due to evaporation of water.
  • one cycle consists of 30 minutes, and 96 cycles (48 hours, 2 days) are called one round, and usually continue in one round, two rounds, three rounds, and the like.
  • the experimental conditions associated with the PMCA experiment were based on Soto C.'s method (Castilla J. et al., Methods Enzymol . 2006, 412, 3-21).
  • Insoluble fractions of brain tissue extracts from hamsters and mice infected with scrapie strains were loaded onto 15% SDS polyacrylamide gel (SDS-PAGE) on a vertical electrophoresis device (Bio-Rad) and electrophoresed at 70 V, 30 mA. This was transferred to nitrocellulose membrane and treated with TBS containing 5% skim milk and 0.1% Tween-20 to prevent nonspecific immune responses, followed by anti-prion antibodies 3F10 (1: 5,000), 3F4 (1 : 5,000) was treated and then treated with TBS containing 0.1% Tween-20 to wash the nitrocellulose membrane (Choi JK, et al., Hybridoma (Larchmt) , 2006, 25, 271-277).
  • mice After treatment with peroxidase-labeled anti-mouse IgG, chemofluorescence was performed to detect the presence and content of brain tissue prion protein in hamsters and mice.
  • the mouse monoclonal 3F4 and 3F10 anti-prion antibodies are antibodies capable of detecting both PrP C and PrP Sc .
  • the PMCA apparatus improved by the present inventors is equipped with a water-cooled heat retainer (constant circulation system) in the horn instead of air cooling as disclosed in Patent No. 1098185, and configured to allow water to enter and exit the cup horn tank (ilsong-). Called PMCA).
  • the ilsong-PMCA is further improved.
  • An improved PMCA device (called ePMCA) of the present invention is shown in FIGS.
  • Conventional PMCA devices (Misonix) all operate in an incubator of an air-cooled heat retainer type maintained at 37 ° C., and sometimes continue to operate for 15 to 30 days. However, the amplification effect is lowered due to the temperature change occurring at this time. Therefore, the present inventors have modified the air-cooled heat retainer method to provide a uniform temperature condition to improve the experimental error caused by the temperature when the conventional PMCA device is put into the air-cooled heat retainer type incubator ( Constant temperature circulation system) was mounted on the horn, and water was introduced into and out of the cup horn tank as shown in FIG.
  • a temperature sensor is installed in the cup horn to check and adjust the temperature in real time. When the temperature exceeds the set temperature range, the protein denatures due to the high temperature.
  • a protection box that maintains the temperature and can suppress the ultrasonic noise is also manufactured in one piece.
  • a composite piezoelectric element material such as PbO, TiO 2 , ZrO 2 , Sb 2 O 3 , Nb 2 O 5 , MnO 2, etc. was used as an ultrasonic wave generating converter, and an ultrasonic room made of metal such as aluminum or stainless steel was used.
  • an ultrasonic room made of metal such as aluminum or stainless steel was used.
  • the material of the horn is titanium, which is stronger than the alloy of aluminum and titanium, which is a horn material of the conventional device, and the converter is also made of titanium so as to transmit a strong horn output.
  • the ilsong-PMCA device was confirmed that the stability is obtained as a result of use.
  • the negative electrode and the positive electrode connected to each of the direct current supply member in a cup horn tank by A more advanced protein misfolding cyclic amplification device was made.
  • the upper cross section of the horn when there is a sample tube containing several samples in the tube rack, ultrasonic waves transmitted to the sample tube through the horn are not uniform, and only strong ultrasonic waves are placed in the center.
  • the upper cross section of the horn was prepared in the form of a concave center. The upper cross section of the horn with a concave center portion actively causes cavitation to transmit stronger ultrasonic waves to the sample, thereby increasing the amplification efficiency.
  • the brain tissue of the experimental animal was secured and the protease K was treated by concentration.
  • Experimental conditions were set using 50, 100 and 200 ⁇ g / ml.
  • Protease K (PK) was treated in a shaker at 45 °C, 150rpm.
  • PK Protease K
  • FIG. 5 Western blots of typical PrP Sc were observed in the brain tissue of experimental animals infected with scrapie strain.
  • concentrations of brain tissues of infected animals were set to 0.1% and 0.5%, and protease K was treated at 50, 100 and 200 ⁇ g / ml, respectively, PrP Sc was not degraded in proportion to the concentration of protease K. Did. That is, the concentration of protease K was strong or weak or the pattern of PrP Sc was the same.
  • FIG. 6b is an experiment using the ilsong-PMCA apparatus, lane 1 was put in 0.001% (10 -5 ) PrP Sc in 100 ⁇ g / ml recHamPrP was carried out 1.5 rounds. Lanes 2 to 8 show the result of performing ePMCA by continuously diluting 0.0001% (10 ⁇ 6 ) PrP Sc by 1/100 to 100 ⁇ g / ml recHamPrP. It was confirmed that ilsong-PMCA was detected at a concentration of 10-18 .
  • Figure 6c is an experiment using the Misonix-PMCA apparatus, lane 1 was carried out 1.5 rounds with 0.001% (10 -5 ) PrP Sc in 100 ⁇ g / ml recHamPrP. Lanes 2 to 8 show the result of performing ePMCA by continuously diluting 0.0001% (10 ⁇ 6 ) PrP Sc by 1/100 to 100 ⁇ g / ml recHamPrP. Experiments with the improved ePMCA and ilsong-PMCA devices were more effective than those with the Misonix-PMCA device. The content of PrP Sc was higher (FIGS. 6A-6C).
  • ePMCA was detected up to a concentration of 10 -30
  • ilsong-PMCA was detected at a concentration of 10 -18
  • Misonix-PMCA was detected at a concentration of 10 -12 .
  • the present invention is useful for diagnosing prion diseases using trace amounts of samples.

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Abstract

La présente invention concerne un procédé de diagnostic infinitésimal d'une encéphalopathie spongiforme transmissible (également connue sous le nom de la maladie à prion) qui utilise un appareil ultrasonore amélioré et, plus spécifiquement, améliore la capacité de détection par amplification d'une protéine prion pathogène infinitésimale par application d'électricité à un appareil ultrasonore amélioré. Il a été confirmé que l'appareil ultrasonore de la présente invention améliore l'amplification d'une protéine prion pathogène par amélioration de la fonction d'amplification cyclique du mauvais repliement des protéines (PMCA) existante, et peut détecter des plus petites quantités par rapport à la condition PMCA existante par modification de la section transversale supérieure d'un flacon et d'une sonde constitué d'un matériau de verre de façon à être une surface incurvée afin d'augmenter la cavitation.
PCT/KR2014/003340 2014-04-17 2014-04-17 Procédé et dispositif de détection de protéine prion pathogène infinitésimale WO2015160007A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030036257A (ko) * 2000-07-07 2003-05-09 어플라이드 리서치 시스템스 에이알에스 홀딩 엔.브이. 입체형태적 질환의 조기 진단
US20060263769A1 (en) * 2005-05-09 2006-11-23 Panomics, Inc. Multiplex capture of nucleic acids
JP2007527504A (ja) * 2003-06-19 2007-09-27 アプライド リサーチ システムズ エーアールエス ホールディング ナームロゼ フェンノートシャップ プリオン変換を調節する因子の使用
EP2280028A1 (fr) * 2008-05-28 2011-02-02 Incorporated Administrative Agency National Agriculture and Food Research Organization Procédé d'amplification efficace d'une protéine prion anormale dérivée de la tremblante du mouton
US20110124843A1 (en) * 2008-05-28 2011-05-26 Incorporated Administrative Agency National Agriculture And Food Research Organization Method for efficiently amplifying abnormal prion protein derived from bse
KR101098185B1 (ko) * 2010-01-14 2011-12-23 한림대학교 산학협력단 극미량 병원성 프리온 단백질 검출방법 및 장치

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030036257A (ko) * 2000-07-07 2003-05-09 어플라이드 리서치 시스템스 에이알에스 홀딩 엔.브이. 입체형태적 질환의 조기 진단
JP2007527504A (ja) * 2003-06-19 2007-09-27 アプライド リサーチ システムズ エーアールエス ホールディング ナームロゼ フェンノートシャップ プリオン変換を調節する因子の使用
US20060263769A1 (en) * 2005-05-09 2006-11-23 Panomics, Inc. Multiplex capture of nucleic acids
EP2280028A1 (fr) * 2008-05-28 2011-02-02 Incorporated Administrative Agency National Agriculture and Food Research Organization Procédé d'amplification efficace d'une protéine prion anormale dérivée de la tremblante du mouton
US20110124843A1 (en) * 2008-05-28 2011-05-26 Incorporated Administrative Agency National Agriculture And Food Research Organization Method for efficiently amplifying abnormal prion protein derived from bse
KR101098185B1 (ko) * 2010-01-14 2011-12-23 한림대학교 산학협력단 극미량 병원성 프리온 단백질 검출방법 및 장치

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