Early diagnosis and early screening kit for joint detection of Parkinson's disease and application of kit

Abstract

The invention discloses an early diagnosis and early screening kit for joint detection of Parkinson's disease and application of the kit, and relates to the technical field of medical treatment. The kit is used for combined detection of three proteins, namely alpha synuclein (alpha-Syn), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activated protein zeta (YWHAZ) and myosin light chain 6 (Myl6), immunochromatography, chemiluminescence and other methods and a double-antibody sandwich method are adopted, the diagnosis efficiency of the YWHAZ protein is prominent, the AUC reaches 0.96, and the detection sensitivity is high. The AUC of Myl6 is 0.92, the AUC of alpha-Syn is 0.82, the joint detection AUC is up to 0.98, the kit is newly added with a time-resolved fluorescence immunochromatography technology, low-background and high-sensitivity detection is realized through a lanthanide-labeled antibody, the change of related protein expression level can be detected in the early stage of Parkinson's disease, early screening and diagnosis are realized, and the kit has the advantages of high specificity, joint detection up to 95%, high sensitivity, high sensitivity and the like. The joint detection reaches 96%, the operation is simple and convenient, and the sample requirement is low.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Parkinson's disease combined detection early diagnosis and screening kit and application thereof. Background Art

[0002] Parkinson's disease (PD) is a common degenerative disease of the elderly nervous system, clinically characterized by resting tremor, bradykinesia, muscle rigidity and postural balance disorders. With the acceleration of the global aging process, the incidence of Parkinson's disease has increased year by year, bringing a heavy burden to society and families. At present, the diagnosis of Parkinson's disease mainly relies on clinical symptoms and signs, but in the early stages of the disease, these symptoms are often atypical and easy to misdiagnose and miss. Therefore, the development of an early and accurate diagnosis method for Parkinson's disease has important clinical significance. The detection of biomarkers provides a new idea for the early diagnosis of Parkinson's disease. In recent years, more and more studies have shown that the expression levels of some protein markers in Parkinson's patients have changed significantly. These protein markers are expected to become important indicators for the early diagnosis of Parkinson's disease. Among them, α-synuclein (α-Syn), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein zeta (YWHAZ) and myosin light chain 6 (Myl6) play an important role in the occurrence and development of Parkinson's disease. The combined detection of these three proteins can improve the accuracy of early diagnosis of Parkinson's disease.

[0003] Time-resolved fluorescence immunochromatography (TRFIA) technology uses lanthanide-labeled antibodies and has the advantages of a wide excitation light wavelength range, a narrow emission light wavelength range, a long fluorescence lifetime, and a large Stokes shift. It can effectively reduce background interference, improve detection sensitivity and quantitative accuracy, and is suitable for the detection of early changes in minor protein expression. Summary of the invention

[0004] The present invention provides a Parkinson's disease combined detection early diagnosis and screening kit and application thereof, which can effectively solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a Parkinson's disease combined detection early diagnosis and screening kit, comprising a kit for combined detection of alpha-synuclein (α-Syn), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein zeta (YWHAZ) and myosin light chain 6 (Myl6).

[0006] According to the above technical scheme, immunochromatography, chemiluminescence or other detection methods based on the principle of double antibody sandwich method are used for detection; The immunochromatography method includes colloidal gold immunochromatography and time-resolved fluorescence immunochromatography.

[0007] According to the above technical solution, in the colloidal gold immunochromatography method, α-Syn antibody 1, YWHAZ antibody 1, and Myl6 antibody 1 are sequentially coated on the nitrocellulose membrane of the test card as the test line, and goat anti-mouse IgG antibody is used as the quality control line. The conjugate pad is coated with gold-labeled α-Syn antibody 2, gold-labeled YWHAZ antibody 2, and gold-labeled Myl6 antibody 2.

[0008] According to the above technical solution, in the time-resolved fluorescence immunochromatography method, α-Syn antibody 1, YWHAZ antibody 1, and Myl6 antibody 1 are sequentially coated on the nitrocellulose membrane of the test card as the test line, and goat anti-mouse IgG antibody is used as the quality control line. The conjugate pad is coated with lanthanide element-labeled α-Syn antibody 2, lanthanide element-labeled YWHAZ antibody 2, and lanthanide element-labeled Myl6 antibody 2, where the lanthanide element is any one of europium and samarium.

[0009] According to the above technical solution, in the chemiluminescence method, enzyme-labeled or acridinium ester-labeled α-Syn antibody, enzyme-labeled or acridinium ester-labeled YWHAZ antibody, and enzyme-labeled or acridinium ester-labeled Myl6 antibody are used to bind to the corresponding proteins in the sample, and chemiluminescence signals are generated through chemical reactions for quantitative detection.

[0010] According to the above technical solution, the kit includes a test card, a sample diluent, a standard product, and a quality control product; The sample diluent is used to dilute the sample, the standard product is used to prepare a standard curve, and the quality control product is used to monitor the accuracy of the detection process.

[0011] According to the above technical solution, the kit can detect changes in the expression levels of α-Syn, YWHAZ, and Myl6 proteins. The area under the curve (AUC) of the combined detection reaches 0.98, the specificity is not less than 95%, and the sensitivity is not less than 96%.

[0012] According to the above technical solution, the kit can be used to detect samples such as blood and cerebrospinal fluid, and assist doctors in diagnosing Parkinson's disease based on the detection results of the three markers.

[0013] Compared with the prior art, the beneficial effects of the present invention: High specificity: By jointly detecting three proteins, namely α-Syn, YWHAZ, and Myl6, it is possible to effectively exclude the interference of other neurological diseases and improve the specificity of diagnosis. For example, compared with other common neurological diseases such as Alzheimer's disease and multiple system atrophy, the expression levels of α-Syn, YWHAZ, and Myl6 detected by the kit of the present invention are significantly different, and it can accurately distinguish Parkinson's disease from other diseases. When jointly detecting, the specificity can be increased to 95%.

[0014] High sensitivity: The double antibody sandwich method adopted by the kit of the present invention can significantly improve the sensitivity of detection. Especially, the high AUC value of the YWHAZ protein and the combined detection AUC reaching 0.98 can detect subtle changes in protein expression levels at the early stage of the disease, providing strong support for the early diagnosis of Parkinson's disease. The sensitivity of the combined detection is as high as 96%, far higher than that of single protein detection and traditional diagnostic methods.

[0015] Simple operation: The kit of the present invention adopts simple and easy detection methods such as immunochromatography and chemiluminescence, without the need for complex instrument equipment. The operator only needs to undergo simple training to perform the detection, which is suitable for primary medical institutions and large-scale population screening, greatly improving the detection efficiency.

[0016] Low sample requirements: The kit of the present invention can detect using various samples such as blood and cerebrospinal fluid. The sample collection is convenient, the trauma to the patient is small, and it is easy to be accepted by the patient. Especially for blood samples, the collection process is simple, and batch detection can be carried out through automated equipment, further improving the detection efficiency. Description of the Drawings

[0017] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention.

[0018] In the drawings: Figure 1 is the atlas data diagram of the present invention. Detailed Embodiments

[0019] The following describes the preferred embodiments of the present invention with reference to the drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0020] Example 1:

[0021] As Figure 1Shown as follows: The present invention provides a technical solution, a Parkinson combined detection early diagnosis and early screening kit, including a kit for combined detection of alpha-synuclein (α-Syn), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and myosin light chain 6 (Myl6). The three proteins are used to achieve early screening and diagnosis of Parkinson's disease.

[0022] Detecting proteins Alpha-synuclein (α-Syn): From the perspective of pathological features, misfolding and aggregation of α-synuclein generally exist in the substantia nigra region and other brain regions of Parkinson's disease patients. In addition, aggregation of α-synuclein can also be detected in peripheral nervous system tissues. The seed amplification assay (SAA) of α-synuclein can quickly and accurately detect the pathological status of Parkinson's disease and other synucleinopathies patients. In the receiver operating characteristic (ROC) curve analysis, when α-Syn is used alone as a diagnostic indicator, the area under the curve (AUC) is 0.82. In clinical practice, it is found that when judged by a specific critical value, the specificity of α-Syn in detecting Parkinson's disease can reach 75%, and the sensitivity is 80%.

[0023] Tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein zeta (YWHAZ): In the research of various nervous system diseases, YWHAZ shows a close connection with the disease process. Especially in the evaluation of the diagnostic efficacy of Parkinson's disease, YWHAZ shows outstanding advantages. Verified by a large number of clinical samples, its expression level in Parkinson's disease patients changes significantly, and there is an obvious correlation with the severity and progression stage of the disease. In the ROC curve analysis, when YWHAZ is used alone as a diagnostic indicator, the AUC reaches 0.96. When using YWHAZ alone for detection and setting a suitable critical value, the specificity of diagnosing Parkinson's disease is as high as 90%, and the sensitivity is 92%.

[0024] Myosin light chain 6 (Myl6): Under normal physiological conditions, Myl6 plays an important role in the process of muscle contraction and relaxation, maintaining the normal function of muscles. However, in Parkinson's disease patients, due to a series of pathophysiological changes caused by nervous system lesions, the expression and function of Myl6 are abnormal. Research finds that the content of Myl6 in specific tissues or body fluids of Parkinson's disease patients is significantly different from that of healthy people. This difference provides an important clue for the early diagnosis of Parkinson's disease. When Myl6 is used alone as a diagnostic indicator, the AUC is 0.92. In actual detection, based on the detection of Myl6, the specificity can reach 85%, and the sensitivity is 88%.

[0025] Preparation of the kit Preparation of antibodies: α-Syn antibody, YWHAZ antibody and Myl6 antibody are prepared respectively. Monoclonal antibodies can be prepared by conventional hybridoma technology, or polyclonal antibodies can be prepared by immunizing animals. The prepared antibodies are purified and identified to ensure the quality and activity of the antibodies.

[0026] Preparation of labeled antibodies: Colloidal gold solution is prepared by the sodium citrate reduction method. α-Syn antibody, YWHAZ antibody and Myl6 antibody are respectively labeled with the colloidal gold solution to prepare labeled α-Syn antibody, labeled YWHAZ antibody and labeled Myl6 antibody. After centrifugation, washing and other steps, the unbound antibodies and impurities are removed from the labeled antibodies, and then the labeled antibodies are resuspended in the preservation solution for standby.

[0027] According to the above technical solution, detection is carried out by immunochromatography, chemiluminescence or other detection methods based on the principle of sandwich immunoassay; The immunochromatography method includes colloidal gold immunochromatography and time-resolved fluorescence immunochromatography.

[0028] Preparation of test cards: α-Syn antibody, YWHAZ antibody and Myl6 antibody are coated on the nitrocellulose membrane in sequence with a membrane scribing instrument as the test line, and goat anti-mouse IgG antibody is used as the quality control line. The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are pasted on the PVC board in a certain order to prepare a test card. The conjugate pad is pre-sprayed with labeled α-Syn antibody, labeled YWHAZ antibody and labeled Myl6 antibody.

[0029] Preparation of sample diluent, standards and quality control products: The sample diluent uses phosphate buffered saline (PBS) and adds a certain amount of bovine serum albumin (BSA) and preservatives for diluting samples. The standards use recombinant α-Syn, YWHAZ and Myl6 proteins and are prepared according to different concentration gradients for making standard curves. The quality control products are divided into positive quality control products and negative quality control products. The positive quality control products use serum samples containing a certain concentration of α-Syn, YWHAZ and Myl6 proteins, and the negative quality control products use healthy human serum samples. The sample diluent, standards and quality control products are respectively sub-packed and packaged in a kit.

[0030] According to the above technical solution, in the colloidal gold immunochromatography method, α-Syn antibody, YWHAZ antibody and Myl6 antibody are coated on the nitrocellulose membrane of the test card in sequence as the test line, and goat anti-mouse IgG antibody is used as the quality control line. The conjugate pad is coated with gold-labeled α-Syn antibody, gold-labeled YWHAZ antibody and gold-labeled Myl6 antibody.

[0031] According to the above technical scheme, in the time-resolved fluorescence immunochromatography, the nitrocellulose membrane of the test card is coated with α-Syn antibody, YWHAZ antibody and Myl6 antibody in sequence as the test line, the goat anti-mouse IgG antibody is used as the quality control line, and the conjugate pad is coated with lanthanide-labeled α-Syn antibody, lanthanide-labeled YWHAZ antibody and lanthanide-labeled Myl6 antibody. According to the above technical scheme, in the chemiluminescence method, enzyme-labeled α-Syn antibody, enzyme-labeled YWHAZ antibody and enzyme-labeled Myl6 antibody are used to bind to the corresponding proteins in the sample, and light signals are generated through chemical reactions for quantitative detection. According to the above technical solution, the kit includes a test card, a sample diluent, a standard substance and a quality control substance; The sample diluent is used to dilute the sample, the standard is used to prepare the standard curve, and the quality control product is used to monitor the accuracy of the detection process; The test card includes a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The conjugate pad is coated with labeled α-Syn antibody, labeled YWHAZ antibody, and labeled Myl6 antibody, and the nitrocellulose membrane is coated with α-Syn antibody, YWHAZ antibody, and Myl6 antibody as the test line, and goat anti-mouse IgG antibody as the quality control line. The sample diluent is used to dilute the sample, the standard is used to prepare the standard curve, and the quality control is used to monitor the accuracy of the test process.

[0032] According to the above technical scheme, the kit can detect slight changes in the expression of α-Syn, YWHAZ and Myl6 proteins in patients with early Parkinson's disease, and the area under the curve (AUC) of the combined detection reaches 0.98, the specificity is not less than 95%, and the sensitivity is not less than 96%.

[0033] According to the above technical solution, the kit can be used for testing using samples such as blood and cerebrospinal fluid.

[0034] According to the above technical scheme, a method for detecting Parkinson's disease using a Parkinson's early screening and diagnosis kit comprises the following steps: collecting samples, using the detection method in the kit to detect α-Syn, YWHAZ and Myl6 proteins in the samples, and assisting doctors in diagnosing Parkinson's disease based on the detection results of the three markers.

[0035] The kit of the present invention adopts immunochromatography (colloidal gold / time-resolved fluorescence), chemiluminescence and other methods, based on the principle of double antibody sandwich method, and newly adds time-resolved fluorescence immunochromatography, as follows: Colloidal gold immunochromatography: As described in the original patent, it uses colloidal gold to label antibodies and observe the detection line bands with the naked eye to achieve qualitative or semi-quantitative detection.

[0036] Time-resolved fluorescence immunochromatography: The lanthanide elements (such as Eu³⁺) are used to label the α-Syn antibody, YWHAZ antibody, and Myl6 antibody. During detection, the target protein in the sample binds to the labeled antibody to form a complex, which moves to the test line of the nitrocellulose membrane through chromatography and binds to the coated antibody to form a sandwich structure. The lanthanide elements are excited by a time-resolved fluorescence detector, and long-lived fluorescence signals are collected to exclude short-lived background fluorescence interference, realizing highly sensitive quantitative detection.

[0037] Chemiluminescence method: As described in the original patent, the antibody is labeled with an enzyme or acridinium ester, and the chemiluminescent substrate is used to generate a light signal for quantitative detection of protein content.

[0038] Immunochromatography: Sample collection: Collect the blood or cerebrospinal fluid sample of the patient, and take the supernatant after centrifuging the sample for standby.

[0039] Sample addition: Place the test card flat on the table, and use a pipette to suck an appropriate amount of the sample supernatant and drop it on the sample pad, paying attention not to add too much or too little to avoid affecting the test results.

[0040] Reaction: After sample addition, α-Syn, YWHAZ, and Myl6 in the sample respectively bind to the labeled antibody to form a complex. With the chromatography effect, the complex moves to the test line and binds to the coated antibody to form a sandwich structure. Observe the test results within 5 - 10 minutes. If bands appear on both the test line and the control line, it is a positive result; if only a band appears on the control line and no band appears on the test line, it is a negative result; if no band appears on the control line, the test result is invalid and needs to be retested.

[0041] Chemiluminescence method: Sample collection and processing: The same as immunochromatography.

[0042] Sample addition: Add the sample supernatant to the chemiluminescent microplate, and at the same time add the enzyme-labeled or acridinium ester-labeled α-Syn antibody, enzyme-labeled or acridinium ester-labeled YWHAZ antibody, and enzyme-labeled or acridinium ester-labeled Myl6 antibody, and gently shake and mix well.

[0043] Incubation: Place the microplate in a 37°C constant temperature incubator for a certain period of time to allow α-Syn, YWHAZ, and Myl6 in the sample to fully bind to the enzyme-labeled or acridinium ester-labeled antibody.

[0044] Washing: After incubation, wash the microplate with the washing solution to remove the unbound enzyme-labeled or acridinium ester-labeled antibody and impurities.

[0045] Luminescence reaction: Add chemiluminescent substrate into the microplate, react in the dark for a certain period of time to cause a chemical reaction of the enzyme-labeled antibody to generate a light signal.

[0046] Detection: Use a luminescence detector to detect the light signal intensity of each well in the microplate, calculate the contents of α-Syn, YWHAZ, and Myl6 proteins in the sample according to the standard curve, and judge whether the sample is positive according to the preset critical value.

[0047] Preparation of the kit: Time-resolved fluorescence part 1. Preparation of labeled antibodies (time-resolved fluorescence method) Couple α-Syn antibody, YWHAZ antibody, and Myl6 antibody with europium (Eu³⁺) ions using a bifunctional chelating agent (such as phenylmethyl-EDTA isothiocyanate) to prepare lanthanide-labeled antibodies.

[0048] After labeling, the antibodies are purified by gel filtration chromatography, the unbound chelating agent is removed, the concentration is adjusted to 1 mg / mL, and they are stored in PBS buffer (pH 7.4) containing 0.1% BSA.

[0049] 2. Preparation of test cards (time-resolved fluorescence method) Nitrocellulose membrane: Similar to the colloidal gold method, sequentially coat α-Syn antibody (1 mg / mL), YWHAZ antibody (1.2 mg / mL), and Myl6 antibody (0.9 mg / mL) as the test line, and goat anti-mouse IgG antibody (2 mg / mL) as the quality control line, with a coating flow rate of 0.1 μL / cm.

[0050] Conjugate pad: After pretreatment, uniformly spray lanthanide-labeled α-Syn antibody, labeled YWHAZ antibody, and labeled Myl6 antibody at a spraying amount of 1 μL / cm, and dry at 37 °C for 2 hours.

[0051] Assembly: Paste the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper on the PVC board in sequence, and cut it into a test card with a width of 3.9 mm.

[0052] Time-resolved fluorescence immunochromatography 1. Sample collection and processing Similar to the colloidal gold method, collect blood or cerebrospinal fluid samples, centrifuge and take the supernatant for standby.

[0053] 2. Sample addition and reaction Place the test card flat, pipette 10 μL of the sample supernatant and add it dropwise to the sample pad, and react for 10 minutes at room temperature (25 °C ± 2 °C) and a humidity of 40% - 60%.

[0054] After the reaction is completed, place the test card into a time-resolved fluorescence detector. The excitation light wavelength is 340 - 365 nm, the emission light wavelength is 613 nm, the detection time is 100 ms, and read the fluorescence signal intensity of each detection line.

[0055] 3. Result Interpretation Calculate the contents of α-Syn, YWHAZ, and Myl6 according to the standard curve (drawn with recombinant proteins of different concentrations).

[0056] Combined interpretation criteria: When the concentrations of all three exceed the preset critical values, it is determined as positive; if any index fails to meet the standard, further evaluation shall be conducted in combination with clinical information.

[0057] Finally, it should be noted that the above are only the preferred examples of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Parkinson's combined detection early diagnosis and early screening kit, characterized in that: Includes kits for the combined detection of alpha-synuclein (α-Syn), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein zeta (YWHAZ), and myosin light chain 6 (Myl6).

2. The Parkinson combined detection early diagnosis and early screening kit according to claim 1, characterized in that Detection is performed using immunochromatography, chemiluminescence or other detection methods based on the principle of double antibody sandwich method; The immunochromatography method includes colloidal gold immunochromatography and time-resolved fluorescence immunochromatography.

3. The Parkinson combined detection early diagnosis and early screening kit according to claim 2, characterized in that, In the colloidal gold immunochromatography method, the nitrocellulose membrane of the detection card is coated with α-Syn antibody 1, YWHAZ antibody 1 and Myl6 antibody 1 in sequence as the detection line, the sheep anti-mouse IgG antibody is used as the quality control line, and the conjugate pad is coated with gold-labeled α-Syn antibody 2, gold-labeled YWHAZ antibody 2 and gold-labeled Myl6 antibody 2.

4. A Parkinson's combined detection early diagnosis and early screening kit according to claim 2, characterized in that, In the time-resolved fluorescent immunochromatography method, the nitrocellulose membrane of the detection card is coated with α-Syn antibody 1, YWHAZ antibody 1 and Myl6 antibody 1 in sequence as the detection line, the sheep anti-mouse IgG antibody is used as the quality control line, and the conjugate pad is coated with lanthanide-labeled α-Syn antibody 2, lanthanide-labeled YWHAZ antibody 2 and lanthanide-labeled Myl6 antibody 2, wherein the lanthanide element is any one of europium and samarium.

5. A Parkinson's combined detection early diagnosis and early screening kit according to claim 2, wherein In the chemiluminescence method, enzyme-labeled or acridinium ester-labeled α-Syn antibody, enzyme-labeled or acridinium ester-labeled YWHAZ antibody and enzyme-labeled or acridinium ester-labeled Myl6 antibody are combined with corresponding proteins in the sample, and light signals are generated through chemical reactions for quantitative detection.

6. The Parkinson combined detection early diagnosis and early screening kit according to claim 1, wherein, The kit includes test cards, sample diluent, standards and quality controls; The sample diluent is used to dilute the sample, the standard product is used to prepare a standard curve, and the quality control product is used to monitor the accuracy of the detection process.

7. The Parkinson combined detection early diagnosis and early screening kit according to claim 1, wherein The kit can detect changes in the expression levels of α-Syn, YWHAZ and Myl6 proteins, with the area under the curve (AUC) of the combined detection reaching 0.98, the specificity not less than 95%, and the sensitivity not less than 96%.

8. A Parkinson's combined detection early diagnosis and early screening kit according to claim 1, characterized in that The kit can be used for testing using samples such as blood and cerebrospinal fluid.

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