WO2015160007A1 - Method and device for detecting infinitesimal pathogenic prion protein - Google Patents

Abstract

The present invention relates to a method for infinitesimal diagnosis of a transmissible spongiform encephalopathy (also known as prion disease) which uses an improved ultrasonic apparatus and, more specifically, improves detection ability by amplifying an infinitesimal pathogenic prion protein by applying electricity to an improved ultrasonic apparatus. It has been confirmed that the ultrasonic apparatus of the present invention improves amplification of a pathogenic prion protein by improving the existing protein misfolding cyclic amplification (PMCA) function, and can detect more minute amounts compared to the existing PMCA condition by modifying the upper cross section of a vial and a horn made of a glass material to be a curved surface in order to increase cavitation.

Description

Trace Pathogenic Prion Protein Detection Method and Apparatus

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.

The diagnosis of Creutzfeldt-Jakob disease (CJD) is accompanied by 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. However, 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. In addition, CJD infection caused by blood transfusions is urgent for early diagnosis of prion disease.

Confirmation of human prion disease in Korea is only achieved by detecting PrP ^Sc in brain tissue after the death of a diseased patient. However, autopsy of patients who died of CJD has been avoided, and PrP ^Sc has not accumulated enough in the early stage of infection with CJD, making it difficult to diagnose. Therefore, researches are underway to develop a high sensitivity and high accuracy diagnostic technology to solve the detection limit problem of trace amount PrP ^Sc . As a result of these studies, the latest detection assays, such as protein misfolding cyclic amplification (PMCA), immuno-PCR, and QUIC-reduced conversion, which amplify pathogenic PrP ^Sc for diagnosis, are being studied (Aguzzi A). et al., Annu Rev Neurosci. , 2008, 31, 439-477).

Since the protein misfolding cyclic amplification (PMCA) technique was published in 2001 by Claudio Soto and colleagues in Nature, research on the PMCA device has been actively conducted (Saborio GP, et al., Nature , 2001, 411, 810-813). Development of diagnostic technology with high sensitivity and accuracy for diagnosis on samples that are easier to obtain than cerebral tissue (cerebrospinal fluid, blood, urine, etc.) has been conducted. Since 2004, a paper on early detection method using PMCA has been published. In addition, it has been reported that early diagnosis is possible in blood of laboratory animals. In 2007, researchers at Yuichi Murayama in Japan and Claudio Soto in 2008 published a paper on the detection of PrP ^Sc from animal urine. Amplification of prion strain using PMCA made it possible to determine and detect a very small amount of human prion agents (Saa P. et al., J. Biol. Chem., 2006, 281, 35245-35252). In 2011, the US-based Baskakov researchers published the PMCAb technique, which improved the amplification of modified prion proteins by increasing the efficiency of PrP ^Sc fragmentation by adding Teflon beads during the PMCA process (Gonzalez MN et. al., PLoS Patohg. , 2011, 7 (2), 1-10). As a result of experiments on the amplification of CWD by PMCAb technique, it was confirmed that the modified prion protein was effectively amplified (Johnson CJ et al., PLoS ONE, 2012, 7 (4), 1-7).

Devices using the PMCA principle are selling automated PMCA models in partnership with Claudio Soto and Misonix in the United States (Castilla J. et al., Methods Enzymol ., 2006, 412, 3-21), and many scientists around the world The company's automated PMCA device is being used for research. Therefore, the present inventors also purchased the 'Misonix Model 4000' and proceeded with the experiment, but problems such as corrosion of the horn, uneven temperature of the cup horn (air-cooled heat retainer method), and output power occurred. .

In order to solve this problem, the researchers registered Korean Patent No. 1098185 under the name "Method and Apparatus for Detecting Trace Trace Pathogenic Prion Protein" in 2011.

It is an object of the present invention to improve the problems of the above-mentioned conventional PMCA device to provide a method and apparatus for detecting trace amounts of prion protein having amplification effect during the culturing process.

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"). Furthermore, in order to increase the cavitation (cavitation) of the ultrasonic waves it was produced 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.

As described above, 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 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.

In addition, 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.

In addition, 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.

Figure 1 schematically shows the protein misfolding cycle amplification apparatus (mixed with ePMCA) of the present invention.

The left side of 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.

5 is a Western blot performed as a preliminary experiment after the preparation of PrP ^C and PrP ^Sc samples for protein misfolding cyclic amplification. PrP ^C and PrP ^Sc were treated with concentration-dependent protease (PK) followed by Western blot. PK: protease, +: treated,-: not treated.

Lane 1, 0.05% PrP ^C , not PK treated;

Lane 2, treated with 10% PrP ^C , 50 μg / ml PK;

Lane 3, treated with 10% PrP ^C , PK 100 μg / ml;

Lane 4, 10% PrP ^C , PK treated with 200 μg / ml;

Lane 5, 0.05% PrP ^Sc , not PK treated;

Lane 6, treated with 0.1% PrP ^Sc , PK 50 μg / ml;

Lane 7, 0.1% PrP ^Sc , treated with 100 μg / ml PK;

Lane 8, 0.1% PrP ^Sc , PK treated with 200 μg / ml;

Lane 9, 0.5% PrP ^Sc , treated with 50 μg / ml PK;

Lane 10, 0.5% PrP ^Sc , PK treated with 100 μg / ml;

Lane 11, 0.5% PrP ^Sc , PK treated with 200 μg / ml.

6 is a comparative analysis experiment using the apparatus of the present invention (ePMCA), the apparatus according to the Korean Patent No. 1098185 (ilsong-PMCA) and Misonix-PMCA apparatus. 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㎍ / ㎖ 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 ㎍ / ㎖ 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 μg / 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 ㎍ / ㎖ 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 μg / 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㎍ / ㎖ 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 μg / ml recHamPrP. Misonix-PMCA was confirmed to be detected at a concentration of 10 ^-12 . PK: protease, +: treated,-: not treated.

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

(a) contacting a sample with an amount of non-pathogenic conformer,

(b) incubating the sample with the non-pathogenic isoform to apply electrical energy;

(c) disintegrating any aggregates formed during step (a) or (b),

(c) 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). In 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,

Horn for transmitting the ultrasonic vibration amplified by the booster to the sample,

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;

And 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),

Provided is 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.

In addition, 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.

In the present invention, the sample tube is characterized in that the glass material. When the sample tube is made of glass, the thermal conductivity is higher than that of the synthetic resin such as polypropylene, so that the amplification efficiency is increased.

In addition, 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.

In addition, the present invention

(a) contacting a sample with an amount of non-pathogenic conformer,

(b) continuously applying direct current electricity between the cathode and the anode during the incubation of the sample and the non-pathogenic isoform,

(c) disintegrating any aggregates formed during step (a) or (b),

(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. At this time, 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.

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.

In addition, 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.

In addition, the present invention is characterized in that the nonionic surfactant is in the range of 0.5 to 2% (w / w).

The improved protein misfolding cyclic amplification (PMCA) method of the present invention

(a) contacting a sample with an amount of non-pathogenic conformer,

(b) continuously incubating the sample and the non-pathogenic isoform while continuously applying direct current electricity between the positive electrode and the negative electrode,

(c) disintegrating any aggregates formed during step (a) or (b),

(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. Conventionally, 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. As 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. Attaching an elongated metal probe to this converter causes vibration to amplify as the vibration passes through the probe. 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.

In the conventional PMCA apparatus, 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. In order to facilitate the amplification of the prion protein, 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). Thus, 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. As shown in FIG. 1, 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. In addition, 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.

As a result of the inventors comparing and verifying prion protein amplification using the conventional PMCA apparatus and the inventors' improved ilsong-PMCA apparatus through Patent No. 1098185, 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. In addition, since 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).

The inventors also found that in 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 Under the conditions, not only Western blotting experiments with the conventional PMCA apparatus and the improved PMCA apparatus were used to examine the results of prion protein detection, but also various buffers and surfactants were added to improve the detection limit of PrP ^{Sc in} the PMCA apparatus. saw. First, an experiment was performed by changing PBS, which is a conversion buffer (CB) applied to the PMCA method, to another buffer. 2- ( N -morpholino) ethanesulfonic acid (MES), piperazine- N, N' -bis (2-ethanesulfonic acid) (PIPES), N -2-acetamido-2-aminoethanesulfonic acid (ACES) , 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxyethylpiperazine- N'-2-propanesulfonic acid (HEPPS) and the like were used. Experiments with buffers such as MES, PIPES, ACES, HEPES, MOPS, and CHES instead of PBS resulted in further improvement in the prion protein detection limit, allowing detection of trace amounts of PrP ^Sc .

We added a surfactant as an additive to further raise the limit of prion protein detection. 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 β- sheet solubility (solubility) is applying a surfactant to the low PrP ^Sc of PrP ^Sc The expected to increase the solubility and small amount of surface active agent by increasing the solubility of PrP ^Sc and to facilitate the contact of normal prion to transformation prion, creating a PrP ^C into contact for the application of PMCA technique to work well is converted to PrP ^Sc gave. Thus, the addition of a small amount of surfactant to increase the solubility of the modified prion protein resulted in the conversion of more PrP ^C to PrP ^Sc in the course of one cycle of incubation followed by ultrasonic grinding. However, at concentrations above the critical micelle concentration (CMC), micelles may act to inhibit the contact between PrP ^C and PrP ^Sc by forming micelles (O / W). Thus, as the concentration of surfactant increases, some of the molecules or ions of the surfactant dissolve to form aggregates and form various associative colloids. This association is called micelles, which are formed at concentrations above CMC. Since the dissolved state of the surfactant changes from solution to associative colloid at the boundary of CMC, the physical and chemical properties of the solution, such as surface tension and general properties, are remarkably changed. 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). .

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples. However, it is obvious to those skilled in the art that the scope of the present invention is not limited by the description of the following examples.

Example 1 Device of the Invention (ePMCA)

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. In order to apply the PMCA principle, rather than putting the PMCA device into the incubator device as in the prior art, 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. In addition, 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.

In addition, 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.

In addition, the inventors replaced the sample tube with a glass tube instead of the existing polypropylene tube.

Example 2: PrP for ePMCA ^C And PrP ^Sc  Sample Manufacturing

As experimental animals, 6-week-old inbred mice (C57BL / 6J, SJL / J, ICR, MB) and golden syrian hamster (SHa) were used by ordering from a central experimental animal. Scrapie strains ME7, 139A, 22L, 87V, 263K, 139H, which cause scrapie disease, were provided by Dr. Alan Dickens of the Neuropathogenesis Unit (edinburgh, scotland).

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 μl in mice and 50 μl in hamsters. I was. 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.

Example 3: ePMCA Experimental Conditions

The PMCA technique is a method consisting of repeated incubation → ultrasonic sonication as one cycle. In this embodiment, 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. Typically, 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).

Example 4: PrP ^C And PrP ^Sc Western blot analysis of

Western blot was first treated with protease K (PK) by normal and modified prion protein concentration to confirm that the modified protein in the brain tissue of the experimental animal causing the prion disease obtained in Example 2 is a pathogenic prion protein. . In addition, the basic condition experiment was carried out by changing the concentration and temperature of protease K, anti-prion antibodies (3F4, 3F10).

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). 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 .

In Pass the brain tissue extracts of hamsters infected with 263K scrapie strain by proteinase K the resulting insoluble fraction was PrP ^Sc detected (Fig. 5, PK: proteinase K, +: treatment should, -: no treatment ).

Result 1: Improved PMCA Device

Device using the principle of PMCA is sold during the automated PMCA models in the United States Misonix four (Castilla, J. et al., Methods Enzymol., 2006, 412, 3-21), automating many of the world's scientists Misonix PMCA Experiment with the equipment. Therefore, the present inventors also purchased the 'Misonix Model 4000' and proceeded with the experiment, it was necessary to improve the PMCA device.

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).

In the present invention, 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. In the ilsong-PMCA device, 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. In addition, a protection box that maintains the temperature and can suppress the ultrasonic noise is also manufactured in one piece.

In addition, 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. When the electric signal of 60Hz is applied by attaching the slope, vibration of 20KHz frequency occurs, and the converter is configured to control the output power from 0 to 2,000W. Thus, according to user's needs, 0 ~ 2,000W output power can be selected and tested. 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.

In addition, in the present invention, by adding a direct current supply member for continuously applying electric energy (electric energy) in order to supply the direct current electricity in the culture process, 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.

In addition, in the present invention, when the upper cross section of the horn is a flat surface instead of a curved surface, 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. In order to solve the problem of transmitting 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.

Result 2: PrP ^C And PrP ^Sc Western blot analysis of

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 μg / ml. Protease K (PK) was treated in a shaker at 45 ℃, 150rpm. As a result, all PrP ^C was decomposed and a clear western blot was seen (FIG. 5). However, Western blots of typical PrP ^Sc were observed in the brain tissue of experimental animals infected with scrapie strain. When the 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 ㎍ / 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.

Outcome 3: comparative experiments using advanced devices (ePMCA, Ilsong-PMCA) and Misonix-PMCA

Misonix-PMCA device and the inventors invented the ePMCA, ilsong-PMCA device was compared. In the case where PMCA was not performed (round 0) as shown in FIGS. 6A to 6C, the Western blot resulted in no PrP ^Sc band. The comparison PrP ^C in the experimental conditions 100㎍ / ㎖ recHamPrP [recHamPrP (recombinant Hamster Prion Protein)] to to put a substrate (substrate), PrP ^Sc are each concentration (10 homogenizing the infected hamster brain 263K, and ^{- 6} ~ 10 ^-30 ) to perform the experiment. Use 100㎍ / ㎖ 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 ㎍ / ㎖ recHamPrP. Lanes 2-15 show the results of the ePMCA was added to keep the dilution 1/100 by a 0.0001% (10 ^-6) PrP ^Sc in 100㎍ / ㎖ 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 ㎍ / ㎖ 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 μg / 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㎍ / ㎖ 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 μg / 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 , and finally, Misonix-PMCA was detected at a concentration of 10 ^-12 . This result confirms that amplification is performed in the order of ePMCA>ilsong-PMCA> Misonix-PMCA. This resulted in the result that the ePMCA of the present invention is the most efficient equipment.

The present invention is useful for diagnosing prion diseases using trace amounts of samples.

This application includes the Ministry of Health and Welfare's R & D project "Health Pathogenesis, Interstitial Transfer, Early Diagnosis and Treatment Development through the Acquisition of Pathogen Samples of Human Prion Diseases in Korea", and Ministry of Future Creation Science It is filed as part of the “Hallim-Uppsala Research Center for the Technology Convergence Study of Related Degenerative Brain Diseases”.

Claims (11)

(a) contacting a sample with an amount of non-pathogenic conformer, (b) incubating the sample with the non-pathogenic isoform to apply electrical energy; (c) disintegrating any aggregates formed during step (a) or (b), (d) measuring the presence or content of pathogenic isoforms in the sample from which the aggregate is degraded, wherein steps (b) and (c) are repeated two or more times prior to the step (d). In 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, Horn for transmitting the ultrasonic vibration amplified by the booster to the sample, 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; And 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, characterized in that it comprises a cathode and an anode connected to the DC electricity supply member, respectively, in a cup horn tank. The method according to claim 1, The constant temperature circulation system protein misfolding cycle amplification apparatus comprising a temperature sensor for measuring the temperature of the water or the sample of the cup horn tank. The method according to claim 1, The protein misfolding cycle amplification apparatus, characterized in that the sample tube is made of glass. The method according to claim 1, The upper section of the horn protein misfolding cycle amplification apparatus, characterized in that formed in a curved surface so as to concave. (a) contacting a sample with an amount of non-pathogenic conformer, (b) continuously applying direct-current electricity in the bath during the incubation of the sample and the non-pathogenic isoform in a cuphorn bath, (c) disintegrating any aggregates formed during step (a) or (b), (d) measuring the presence or content of pathogenic isoforms in the sample from which the aggregate is degraded, wherein steps (b) and (c) are repeated two or more times prior to the step (d). A method for detecting pathogenic prion proteins by protein misfolding cyclic amplification. The method according to claim 5, The direct current is a method for detecting a pathogenic prion protein, characterized in that the range of DC 5 ~ 50V and 10 ~ 100W The method according to claim 5, (A) A method for detecting a pathogenic prion protein, comprising adding a sulfonic acid-containing buffer having a pH of 7.0 to 8.0 as a buffer in contacting a sample with a predetermined amount of non-pathogenic conformer. The method according to claim 7, The sulfonic acid containing buffer is 2- ( N -morpholino) ethanesulfonic acid (MES), piperazine- N, N' -bis (2-ethanesulfonic acid) (PIPES), N -2-acetamido-2-amino Ethanesulfonic acid (ACES), 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and N-2-hydride A method for detecting a pathogenic prion protein, characterized in that at least one selected from the group consisting of oxyethylpiperazine-N'-2-propanesulfonic acid (HEPPS). The method according to claim 7, A non-ionic surfactant is added to the buffer at or below the critical micelle concentration. The method according to claim 9, The nonionic surfactant is a method for detecting a pathogenic prion protein, characterized in that at least one selected from the group consisting of Tween 80, Triton X-100, Brij, Lubrol and polyethylene glycol. The method according to claim 9, The nonionic surfactant is added to the range of 0.5 ~ 2% (w / w) method for detecting a pathogenic prion protein.

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