KR101483438B1 - Ultra-efficient Replication Method of Infectious Prions and Device thereof - Google Patents
Ultra-efficient Replication Method of Infectious Prions and Device thereof Download PDFInfo
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- KR101483438B1 KR101483438B1 KR20140045783A KR20140045783A KR101483438B1 KR 101483438 B1 KR101483438 B1 KR 101483438B1 KR 20140045783 A KR20140045783 A KR 20140045783A KR 20140045783 A KR20140045783 A KR 20140045783A KR 101483438 B1 KR101483438 B1 KR 101483438B1
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Abstract
The present invention relates to a method for diagnosing infectious spongiform encephalopathy (aka, prion disease) using improved ultrasound equipment, more specifically, by amplifying a trace amount of pathogenic prion protein by applying electricity to an improved ultrasonic apparatus, . The ultrasonic device of the present invention improves the existing PMCA (Protein misfolding cyclic amplification) function to improve the amplification of the pathogenic prion protein. In addition, in order to increase the cavitation, the vial and horn section of the glass are curved And it is confirmed that it is possible to detect more minute amounts than the existing PMCA conditions.
Description
The present invention relates to a method for diagnosing infectious spongiform encephalopathy (prion disease) using an improved ultrasonic apparatus, and more particularly, to a method for amplifying and detecting a trace amount of pathogenic prion protein through an improved ultrasonic apparatus.
CJD (CJD) diagnosis is performed by observing brain waves / cerebrospinal fluid / radiology and clinical progress along with characteristic clinical symptoms, and performing confirmation through laboratory test method. Experimental diagnoses include 14-3-3 protein detection in cerebrospinal fluid, PrP Sc detection in brain and tonsillar tissues of CJD patients, nucleotide sequence analysis and histopathological examination of prion genes. However, all of these diagnostic methods are diagnostic methods for the diagnosis and confirmation after the onset of the disease, and it is impossible to make early diagnosis for prevention. In addition, CJD infection caused by blood transfusion highlighted the urgent need for early diagnosis of prion disease.
In Korea, confirmation of human prion disease is only made by detecting PrP Sc in brain tissue after the death of a diseased patient. However, the autopsy of patients who die from CJD is avoided, and early diagnosis of CJD infection is difficult because of insufficient accumulation of PrP Sc . Therefore, in order to solve the problem of the detection limit of the trace PrP Sc , studies are being conducted to develop diagnostic techniques with high sensitivity and accuracy. As a result of these studies, up-to-date detection methods such as PMFA (protein misfolding cyclic amplification), immuno-PCR, and QUICK-induced conversion have been studied to amplify pathogenic PrP Sc (Aguzzi et al., Annu Rev Neurosci . , 2008, 31, 439-477).
The PMCA (protein misfolding cyclic amplification) technique has been actively pursued with the PMCA device since its publication in Nature in 2001 by Claudio Soto et al. (Saborio GP, et al., Nature , 2001, 411, 810-813). The development of highly sensitive and accurate diagnostic technology has been carried out in order to be able to diagnose the samples (cerebrospinal fluid, blood, urine, etc.) that are easier to obtain than the brain tissue. Since 2004, a paper on the early diagnosis detection method using PMCA has been published , Early diagnosis is possible in the blood of experimental animals. In 2007, Yuichi Murayama of Japan and Claudio Soto of 2008 published a paper on PrP Sc detection from laboratory animal urine. Amplification of the modified prion using PMCA has made possible identification and trace detection of human prion pathogens (Saa P. et al., J. Biol . Chem . , 2006, 281, 35245-35252). In 2011, Baskakov's team in the US announced a PMCAb technique that improved the amplification of modified prion proteins by increasing the efficiency of PrP Sc fragmentation by adding Teflon beads in the PMCA process (Gonzalez MN et al., PLoS Patohg . , 2011, 7 (2), 1-10). As a result of the PMCAb technique for the amplification of CWD, it was confirmed that the modified prion protein was effectively amplified (Johnson CJ et al., PLoS ONE , 2012, 7 (4), 1-7).
The PMCA principle is based on an automated PMCA model (Castilla J. et al., Methods Enzymol ., 2006, 412, 3-21) in collaboration with Claudio Soto and the US Misonix, The company's automated PMCA equipment is used for research. Therefore, the inventors of the present invention purchased the Misonix Model 4000 and conducted experiments. However, the inventors of the present invention encountered problems such as corrosion of the horn, temperature unevenness of the cup horn (air-cooled heat storage), and output power .
In order to solve this problem, we have registered Korea Patent No. 1098185 under the title of "Method and apparatus for detection of trace pathogenic prion proteins" in 2011.
SUMMARY OF THE INVENTION An object of the present invention is to provide a method and apparatus for detecting a pathogenic prion protein having an improved amplification effect during a culture process by improving the problems of the conventional PMCA apparatus mentioned above.
Another object of the present invention is to effectively amplify a pathogenic prion protein more than the existing PMCA by selecting glass as a vial material and forming a horn section with a curved surface in order to increase cavitation during the ultrasonic treatment process.
The inventors of the present invention improved the amplification of pathogenic prion proteins by applying electric energy to the cathode and the anode during the culture process in the course of the PMCA culture and the ultrasonic treatment cycle. Energy mixing technology with "ePMCA"). In addition, to increase the cavitation of ultrasound, glass vials and horn sections were made with curved surfaces. By the appropriate combination of buffers and surfactant additives, detection of the trace of pathogenic PrP Sc .
The present invention
(I) contacting a sample with a quantity of non-pathogenic conformer,
(Ii) applying electric energy in incubating the sample and the non-pathogenic dimorphism by incubation,
(Iii) decomposing any aggregates formed during step (i) or (ii)
(Iv) measuring the presence or content of a pathogenic dendritic cell in the sample, wherein steps (ii) and (iii) are performed by repeating the protein misfolding cycle amplification A 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 electric energy for generating ultrasonic waves to perform the step of decomposing the aggregate of (iii)
A converter including a piezoelectric element for converting electric energy provided by the ultrasonic generator into ultrasonic vibration,
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,
A cupfish water tank coupled to an upper portion of the horn and having a constant temperature water tank formed therein,
A tube rack for placing a sample tube containing the sample on the upper part of the cup-
A constant-temperature circulation system for supplying and discharging water to the cup-
A DC electric supply member for continuously applying electric energy to supply DC electricity in the culturing process of the step (ii)
The present invention provides a protein misfolding cyclic amplification apparatus, comprising a cathode and an anode, each of which is connected to the DC electricity supplying member in a cupophone water tank.
Further, in the present invention, the thermostatic circulation system includes a temperature sensor for measuring the temperature of water or a sample of the cupophone water tank.
Further, in the present invention, the sample tube is a glass material. When the sample tube is made of glass, the thermal conductivity is higher than that of a synthetic resin material such as polypropylene, and the amplification efficiency is increased.
Further, in the present invention, the upper end surface of the horn is formed to be curved so that the central portion thereof is concave. If the upper section of the horn is a flat rather than a curved surface, if there is a sample tube containing several samples in the tube rack, the ultrasonic wave transmitted to the sample tube through the horn is not uniform and strong ultrasound is transmitted only to the sample tube located at the center. In order to solve the problem, the upper section of the horn was made concave at the center. The upper section of the concave horn with the central part is actively cavitated and can transmit stronger ultrasound to the sample, thereby increasing the amplification efficiency.
In addition,
(I) contacting a sample with a quantity of non-pathogenic conformer,
(Ii) continuously applying DC electricity between the cathode and the anode in the incubation process of incubating the sample and the non-pathogenic dimorphism,
(Iii) decomposing any aggregates formed during step (i) or (ii)
(Iv) measuring the presence or content of a pathogenic dendritic cell in the sample, wherein steps (ii) and (iii) are performed by repeating the protein misfolding cycle amplification protein misfolding cyclic amplification method to detect pathogenic prion protein. The DC current is preferably in the range of 5 to 50 V DC and 10 to 100 W, and most preferably 24 V / 30 W. If the DC electric energy is too strong, the equipment is likely to corrode, and if it is too weak, the amplification efficiency will decrease.
Further, the present invention provides a method for detecting a pathogenic prion protein, characterized by adding a sulfonic acid-containing buffer solution having a pH of 7.0 to 8.0 as a buffer in the above method.
In addition, the present invention is the 2-sulfonic acid-containing buffer solution (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) And N-2-hydroxyethylpiperazine-N'-2-propanesulfonic acid (HEPPS).
In addition, the present invention provides a method for detecting a pathogenic prion protein, wherein a non-ionic surfactant is added to the buffer solution at a concentration lower than or equal to the critical micelle concentration.
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
(I) contacting a sample with a quantity of non-pathogenic conformer,
(Ii) continuously applying DC electricity between the anode and the cathode while incubating the sample and the non-pathogenic heterogeneous body at a constant temperature,
(Iii) decomposing any aggregates formed during step (i) or (ii)
(Iv) measuring the presence or content of the pathogenic dendritic cells in the sample. Usually, the step (ii) and the step (iii) are repeated two or more times before the step (iv). DC was continuously applied during the incubation. The aggregate decomposing step is generally performed using ultrasonic waves. Conventional PMCA devices also employ a method of decomposing aggregates using ultrasonic waves.
The improved PMCA apparatus of the present invention comprises an ultrasonic generator for providing electric energy for generating ultrasonic waves in order to perform the step of decomposing the aggregate of (iii), an ultrasonic generator for converting the electric energy provided by the ultrasonic generator into ultrasonic vibration 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, a horn connected to the upper part of the horn and having a constant temperature water tank, And a tube rack for fixing the sample tube on the upper side of the cupphoon water tank.
The present inventors have selected a piezoelectric device which is excellent in durability and capable of producing high output, because the electromechanical coupling factor and the mechanical quality factor are large as a piezoelectric device to be used in a converter. The piezoelectric element is made of PbO, TiO 2 , ZrO 2 , Sb 2 O 3 , Nb 2 O 5 And MnO 2 And the like were selected. An ultrasonic wave emitting surface made of metal such as aluminum or stainless steel is adhered to a composite piezoelectric element and an electric signal of 60Hz is applied normally so that a vibration of a frequency of 20KHz occurs and a power control function of 0 ~ A converter was constructed. When the probe is attached to the converter with a long metal probe, the vibration is amplified as the probe moves along the probe. The booster and the horn are made of titanium material, As a material for amplifying the output of the test piece. The ultrasonic waves amplified by the booster were designed to spread out into the solution in the cup horn. The detailed drawing is shown in Fig.
Conventionally, the PMCA apparatus is set to a cycle in which 40 seconds of potency 40 to 60% ultrasound is applied for 40 seconds while maintained at 37 占 폚 for 30 minutes. Usually, 96 cycles are performed for one day in two rounds. All PMCA devices from Misonix are housed in an incubator maintained at 37 ° C to provide 1, 2, 3, ... Continue the round and experiment, sometimes cycling continuously from 15 to 30 days. Misonix's PMCA equipment maintains its temperature with an air-cooled thermal hold. Supattapone scientists performed the PMCA experiments at 4 ° C, 25 ° C and 37 ° C to obtain the best amplification results at 37 ° C (Lucassen R. et al., Biochemistry , 2003, 42, 4127-4135). Therefore, the present inventors have attempted to improve the experimental error caused by the temperature change that may occur when the PMCA device of Misonix Co., Ltd. is put into an incubator. In order to provide uniform temperature condition, a constant temperature circulating system of a water- Or cupholing. As shown in FIG. 1, a water inlet pipe and a water outlet pipe are formed outside the cuphole water tank, and water is continuously supplied from the middle part of the horn to the surface of the cuphole water tank by closely contacting the lower part of the horn. It was designed so that it does not affect the sample during ultrasonic wave generation, and the experiment error due to temperature was reduced. In addition, a temperature sensor can be attached to the cup water tank to check the temperature of the water in the cup water tank in real time, so that the protein denaturation due to high temperature can be suppressed. In addition, a protection box for suppressing the ultrasonic noise, Can be added so that they can move integrally. Also, in the present invention, the angle of the horn shape of the plane is adjusted to be spherical so as to uniformly transmit ultrasonic waves.
The present inventors have conducted an experiment to compare and verify the prion protein amplification using the PMCA apparatus and the ilsong-PMCA apparatus improved by the present inventors through the patent No. 1098185. As a result, the ilsong-PMCA apparatus was detected from the existing Misonix apparatus (See Japanese Patent No. 1098185). This is because the improved PMCA device keeps the uniform temperature and immediately changes the temperature from the high temperature generated after the application of the ultrasonic waves to the user-set temperature, thereby suppressing protein denaturation due to high temperature. In addition, since the ultrasonic intensity can be varied from 0 to 2000 W in the piezoelectric element of the converter, it is possible to apply a stronger ultrasonic wave than the conventional PMCA device (up to 600 W power output).
The present inventors have also found that in the patent publication No. 1098185, the same effect as in the case of adding the CB (conversion buffer) (PBS, 0.15M NaCl, 1% Triton X-100, complete protease inhibitor cocktail, pH 7.0 to 7.3) In addition to examining the results of detection of prion protein by Western blotting with the conventional PMCA device and the improved PMCA device under the condition, various experiments were carried out by adding buffers and surfactants to improve the detection limit of PrP Sc in PMCA equipment saw. First, the experiment was conducted by replacing the PBS (conversion buffer), CB, which was preferentially applied to the PMCA method, with another buffer solution. 2 - (N - morpholino) ethanesulfonic acid (MES), piperazine - N, N '- bis (2-ethanesulfonic acid) (PIPES), N-2-acetamido-2-aminoethanesulfonic acid (ACES) , N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxyethylpiperazine- N'-2-propanesulfonic acid (HEPPS). As a result of experiments using buffer solutions such as MES, PIPES, ACES, HEPES, MOPS and CHES instead of PBS, the detection limit of prion protein was further improved and a trace amount of PrP Sc could be detected.
The present inventors added a surfactant as an additive to further increase the detection limit of prion protein. The surfactant is a substance that adsorbs at the interface and significantly lowers the interfacial tension, and is called an emulsifier, a solubilizer, a wetting agent, and a detergent 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 By increasing the solubility of PrP Sc by adding a small amount of surfactant, which is expected to increase the solubility, it facilitates the contact between normal and modified prions and makes the contacted PrP C convert to PrP Sc during the application of PMCA technique gave. Therefore, when a small amount of surfactant was added to increase the solubility of modified prion protein, more PrP C was converted to PrP Sc during one cycle of culture → ultrasonic pulverization. However, micelles in the form of water-in-oil (O / W) form at micelle concentration above critical micelle concentration (CMC), and micelles can inhibit the contact between PrP C and PrP Sc . Thus, as the concentration of the surfactant increases, some molecules or ions of the surfactant form and aggregate and form various associative colloids. This association is called micelle, which is formed at concentrations above CMC. Since the solubility of the surfactant changes from solution to colloidal solution to CMC, the physicochemical properties of the solution such as surface tension and general properties change significantly. The present inventors obtained experimental results that the conversion of PrP C to PrP Sc is inhibited when a surfactant at a CMC concentration or higher is added. This suggests that surfactants that are added in excess of CMC may inhibit contact between normal and modified prion proteins because they form micelles. It was confirmed that the addition of 1% to 2% of nonionic surfactant such as Tween 80, Triton X-100, Brij, Lubrol and PEG improves the detection limit of prion protein (see Patent Registration No. 1098185) .
As described above, the present invention has made efforts to detect a micro-pathogenic prion protein using an automated ePMCA device. As a result, it has been found that the present PMCA device can generate ultrasound such as a piezoelectric element, a horn, a booster, In order to improve cavitation, the sample tubes and horn sections of glass were modified to curved surfaces. During the incubation, electrical energy was continuously applied to improve the amplification efficiency and water-cooled heat oil circulation system Thereby facilitating temperature control and improving the detection limit of prion protein.
In addition, the present invention uses at least one of MES, PIPES, ACES, MOPSO, TES, HEPES and HEPPS as a buffer, and the detection limit of the prion protein is further improved by using the concentration of 50 to 500 mM.
In addition, the present invention can detect a trace amount of pathogenic prion protein by adding 0.5 to 2% of nonionic surfactant such as Tween 80, Triton X-100, Brij, Lubrol and PEG.
Figure 1 schematically shows a protein misfolding cycle amplification device of the present invention (mixed with ePMCA).
2 shows the ultrasonic converter system of the protein misfolding cycle amplifying apparatus according to the present invention's patent No. 1098185, and the right side shows a horn-side end surface formed with a concave curved surface at the center.
The left side of FIG. 3 shows the ultrasonic converter system of the protein misfolding cycle amplifying apparatus according to the patent No. 1098185 of the present inventors. On the right side, the cross section of the horn side is formed into a concave curved surface at the center, And a negative electrode and a positive electrode are provided so as to be able to perform the same.
Figure 4 shows a schematic diagram of the thermostatic circulation system of the protein misfolding cycle amplification device (ePMCA) according to the present invention.
FIG. 5 is a Western blot performed as a preliminary experiment after preparation of PrP C and PrP Sc samples for protein misfolding cyclic amplification. PrP C and PrP Sc were treated with concentration-dependent proteolytic enzymes (PK) and then Western blotted. PK: protease, +: treated, -: not treated.
6 is a comparative analysis experiment using the apparatus (ePMCA) of the present invention, the apparatus according to Korean Patent No. 1098185 (ilsong-PMCA) and the Misonix-PMCA apparatus of the present invention. PrP C used recHamPrP (recombinant Hamster Prion Protein). PrP Sc was used to homogenize 263K-infected hamster brain tissue and make experiments at each concentration (10 -6 to 10 -30 ). EPCMC, ilsong-PMCA and Misonix-PMCA were performed by adding PrP C to 100 μg / mL recHamPrP and diluting PrP Sc to 1/100 of the concentration to PrP C. 6A is an experiment using an ePMCA device.
7 is a photograph of a prototype of the ePMCA device of the present invention. The upper right and lower two photographs and the lower left photograph are all enlarged photographs of the upper side of the tube rack for inserting the sample tube, showing that the cathode and the anode are installed.
Hereinafter, the configuration of the present invention will be described in more detail with reference to examples. However, it is apparent to those skilled in the art that the scope of the present invention is not limited by the description of the embodiments below.
Example 1: Device of the present invention ( ePMCA )
In order to use the piezoelectric transducer as an ultrasonic transducer, PbO, TiO 2 , ZrO 2 , Sb 2 O 3 , Nb 2 O 5 And MnO 2 And the like were selected. When the electric signal of 60Hz is applied, the frequency is 20KHz and the converter is configured to adjust the output power from 0 to 2000W. Booster and horn are made of titanium material. In order to apply the PMCA principle, the PMCA apparatus is inserted into the incubator apparatus to carry out the culturing process, but the constant temperature circulation system is applied so that the desired temperature can be uniformly applied in the cupphoon water tank, and the ilsong-PMCA apparatus Respectively. The water supply and drainage pipes were connected to the cup water tank and the flow rate was adjusted. In addition, the temperature sensor was mounted on the cuphoon tank to minimize the temperature change and the water temperature of the tank was measured in real time. Since the sample is contained in a water tank, the temperature of the water in the water tank and the temperature of the sample are the same. In practice, the temperature of the sample after application of ultrasonic waves is high (50 to 60 ° C). Therefore, we tried to solve problems such as denaturation of proteins and non-uniform amplification due to evaporation of water at high temperature.
The present inventors have further improved the ilsong-PMCA device so that the cathode and the anode are positioned in the cuphole and the DC supply member is connected to the cathode and the anode as shown in the right side of FIG. 3, Respectively.
In order to uniformly transmit ultrasonic waves to a plurality of sample tubes that are inserted into a tube rack, the present inventors formed concave upper surfaces of the horns with concave central portions.
Further, the present inventors replaced the sample tube with a glass tube instead of the conventional polypropylene tube.
Example 2: ePMCA for PrP C Wow PrP Sc Sample preparation
6 weeks old inbred mice (C57BL / 6J, SJL / J, ICR, MB) and golden syrian hamster (SHa) were ordered from the experimental animals. The scrapie strain ME7, 139A, 22L, 87V, 263K, and 139H, which lead to scrapie disease, were received from Dr. Alan Dickins of the neuropathogenesis unit, edinburgh, scotland.
The experiment was divided into a control group consisting of a scrapie-infected experimental group and a normal animal of the same age. ME7 and 139A scrapie strains were infected with SJL, ME7, ICR mice and 22L scrapie strains were infected with C57BL mice. 87V scrapie strain was infected with MB mouse, 263K and 139H scrapie strain with hamster. Infection was performed by injecting 0.01 M phosphate buffer (PBS, pH 7.4) containing 1% (w / v) brain homogenate infected with scrapie strain, 30 μl for mouse and 50 μl for hamster . Scrapie Strain 263K-infected hamsters apparently developed 70 days after infection, 152 days for scrapie strain 22L mice, 158 days for experimental animals infected with scrapie strain ME7, 139A and 139H, and 287 days for experimental animals infected with scrapie strain 87V And brain tissue was sacrificed when there were clinical symptoms. Brain tissue from each scrapie-infected and uninfected control group used for harvest was stored at -70 ° C.
Each of the extracted brain tissues was homogenized using a homogenizer to a concentration of 10% (w / v). Then, centrifugation was carried out using a centrifuge at 1,500 rpm for 30 seconds, and the supernatant was separated into a pellet. At this time, the supernatant was carefully taken and used in the experiment. The brain tissue of the infected animal was amplified well by using brain tissue immediately after experiment.
Example 3: ePMCA experimental conditions
The PMCA technique is repeatedly constructed by incubating one cycle of ultrasonication. In this embodiment, DC 24V / 30W electricity was applied during the incubation process for 29 minutes and 20 seconds, Ultrasonic milling was performed for a second. Ultrasonic pulverization power was optimized between 40 and 60% for the experiment. The temperature was best maintained at 37 ° C during PMCA. At 37 ° C and above, the water evaporated into the tube lid. Therefore, the state of the tube containing the test sample is not uniform, and the amplification efficiency is lowered because the concentration of the bottom portion to which the ultrasonic wave is applied is increased due to moisture evaporation. Usually one cycle consists of 30 minutes, and 96 cycles (48 hours, 2 days) are called 1 round, and usually proceed in 1st round, 2nd round and 3rd round. Experimental conditions related to the PMCA experiment used the method of Soto C. (Castilla J. et al., Methods Enzymol . 2006, 412, 3-21).
Example 4: PrP C Wow PrP Sc of Western Blot analysis
Western blot was preferentially treated with proteinase K (PK) for the normal and modified prion protein concentration to confirm that the modified protein in the brain tissue of the experimental animal that induced the prion disease obtained in Example 2 was a pathogenic prion protein . The basal condition experiment was carried out by changing the concentration and temperature of the protease K and the anti-prion antibody (3F4, 3F10).
The insoluble fraction of the brain tissue extract of hamster and mouse infected with scrapie strain was loaded on a 15% SDS polyacrylamide gel (SDS-PAGE) on a vertical electrophoresis apparatus (Bio-Rad) and electrophoresed at 70 V and 30 mA. Prion antibodies 3F10 (1: 5,000), 3F4 (1: 5,000) were prepared after transferring them to a nitrocellulose membrane and treating them with TBS containing 5% skim milk and 0.1% Tween-20 to prevent nonspecific immune responses. : 5,000), followed by reaction with TBS containing 0.1% Tween-20 to wash the nitrocellulose membrane (Choi JK, et al., Hybridoma ( Larchmt ) , 2006, 25, 271-277). Then, the peroxidase-labeled anti-mouse IgG was treated and chemiluminescence was performed to detect the presence and content of brain tissue prion proteins in hamsters and mice. The mouse monoclonal 3F4, 3F10 anti-prion antibody is an antibody capable of detecting both PrP C and PrP Sc .
PrP Sc was detected in the insoluble fraction obtained after treating the brain tissue extract of 263K scrapie strain-infected hamster with protease K (Fig. 5, PK: protease K, +: treated, -: not treated ).
Result 1: Improved PMCA Device
The device using the PMCA principle is being sold on the automated PMCA model at Misonix, USA (Castilla, J. et al., Methods Enzymol ., 2006, 412, 3-21), and many scientists around the world are experimenting with automated PMCA equipment from Misonix. Therefore, the present inventors also purchased the Misonix Model 4000 to conduct experiments, but it was necessary to improve the PMCA device.
The first improved PMCA apparatus by the present inventors is a system in which a water-cooled heat oil circulation system (constant-temperature circulation system) is mounted on the horn and water is introduced into and out of the cupphoon water tank as described in Japanese Patent No. 1098185 (ilsong- PMCA).
In the present invention, the ilsong-PMCA is further improved. The improved PMCA device (referred to as ePMCA) of the present invention is shown in Figures 1-3. Conventional PMCA devices (Misonix) are all put into an incubator in an air-cooled, heat-retaining system maintained at 37 ° C, sometimes operating from 15 to 30 days. However, the amplification effect is deteriorated due to the temperature change occurring at this time. Thus, the present inventors have proposed a water-cooling type heat exchanger (hereinafter referred to as a " heat exchanger ") which can provide a uniform temperature condition by modifying an air-cooling type heat exchange method in order to improve the experimental error caused by the temperature when the PMCA device is put into an incubator of an air- A thermostatic circulation system) was mounted on the horn, and water was introduced into and out of the cup water tank as shown in FIG. The ilsong-PMCA device is equipped with a temperature sensor in the cuphole to check and adjust the temperature in real time. When the temperature exceeds the set temperature range, the protein is denatured due to the high temperature. Therefore, the temperature of the supplied water is lowered , A protection box which can keep the temperature and suppress the ultrasonic noise is also made as one body.
In addition, the converter for generating ultrasonic PbO, TiO 2, ZrO 2, Sb 2
In addition, in the present invention, in order to supply DC electricity during the culturing process, a DC electricity supplying member for continuously applying electric energy is provided, and a cathode and an anode connected to the DC electricity supplying member are added A more improved protein misfolding cyclic amplification device was constructed.
In the present invention, when the upper end surface of the horn is a flat surface rather than a curved surface, when there is a sample tube containing several samples in the tube rack, the ultrasonic waves transmitted to the sample tube through the horn are not uniform, In order to solve this problem, the upper section of the horn was made concave at the center. The upper section of the concave horn with the central part is actively cavitated and can transmit stronger ultrasound to the sample, thereby increasing the amplification efficiency.
Result 2: PrP C Wow PrP Sc of Western Blot analysis
The brain tissue of the experimental animals was secured and proteolytic enzyme K was treated by concentration. 50, 100 and 200 / / ml, respectively. Proteinase K (PK) was treated in a shaker at 45 ° C and 150 rpm. As a result, PrP C was completely decomposed and a clean Western blot was observed (FIG. 5). However, in the brain tissue of the experimental animals infected with scrapie strain, western blot of a typical PrP Sc was observed when treated as above. When the brain tissue concentrations of infected experimental animals were set at 0.1% and 0.5%, and the protease K was treated at 50, 100 and 200 μg / ml, respectively, PrP Sc was decomposed in proportion to the concentration of proteinase K I did not. That is, the concentration of protease K was strong or weak or the pattern of PrP Sc was the same.
Result 3: Improved device ( ePMCA , Ilsong - PMCA )Wow Misonix - PMCA Comparative experiment using
The Misonix-PMCA device and the ePMCA, ilsong-PMCA device invented by the present inventors were compared and compared. As shown in Figs. 6A to 6C, no PMCA was performed (0 round), and the PrP Sc band did not appear as a result of western blotting. The comparison PrP C in the
Claims (11)
(Ii) applying electric energy in incubating the sample and the non-pathogenic dimorphism by incubation,
(Iii) decomposing any aggregates formed during step (i) or (ii)
(Iv) measuring the presence or content of a pathogenic dendritic cell in the degraded sample, wherein the step (ii) and the step (iii) are performed by repeating the step A protein misfolding cycle amplification apparatus comprising an ultrasonic generator, a converter, a booster and a horn for performing misfolding cyclic amplification,
An ultrasonic generator for providing electric energy for generating ultrasonic waves to perform the step of decomposing the aggregate of (iii)
A converter including a piezoelectric element for converting electric energy provided by the ultrasonic generator into ultrasonic vibration,
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,
A cupfish water tank coupled to an upper portion of the horn and having a constant temperature water tank formed therein,
A tube rack for placing a sample tube containing the sample on the upper part of the cup-
A constant-temperature circulation system for supplying and discharging water to the cup-
A DC electric supply member for continuously applying electric energy to supply DC electricity in the culturing process of the step (ii)
And a cathode and an anode which are respectively connected to the DC electricity supplying member in the cupophone water tank, and a protein misfolding cyclic amplification device.
Wherein the thermostatic circulation system comprises a temperature sensor for measuring the temperature of water or a sample of the cupophone water tank.
Wherein the sample tube is made of glass.
Wherein an upper end surface of the horn is formed as a curved surface so that a central portion thereof is concave.
(Ii) Continuous application of DC electricity in the water tank during the incubation of the sample and the non-pathogenic dimorphism in the incubation tank in a cup-
(Iii) decomposing any aggregates formed during step (i) or (ii)
(Iv) measuring the presence or content of a pathogenic dendritic cell in the degraded sample, wherein the step (ii) and the step (iii) are performed by repeating the step A method for detecting a pathogenic prion protein by a misfolding cyclic amplification method.
Wherein the direct current is in the range of DC 5 ~ 50V and 10 ~ 100W.
A method for detecting a pathogenic prion protein, comprising: (i) adding a sulfonic acid-containing buffer solution having a pH of 7.0 to 8.0 as a buffer in a step of bringing a sample into contact with a predetermined amount of a non-pathogenic conformer.
The 2-sulfonic acid-containing buffer, the (N - morpholino) ethanesulfonic acid (MES), piperazine - N, N '- bis (2-ethanesulfonic acid) (PIPES), N-2-acetamido-2-amino N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) and N-2-hydroxymethylsulfonic acid (ACES) Wherein the protein is selected from the group consisting of N-2-hydroxypropyl-N'-2-propanesulfonic acid (HEPPS).
Wherein the non-ionic surfactant is added to the buffer solution at a concentration lower than or equal to the critical micelle concentration.
Wherein the non-ionic surfactant is at least one selected from the group consisting of Tween 80, Triton X-100, Brij, Lubrol, and polyethylene glycol.
Wherein the non-ionic surfactant is added in a range of 0.5 to 2% (w / w).
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| PCT/KR2014/003340 WO2015160007A1 (en) | 2014-04-17 | 2014-04-17 | Method and device for detecting infinitesimal pathogenic prion protein |
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| KR20190089329A (en) | 2018-01-22 | 2019-07-31 | 대한민국(농림축산식품부 농림축산검역본부장) | Detecting method of abnormal prion from soil |
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| KR20030036257A (en) * | 2000-07-07 | 2003-05-09 | 어플라이드 리서치 시스템스 에이알에스 홀딩 엔.브이. | Early diagnosis of conformational diseases |
| US20060263769A1 (en) | 2005-05-09 | 2006-11-23 | Panomics, Inc. | Multiplex capture of nucleic acids |
| JP2007527504A (en) | 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 | Use of factors that regulate prion conversion |
| 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 |
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| WO2009145194A1 (en) * | 2008-05-28 | 2009-12-03 | 独立行政法人農業・食品産業技術総合研究機構 | Method for efficiently amplifying abnormal prion protein derived from sheep scrapie |
| KR101098185B1 (en) * | 2010-01-14 | 2011-12-23 | 한림대학교 산학협력단 | Ultra-efficient Replication Method Infectious Prions and Device thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20030036257A (en) * | 2000-07-07 | 2003-05-09 | 어플라이드 리서치 시스템스 에이알에스 홀딩 엔.브이. | Early diagnosis of conformational diseases |
| JP2007527504A (en) | 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 | Use of factors that regulate prion conversion |
| US20060263769A1 (en) | 2005-05-09 | 2006-11-23 | Panomics, Inc. | Multiplex capture of nucleic acids |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20190089329A (en) | 2018-01-22 | 2019-07-31 | 대한민국(농림축산식품부 농림축산검역본부장) | Detecting method of abnormal prion from soil |
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