JP6273338B1 - How to identify Parkinson's disease dementia from Parkinson's disease - Google Patents

Abstract

Provided is a method for identifying Parkinson's disease or Parkinson's disease dementia with normal cognitive ability in a subject. An in vitro immunomagnetic decrease (IMR) signal of α-synuclein in a blood sample of a subject is detected, wherein (a) the IMR signal binds to a magnetic nanoparticle comprising an antibody against α-synuclein. (B) applying the IMR signal of α-synuclein detected in step (a) to the logistic function (I), and calculating the concentration of α-synuclein in the blood sample, (C) The α-synuclein concentration obtained in step (b) is compared with a predetermined threshold range. If the concentration is higher than the predetermined threshold range, Parkinson's disease dementia is normal. Parkinson's disease of cognitive ability, and a process of indicating health when lower than a predetermined threshold range. [Selection figure] None

Description

    The present invention relates to a method for identifying Parkinson's disease dementia from Parkinson's disease.

  Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Over 1% of elderly people over the age of 66 suffer from PD. About 10 million people around the world live with PD. Direct or indirect medical costs per PD patient are estimated at approximately US $ 100,000 per year. Many countries, especially the United States, Canada, Europe and Australia, are worried about an unsustainable increase in health care costs. Much resources and efforts are devoted to the diagnosis, treatment, and vaccine development of PD.

  Clinical criteria for PD diagnosis are observation of movement disorders such as slow movement, gear-like stiffness, resting tremor, and unstable posture. Although these clinical features are commonly used, there are several serious problems in the diagnosis of PD. For example, other movement disorders (eg, multiple system atrophy, cerebral cortex basal ganglia degeneration, or progressive supranuclear paralysis) may overlap with the clinical symptoms of PD, reducing the accuracy of PD diagnosis. Furthermore, clinical symptoms have been reported to appear after more than 50% degeneration of dopaminergic neurons in the basal ganglia, particularly the substantia nigra. Early stage PD diagnosis using observation of clinical movement disorders is very difficult. Gene sequence analysis may be a better method for early stage PD diagnosis. Still, only 10% of PD patients are inherited. 90% of PD patients are sporadic.

  Cognitive dysfunction called Parkinson's disease dementia (PDD) and the onset of dementia are common in PD. Predicting dementia progression in PD is difficult and has a significant impact in the field. Researchers are currently working to achieve biomolecular diagnostics that distinguish PD and PDD. α-synuclein is the most recognized biomarker for PD or PDD. Alpha-synuclein molecules are phosphorylated, phosphorous-alpha-synuclein molecules that aggregate easily with each other to form Lewy bodies in dopaminergic neurons. Dopaminergic neurons with Lewy bodies degenerate and lose the ability to express dopamine. Nervous cells in the motor cortex of the brain are damaged due to lack of dopamine, and movement disorders are promoted.

  Numerous findings have shown that the formation of Lewy bodies reduces the concentration of α-synuclein in cerebrospinal fluid (CSF) in PD or PDD patients compared to healthy subjects. However, the results reported for variability in blood α-synuclein concentrations are inconsistent. The main reason for the inconsistency in the analysis results for plasma α-synuclein is that the low concentration detection limit of this analysis method is not excellent. According to these reports, enzyme immunoassay (ELISA) has been used for the analysis of α-synuclein in CSF or plasma. Alpha-synuclein is expressed in the brain and spinal cord and is abundant at these sites, but is very low in the peripheral blood system. ELISA cannot accurately detect very low concentrations of protein, such as α-synuclein in plasma. For this reason, CSF is more suitable as a substitute for plasma for analysis of α-synuclein in PD or PDD biomolecular diagnosis using ELISA.

  CSF is usually collected by lumbar puncture, which is risky and uncomfortable. Early diagnosis by analysis of α-synuclein in CSF is not widely accepted by the general population. Instead, blood can be collected much more easily at a medical institution. Therefore, a highly sensitive detection technique is required to realize analysis of ultra-low α-synuclein in plasma.

  It is an object of the present invention to provide a method for identifying normal cognitive Parkinson's disease or Parkinson's disease dementia in a subject.

A method for identifying normal cognitive Parkinson's disease or Parkinson's disease dementia in a subject of the present invention comprises (a) generating an in vitro immunomagnetic reduction (IMR) signal of α-synuclein in a blood sample of a subject. Detecting, wherein the IMR signal is generated by α-synuclein bound to magnetic nanoparticles containing antibodies against α-synuclein, and (b) the IMR signal of α-synuclein detected in step (a) Is applied to the logistic function (I) to calculate the concentration of α-synuclein in the blood sample,
(I)
Of these, IMR (%) is the IMR signal of α-synuclein, φ _α-syn is the concentration of α-synuclein, fitting parameter A is the background value, B is the maximum value, and φ _o is the IMR. The concentration of α-synuclein when the signal is equal to ((A + B) / 2), γ is the data point φ _o of the curve when φ _α-syn is the x axis and IMR (%) is the y axis (C) comparing the concentration of α-synuclein obtained in step (b) with a predetermined threshold range, and when the concentration of α-synuclein is higher than the predetermined threshold range, When it is Parkinson's disease dementia and the concentration of α-synuclein is within a predetermined threshold range, it indicates that the disease is normal cognitive Parkinson's disease, and the concentration of α-synuclein is lower than the predetermined threshold range. Low Optionally comprises a step of indicating a health.

2 is a graph showing a bioactivity test against antis immobilized on magnetic Fe ₃ O ₄ nanoparticles using immunomagnetic reduction. It is a graph which shows (alpha)-synuclein density | concentration dependence IMR signal (solid line) in 450 nm and OD450nm, and an optical-absorbance (optical absorptivity density) (broken line). is a graph showing the converted α- synuclein concentration phi _alpha-syn, and _IMR pair spiked α- synuclein concentration phi _alpha-syn in PBS solution for dynamic range analysis for α- synuclein measurement. FIG. 6 is a graph showing plasma α-synuclein concentrations detected using IMR for healthy subjects, PD patients, and PDD patients.

  The inventors have developed an immunoassay technique called immunomagnetic reduction (IMR) for quantitatively detecting biomolecules at ultra-low concentrations such as 1-10 pg / ml or less. The main contribution to the ultra-high sensitivity of IMR is the use of antibody functional magnetic nanoparticles. These magnetic nanoparticles are well dispersed in the reagent and can capture target biomolecules anywhere in the test sample. In addition, since the particles are nano-sized, the total bond exemption is very large. Therefore, antibodies immobilized on the surface of magnetic nanoparticles can bind to target biomolecules very efficiently, and an ultrasensitive immunoassay using IMR has been established. IMR has been shown to be applicable to the analysis of very low concentrations of β-amyloid and tau protein in human plasma. A clear distinction between healthy subjects and patients with mild cognitive impairment due to Alzheimer's disease was revealed by analysis of plasma β-amyloid and tau protein. These results analyze the very low levels of α-synuclein in human plasma and investigate the feasibility of achieving PD or PDD biomolecular diagnostics or distinguishing PD and PDD by plasma α-synuclein concentration Got the motivation to do. In this work, a reagent for α-synuclein analysis using IMR is prepared. Characterization of reagents and α-synuclein analysis. For further comparison, the analytical properties for α-synuclein using ELISA are examined. Finally, we report the initial results on the differentiation of PD patients, PDD patients and healthy subjects by analysis of plasma α-synuclein. Although cross-sectional studies conducted in this work cannot address the prediction of PDD progression in PD, the results may suggest the feasibility of using this method of plasma α-synuclein determination in PDD and PD is there.

A method for identifying normal cognitive Parkinson's disease or Parkinson's disease dementia in a subject of the present invention comprises (a) generating an in vitro immunomagnetic reduction (IMR) signal of α-synuclein in a blood sample of a subject. And wherein the IMR signal is generated by α-synuclein bound to magnetic nanoparticles containing antibodies against α-synuclein, and (b) the IMR signal of α-synuclein detected in step (a) Is applied to the logistic function (I) to calculate the concentration of α-synuclein in the blood sample,
(I)
Of these, IMR (%) is the IMR signal of α-synuclein, φ _α-syn is the concentration of α-synuclein, fitting parameter A is the background value, B is the maximum value, and φ _o is the IMR. The concentration of α-synuclein when the signal is equal to ((A + B) / 2), γ is the data point φ _o of the curve when φ _α-syn is the x axis and IMR (%) is the y axis (C) comparing the concentration of α-synuclein obtained in step (b) with a predetermined threshold range, and when the concentration of α-synuclein is higher than the predetermined threshold range, When it is Parkinson's disease dementia and the concentration of α-synuclein is within a predetermined threshold range, it indicates that the disease is normal cognitive Parkinson's disease, and the concentration of α-synuclein is lower than the predetermined threshold range. Low If, and a step of indicating a health.

In a preferred embodiment, the magnetic nanoparticle material is selected from the group consisting of Fe ₃ O ₄ , Fe ₂ O ₃ , MnFe ₂ O ₄ , CoFe ₂ O ₄ , NiFe ₂ O ₄ .

In another preferred embodiment, the magnetic nanoparticle material is selected from the group consisting of Fe ₃ O ₄ .

  In a further preferred embodiment, the blood sample is plasma.

  The following examples are merely illustrative of various elements and features of the invention and do not limit the invention.

  Method

Reagents for α-synuclein analysis include magnetic Fe ₃ O ₄ nanoparticles (MF-DEX-0060, MagQu), a functional monoclonal antibody against α-synuclein (sc-12767, Santa Cruz Biotech.). Detailed processes for antibody immobilization on magnetic Fe ₃ O ₄ nanoparticles are described in Horn et al. (IEEE Trans Appl Supercond. 2005; 15: 668-671) and Yang et al. (J Magn Magn Mater. 2008; 320: 2688). ~ 2691). Antibody functional magnetic _Fe 3 _{O 4} nanoparticles are dispersed in pH-7.2 phosphate-buffered saline (PBS) solution. The particle size distribution is analyzed by dynamic laser light scattering (Nanotrac-150, Microtrac). The magnetic concentration of the reagent is measured using a vibrating sample magnetometer (HysterMag, MagQu). The biological activity of the antibody on the magnetic nanoparticles is measured using an IMR analyzer (XacPro-S, MagQu). The IMR analyzer is an AC magnetic susceptometer equipped with a high-temperature superconducting quantum interferometer (High-Tc SQUID) magnetometer as a magnetic sensor. Details of the AC magnetic susceptibility meter are described in Yang et al. (IEEE Trans Appl Super Super. 2013; 23: 160064-1600607) and Chieh et al. (J Appl Phys 2008, 103: 014703-1-6). Yes. To establish the relationship between the IMR signal and the concentration of α-synuclein, α-synuclein spiked with PBS solution (ab51189, Abcam) was prepared. For each measurement of IMR signal, 80 μl of reagent was mixed with 40 μl of α-synuclein solution, followed by detection of IMR signal using an IMR analyzer (XacPro-S, MagQu). The same measurements were made on the IMR signal at each concentration of α-synuclein solution. In addition to the measurement of the IMR signal, a commercially available ELISA kit (KHB0061, Novex) ga was used to find the α-synuclein concentration-dependent absorbance unit.

  Volunteers who participated in the present invention were given medical checklists for major systemic diseases, surgery, and hospitalization. Volunteers who reported uncontrolled disease, including heart failure, recent myocardial infarction (past 6 months), malignancy (past 2 years), or poorly managed diabetes (HbA1C> 8.5) were excluded . Applicants were also given a medical examination. The present invention was approved by the University Hospital Ethics Committee and the Clinical Trial Review Board.

  Participants were required to provide 10 ml of non-fasting venous blood (K3 EDTA, lavender cap blood collection tubes). Each sample was assigned a registration number according to the sampling order. Therefore, laboratory staff were not informed of the subject's clinical status and demographic data. Blood samples were centrifuged within 1 hour of collection (2500 g, 15 minutes), plasma was divided into cryotubes, stored at −80 ° C. for up to 3 months, and then thawed for measurement by IMR. For determination of α-synuclein concentration by IMR, 80 μl of reagent was mixed with 40 μl of plasma. Similar measurements were made for each plasma sample.

  9 human plasma samples from healthy subjects aged 38-73, 9 human plasma samples from PD patients (38-85 years old), 14 human plasma samples from PDD patients (60-81 years old), Used for α-synuclein analysis using IMR. PD and PDD patients were identified using clinical symptoms. Note that PD patients are cognitively normal. Prior to performing the procedure, informed consent was provided to all participating patients and the present invention was approved by the National Taiwan University Hospital Research Ethics Committee.

The average value of the hydrodynamic diameter of the antibody functional magnetic Fe ₃ O ₄ nanoparticles was 55.5 nm, and the standard deviation of the particle hydrodynamic diameter was 12.7 nm. Using a scanning electron microscope, the average value of the diameter of the antibody functional magnetic Fe ₃ O ₄ nanoparticles was obtained as ˜40 nm. The reagent is superparamagnetic with a saturation magnetization of 0.3 emu / g. According to a previously published document (J Appl Phys 2013, 144903-1-5), the number of antibody functional nanoparticles in 1 ml of 0.3 emu / g reagent is approximately 10 ¹³ . The total surface area of antibody functional magnetic nanoparticles in 1 ml of reagent is about 1000 cm ² . In the experiment, 80 μl of reagent is used. The total surface area of antibody functional magnetic nanoparticles in 80 μl of reagent for each assay is about 80 cm ² . When compared to a 96 well ELISA plate, the binding area between the antibody and target biomolecule in each well is 0.45 cm ² . Therefore, the bonding area in IMR is nearly 180 times larger than that in ELISA.

The biological activity of the antibody immobilized on the magnetic nanoparticle is examined by measuring the IMR signal for binding between α-synuclein and the antibody on the magnetic nanoparticle. As shown in FIG. 1, the time-dependent ac magnetic susceptibility χ _ac of the reagent after mixing the reagent and the test solution was recorded. Two test samples were prepared: a pure PBS solution and an α-synuclein solution of 3.1 fg / ml. The broken line in FIG. 1 represents the time-dependent ac magnetic susceptibility χ _ac of the mixture of reagent and PBS solution. Obviously, the dashed χ _ac over time remains almost unchanged. However, for the solid line corresponding to the mixed solution of the reagent and α-synuclein 3.1 fg / ml, the χ _ac with time decreases in 45 minutes and reaches a lower level. A significant decrease in the reagent's ac magnetic susceptibility χ _ac is observed due to binding between α-synuclein and antibodies on the magnetic nanoparticles.

To quantify the decrease in reagent ac magnetic susceptibility χ _ac, the first / last χ _ac before / after binding between α-synuclein and the antibody on the magnetic nanoparticle was calculated according to the time course χ _ac shown in FIG. The As mentioned in a previously published document (ACS Chem Neurosci. 2011; 2: 500-505; J Appl Phys 2010, 107: 074903-1-5), the reduction in the ac magnetic susceptibility χ _ac of the reagent. The confidence interval for determination is within the first and last 40 to 50 minutes of the time-dependent ac susceptibility χ _ac shown in FIG. In the present invention, the data of ac susceptibility χ _ac of the reagent within the first and last 45 minutes is used to determine the decrease in χ _ac .

In FIG. 1, the p value of the magnetic susceptibility χ _ac between the first and last 45 minutes was 0.046 in the case of the PBS solution. A slight decrease is observed in the ac magnetic susceptibility χ _ac of the reagent mixed with PBS. In the case of the 3.1 fg / ml α-synuclein solution, the p value of the magnetic susceptibility χ _ac during the first and last 45 minutes was 0.007. A clear decrease in the time-dependent ac magnetic susceptibility χ _ac of the reagent after mixing with α-synuclein solution was demonstrated.

The initial χ _ac which is the average value of χ _ac within the first 45 minutes is represented as χ _{ac, o} . The final χ _ac which is the average value of χ _ac within the last 45 minutes is expressed as χ _{ac, φ} . The decrease in the ac susceptibility χ _ac of the reagent, ie the IMR signal, is obtained by the following equation (1).
IMR (%) = (χ _{ac, o} -χ _{ac, φ} ) / χ _{ac, o} × 100% (1)

  Equation (1) calculates the IMR signal for the dashed and solid lines in FIG. 1 to be 1.56 and 2.13%, respectively. The results shown in FIG. 1 show the background level of the IMR assay. Such background levels are mainly due to the electronic noise of the assay system. According to similar measurements, the IMR signal of the PBS solution is 1.56 and 1.65%. Therefore, the background level of the IMR signal is 1.61% with a standard deviation of 0.06%.

The IMR signal as a function of the concentration of α-synuclein, ie, the IMR (%)-φ _α-syn curve is shown in FIG. As the α-synuclein concentration φ _α-syn increases from 3 × 10 ⁻⁴ pg / ml (= 0.3 fg / ml), the IMR signal increases. It turned out that (phi) ( _alpha ) _-syn dependence IMR (%) follows the logistic function represented by following Formula (2).
(2)
Of these, A, B, φ _o , and γ are fitting parameters. By fitting the data points in FIG. 2 to Equation (2), the fitting parameters are acquired as A = 1.94, B = 3.95, φ _o = 49.7, and γ = 0.26. The fitting curve is shown by a solid line in FIG. Corresponding coefficient of determination ^{R 2} is 0.998. The fact that R ² is very close to 1 suggests that the φα _-syn- dependent IMR (%) conforms to the logistic function.

Parameter A in equation (2) is the value of IMR (%) when extrapolating φα _-syn to zero. Usually, the value A is slightly higher than the background level. For example, A is 1.94%, and the background level here is 1.61%. The difference between A and the background level is mainly due to noise caused by dynamic equilibrium in the binding between protein molecules and antibody functional magnetic nanoparticles. However, A is not used as a low-detection limit. Traditionally, the lower detection limit is defined as the concentration that exhibits an IMR signal that is three times higher than the standard deviation of the IMR signal for a low concentration test (ie, 3-σ criterion) than A. In this experiment, the standard deviation of the low concentration test is 0.028%. Thus, the low concentration limit is the concentration at which the IMR signal is 2.02%. From formula (2), it can be seen that the lower limit of detection of the α-synuclein assay is 0.3 fg / ml.

The α-synuclein concentration-dependent absorbance at 450 nm and OD 450 nm using ELISA is shown in FIG. The experimental data is fitted to the next logistic function.
(3)

The fitting parameters of A ^′ , B ^′ , φ _o ^′ , γ ^{′ in} the equation (3) are 0.189, 5.070, 13566.08, and 1.44. The logistic function of equation (3) is indicated by a broken line in FIG. × coefficient of determination ^{R 2} between marks and the broken line shows the 0.999. By utilizing the 3-σ criterion, the lower limit of detection of the α-synuclein assay using ELISA is 79.04 pg / ml. It is clear that IMR is 250,000 times more sensitive than ELISA for α-synuclein assays. As described above, when the reaction surface is taken into account, the detection sensitivity of IMR is 200 times higher than that of ELISA. The additional 1250 times may be due to an ultra-low noise magnetic sensor, a high temperature superconducting quantum interferometer (High-Tc SQUID) magnetometer. A High-Tc SQUID magnetometer noise level of 50 fT / Hz ^1/2 is shown, which is three orders of magnitude lower than the magnetic signal produced by a single nanoparticle. This suggests that a decrease in ac magnetic signal due to a single magnetic nanoparticle due to binding to the target biomolecule may be detectable with a High-Tc SQUID magnetometer. For this reason, the ultra-low noise High-Tc SQUID magnetometer is very sensitive to the decrease in the ac magnetic signal of the reagent and very sensitive to biomolecular assays.

In addition to the lower detection limit, the dynamic range of α-synuclein assays using IMR is an important characteristic. To examine the dynamic range, the experimental IMR signal shown in FIG. 2 is converted to α-synuclein concentration by equation (2). The concentration of converted α-synuclein is expressed as φ _{α-syn, IMR} . As shown in FIG. 3, the correlation between [phi] [ _{alpha] -syn, IMR} and [phi] [ _{alpha] -syn} is examined. In FIG. 3, linearity can be obtained between φ _{α-syn, IMR} and φ _α-syn . According to regulations promulgated by the US Food and Drug Administration (FDA), the linear slope in FIG. 3 should be between 0.90 and 1.10. In FIG. 3, when φ _{α-syn, IMR} is used for _α- synuclein concentrations φ _α-syn of 0.31 fg / ml to 31 ng / ml, φ _α-syn, The slope of the _IMR −φ _α-syn curve is 0.77, and the determination coefficient R ² is 0.991. The dotted slope in FIG. 3 does not meet US FDA requirements. It is necessary to narrow the α-synuclein concentration range in order to examine the dynamic range of the assay. For this reason, the highest φ _{α-syn, IMR} in FIG. 3, that is, 31 μg / ml φ _α-syn is ignored. 0.31~3.1ng / ml range phi _alpha-syn, linear curves between _IMR and phi _alpha-syn are indicated by broken lines in FIG. By dashed lines inclined 1.48, the coefficient of determination ^{R 2} is 0.999. The slope of the dashed line is much higher than the requirements of the US FDA. In FIG. 3, the second highest φ _{α-syn, IMR} is likely to be ignored. 0.31fg / ml~310pg / ml range phi _alpha-syn, linear curves between _IMR and phi _alpha-syn shown in solid lines in FIG. A solid line of slope 0.93, the coefficient of determination ^{R 2} is 0.999. Note that the slope of the solid line meets US FDA requirements. Therefore, the dynamic range of α-synuclein concentration in the IMR assay is 0.3 fg / ml to 310 pg / ml.

The data shown in FIG. 2 demonstrates that the IMR assay is extremely sensitive and may detect α-synuclein in human plasma. Plasma samples provided by 9 healthy subjects, 9 PD patients, and 14 PDD patients were collected for preliminary studies on the distinction between healthy subjects and PD patients and PDD patients using IMR. It was. Table 1 shows the demographic information of the 33 subjects collected. FIG. 4 shows the detected concentration φ _{α-syn, IMR} of _α- synuclein in human plasma. Plasma φα _{-syn, IMR} of PDD patients ranged from 0.1 to 100 pg / ml, while plasma φα _{-syn, IMR} of healthy subjects was much lower than 0.1 pg / ml. The plasma φ _{α-syn, IMR} of PD patients was distributed between those of healthy subjects and those of PDD patients. The p value for plasma φ _{α-syn, IMR} between healthy subjects and PD patients is 0.005, revealing the fact that PD patients show higher plasma α-synuclein concentrations compared to healthy subjects . In FIG. 4, a clear distinction in plasma φ _{α-syn, IMR} between PD and PDD patients was observed (p <0.001). According to the results of FIG. 4, plasma α-synuclein concentrations continue to rise as healthy subjects suffer from PD and progress to PDD. It should be noted that the age was matched between healthy subjects and PD patients (p> 0.05) and between PD patients and PDD patients (p> 0.05).

  In previous studies, α-synuclein is released from nerve cells by exocytosis into body fluids including CSF and plasma, which may contribute to cell-to-cell transmission of α-synuclein pathological changes in the brain. It is shown. Numerous studies have focused on checking total or oligomeric α-synuclein levels in plasma samples taken from PD patients and comparing them to healthy controls, but the results are inconsistent. Since phosphorylated and fibrotic α-synuclein is the major pathologic variant of the protein, one recent study showed that phosphorylated α-synuclein was found in early PD samples without dementia compared to the control group. Higher plasma levels were observed. Through these observations, it is possible that plasma levels of α-synuclein (total or oligomeric or phosphorylated forms) may partially reflect the pathological changes of α-synuclein in the brain of PD patients. Suggests feasibility and potential. Furthermore, cortical Lewy body / neurite lesions are more extensive in PDD than in PD without dementia, suggesting that α-synuclein deposition in plasma is more severe in PDD than in PD doing. Our results supported this hypothesis that plasma levels of α-synuclein are only slightly higher in normal cognitive PD than in healthy controls, but much higher in PDD. In order to better understand the pathophysiology of PDD, amyloid β plaques and tau neurofibrillary tangles that are characteristic pathologies of Alzheimer's dementia were also observed and correlated with cognitive status in PDD patients. Future research is needed that incorporates the evaluation of phosphorylated-α-synuclein, amyloid β protein, total and phosphorylated tau at plasma levels.

  In plasma samples, heterophile antibodies are a major confounding factor and interfere with assay results by sandwich ELISA. According to the guidance of the Clinical and Laboratory Standards Institute (CLSI-EP-A2: Interference Testing of Clinical Chemistry), heterophilic antibodies (HA) are common in immunoassays. It is defined as one of the interfering substances. The IMR method shows low interference and high specificity when compared to ELISA through previous studies. The mechanism of selection is based on the centrifugal force contributed by the vibrating magnetic nanoparticles in the reagent. Details are explained in previous studies. In fact, the mechanism of selection prevents binding of naturally occurring biomolecules of drugs frequently used in plasma as well as HA to magnetic nanoparticles. This characterizes IMR as a highly specific method for clinical analysis of plasma biomarkers of Parkinson's disease.

  Clinically, patients are first diagnosed with PD and may develop dementia at a later stage of the disease, thereby making a diagnosis of PDD. Therefore, a biomarker that can be diagnosed at an early stage of PDD prediction or progression in PD patients has clinical significance. According to the results of FIG. 4, plasma α-synuclein in PDD patients shows a clearly higher level than PD patients (p <0.001). This suggests the promising use of plasma α-synuclein as a clinical parameter to monitor progression to PDD in PD patients.

  By immobilizing antibodies against α-synuclein to magnetic nanoparticles, reagents for assaying α-synuclein are developed. By utilizing immunomagnetic reduction (IMR) with the assistance of a High-Tc SQUID magnetometer, the dynamic range of the α-synuclein assay is 0.3 fg / ml to 310 pg / ml. An ultrasensitive SQUID-based IMR is applied to human plasma α-synuclein assays. The rudimentary results indicate that there is a clear difference in plasma α-synuclein concentrations in healthy subjects, PD patients, and PDD patients. This method shows the promise of applying IMR to the diagnosis of PD and PDD by assaying plasma α-synuclein.

Nothing

Claims (4)

A method of identifying normal cognitive Parkinson's disease or Parkinson's disease dementia in a subject comprising:
(A) In vitro immunomagnetic reduction (IMR) signal of α-synuclein in a blood sample of a subject was detected, and the IMR signal was bound to magnetic nanoparticles containing an antibody against α-synuclein. a step produced by α-synuclein;
(B) fitting the IMR signal of α-synuclein detected in step (a) to the logistic function (I) to calculate the concentration of α-synuclein in the blood sample;
(I)
Of these, IMR (%) is the IMR signal of α-synuclein, φ _α-syn is the concentration of α-synuclein, fitting parameter A is the background value, B is the maximum value, and φ _o is the IMR. The concentration of α-synuclein when the signal is equal to ((A + B) / 2), γ is the data point φ _o of the curve when φ _α-syn is the x axis and IMR (%) is the y axis A process that is inclined at
(C) The α-synuclein concentration obtained in step (b) is compared with a predetermined threshold range, and when the α-synuclein concentration is higher than the predetermined threshold range, it is confirmed that the disease is Parkinson's disease dementia. If the concentration of α-synuclein is between a predetermined threshold range, it indicates normal cognitive Parkinson's disease, and if the concentration of α-synuclein is lower than the predetermined threshold range, it indicates that the subject is healthy. A process of showing,
A method for identifying Parkinson's disease or Parkinson's disease dementia with normal cognitive power, comprising: The material of the magnetic nanoparticles is selected from the group consisting of Fe ₃ O ₄ , Fe ₂ O ₃ , MnFe ₂ O ₄ , CoFe ₂ O ₄ , NiFe ₂ O _4. A method for identifying Parkinson's disease or Parkinson's disease dementia with normal cognitive ability as described in 1. The method for identifying Parkinson's disease of normal cognitive ability or Parkinson's disease dementia according to claim 1, wherein the magnetic nanoparticle material is Fe ₃ O ₄ .   The method for identifying Parkinson's disease or Parkinson's disease dementia of normal cognitive ability according to claim 1, wherein the blood sample is plasma.