α-Synuclein antigenic epitope peptide, kit for detecting SNCA in saliva, and application thereof in the diagnosis of Parkinson's disease

Abstract

The present invention provides an SNCA-specific antibody, which is prepared by immunizing an animal with an SNCA-specific antigenic epitope peptide conjugated to a carrier protein, wherein the amino acid sequence of the SNCA-specific antigenic epitope peptide is one of the following two sequences: Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala. The present invention further discloses an α-synuclein assay kit, comprising: a solid-phase carrier, an SNCA-specific antibody, a label, and an SNCA calibrator. The present invention also discloses the application of the kit provided by the present invention in the preparation of products for diagnosing Parkinson's disease. The kit prepared by the present invention needs to have good diagnostic sensitivity, specificity, and repeatability, meeting the requirements of clinical applications.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an α-synuclein antigenic epitope peptide, a specific antibody prepared with the antigenic epitope peptide, a kit for detecting α-synuclein in saliva prepared with the antibody, and the application of the kit in the diagnosis of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease in the middle-aged and elderly. It is mainly characterized by the pathological changes of progressive degeneration of substantia nigra dopaminergic neurons and the formation of Lewy bodies, the biochemical changes of decreased dopamine neurotransmitter in the striatum area and the imbalance between dopamine and acetylcholine neurotransmitters, and the clinical manifestations of motor symptoms such as tremor, muscle rigidity, bradykinesia, and postural balance disorder, as well as non-motor symptoms such as sleep disorder, olfactory disorder, autonomic dysfunction, cognitive and mental disorders. Epidemiological investigation and research show that the prevalence of Parkinson's disease in people over 60 years old in European and American countries reaches 1%, and over 4% in people over 80 years old. The prevalence of people over 65 years old in China is 1.7%, which is similar to that in European and American countries. China is the most populous country in the world. In the future, the number of Parkinson's disease patients in China will increase from 1.99 million in 2005 to 5 million in 2030, almost accounting for half of the global number of Parkinson's disease patients. As the disease progresses, the motor and non-motor symptoms of Parkinson's disease will gradually worsen. On the one hand, it will damage the daily activities of the patients themselves. On the other hand, it will also bring a huge social and medical burden.

[0003] The current diagnostic criteria for Parkinson's disease include "Diagnostic Criteria for Parkinson's Disease in China (2016 Edition)" and "MDS Clinical Diagnostic Criteria for Parkinson's Disease of the Movement Disorder Society (MDS-2015)", both of which are improvements based on the "Clinical Diagnostic Criteria for Parkinson's Disease" formulated by the UK Parkinson's Disease Brain Bank in 1997. The above diagnostic criteria for Parkinson's disease mainly follow a three-step rule of inclusion, exclusion / warning, and support. Clinically diagnosing Parkinson's disease (PD) requires first determining whether the patient has one of the two main signs of bradykinesia (slow initiation of voluntary movement, progressive reduction in the speed and amplitude of repetitive movements) or resting tremor (4-6 Hz) / rigidity, and at the same time meeting the conditions of not meeting the absolute exclusion criteria, having at least two supportive criteria, and having no warning signs. The exclusion criteria and warning signs include the therapeutic effect of dopamine drugs, the patient's function, and the results of imaging tests. The supportive criteria include a clear and significant effective response to dopaminergic drug treatment, the appearance of levodopa-induced dyskinesia, the resting tremor of a single limb recorded in the clinical physical examination (previous or current examination), and the presence of olfactory loss or cardiac denervation shown by cardiac MIBG scintigraphy. The examination of all core main signs must be carried out according to the methods described in the MDS-Unified Parkinson's Disease Rating Scale (MDS-UPDRS).

[0004] Due to the lack of objective examination methods, the current diagnosis of PD still relies on the long-term observation of clinical symptoms and the effectiveness of taking dopaminergic drugs. Moreover, there are many overlaps in the clinical symptoms and pathological bases between PD symptoms and multiple system atrophy, progressive supranuclear palsy, and corticobasal ganglionic degeneration diseases. Using clinical symptoms as the key diagnostic index is unstable, and the misdiagnosis and missed diagnosis rates are relatively high, which brings great inconvenience to the diagnosis and treatment of PD.

[0005] In view of the current situation of Parkinson's disease diagnosis, clinically, efforts have been continuously made to find a stable, reliable biological marker that can objectively diagnose Parkinson's disease and evaluate its curative effect. Currently, more research has been focused on brain imaging and body fluid biomarkers.

[0006] Parkinson's disease belongs to neurodegenerative diseases, and its occurrence is closely related to the changes in the structure and function of the brain. Brain imaging is an important tool for the study of neurodegenerative diseases. Among them, transcranial ultrasound, magnetic resonance, and positron emission computed tomography can all detect the key changes in the pathological characteristics of Parkinson's disease, and play a certain role in the diagnosis and differential diagnosis of Parkinson's disease. Transcranial ultrasound is non-invasive and easy to operate. Studies have found that more than 90% of Parkinson's patients have abnormalities in the substantia nigra, that is, the phenomenon of hyperechogenicity (SN+). However, it only targets people with a relatively high probability of PD, and the above-mentioned clinical diagnosis still needs to be prioritized. The differences in excitability between PD patients and normal people shown by blood oxygenation level-dependent functional magnetic resonance imaging lack specific quantitative criteria and are still affected by the subjective consciousness of the diagnostician. However, due to the current magnetic resonance technology being restricted by many influencing factors, such as the heterogeneity of the disease; the lack of imaging standards for the normal brain; the high sensitivity of magnetic resonance to changes in the physiological or exogenous chemical substances of the subjects (such as excitement, coffee, smoking, temperature, etc.); the limitations of statistical analysis methods; the limitations of measured variables, etc., and due to individual differences, the excitability of the cerebellum-thalamus-cortex circuit and the striatum-thalamus-cortex circuit among different individuals is affected by many external factors, which brings difficulties to the diagnosis of PD, and its performance is still far from meeting the requirements of clinical applications. PET presynaptic membrane dopamine transporter is currently considered the most sensitive PD biomarker, with high sensitivity, can be used as an evaluation index of striatal function status, can greatly reduce the misdiagnosis rate of Parkinson's disease, and also plays a key role in the differential diagnosis of Parkinson's disease. However, due to the long scanning time, high cost, and high requirements for the operator's technology and equipment of this operation, in addition, PET tracers may increase the genotoxicity of ionizing radiation. Therefore, imaging cannot be used as a diagnostic index for Parkinson's disease in clinical practice.

[0007] So far, the exact neuropathological basis of Parkinson's disease is still controversial. Over the years, the academic community has put forward various hypotheses to explain the pathogenesis of Parkinson's disease. Current research has found that the pathological feature of Parkinson's disease is Lewy bodies, that is, a large number of dopaminergic neurons in the substantia nigra pars compacta degenerate and are lost, and eosinophilic inclusions appear in the cytoplasm of the remaining neurons. Alpha-synuclein (SNCA) is an important component of Lewy bodies and is a marker protein of Parkinson's disease pathology. Therefore, researchers hope to analyze the potential of SNCA as a biomarker for Parkinson's disease in cerebrospinal fluid and blood. However, due to the difficulty in obtaining cerebrospinal fluid samples, relevant studies often come from small-sample clinics. The results of the corresponding cerebrospinal fluid SNCA levels and structural imaging showing Parkinson's disease are interrupted from the overall disease course, and more longitudinal studies are still needed to support their potential value. The total blood SNCA level is affected by SNCA in red blood cells and exosomal SNCA, lacking the ability to distinguish, making it unable to be used as an individual biomarker for the diagnosis of Parkinson's disease, with low diagnostic accuracy and unable to be considered a single diagnostic or prognostic biomarker for PD.

[0008] With the in - depth research, it is found that SNCA is not limited to the central nervous system and has also been reported in peripheral tissues. Researchers such as Devic detected the presence of α - synuclein in saliva in 2011. The parasympathetic nerves that innervate the salivary glands originate from the brainstem, creating a common link in the transmission of SNCA pathology. SNCA in saliva may be a biomarker for the histopathological identification of the pre - motor stage of PD. Moreover, compared with the lacrimal gland and olfactory mucosa that share parasympathetic nerves, the aggregates of SNCA in saliva have the highest overall sensitivity and specificity.

[0009] Neuropathological studies have shown that in the early stage of PD, cells that aggregate α - synuclein are mainly distributed in the brainstem nuclei including the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve, or in the superior and inferior salivary nuclei and parasympathetic salivary ganglia. This suggests that α - synuclein may diffuse out of the neuronal cell bodies of salivary neurons, along the axons to the synaptic terminals around the salivary gland epithelial cells, and aggregate in the salivary glands and saliva. The decrease in the concentration of α - synuclein in the saliva of Parkinson's disease patients may be due to the intracellular and axonal aggregation of α - synuclein in the neurons of the salivary nuclei or salivary ganglia. In terms of the ROC curve for diagnosing PD using salivary α - synuclein, the sensitivity, specificity, and positive predictive value are all higher than those of blood α - synuclein, making it more suitable as an indicator for diagnosing PD. Saliva specimens not only have the characteristics of being non - invasive and easy to collect, but also recent studies have shown that the submandibular gland that secretes saliva is a target organ affected in the early stage of PD, suggesting that salivary SNCA may become a potential biomarker for early screening of PD.

[0010] Although there are currently preliminary studies on the use of salivary SNCA for the auxiliary diagnosis of Parkinson's disease, due to the great difficulty of clinical trials for the diagnosis of Parkinson's disease, there is no kit for measuring α - synuclein in saliva for the diagnosis of Parkinson's disease. Therefore, there is a need in this field to develop a kit that can measure the concentration level of α - synuclein in human saliva to overcome the deficiencies of subjective diagnosis of early Parkinson's disease clinically and provide a more accurate and reliable objective indicator for the auxiliary diagnosis and early screening of Parkinson's disease. Summary of the Invention

[0011] To solve the problems existing in the prior art, the present invention screened and obtained a SNCA - specific antigen - epitope peptide, which is characterized in that the amino acid sequence is as follows:

[0012] Tyr - Lys - Thr - Lys - Gln - Gly - Val - Ala - Glu - Ala - Ala - Gly - Lys - Thr - Lys - Glu(1) or

[0013] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala(2).

[0014] The present invention thus provides an SNCA-specific antibody, which is a polyclonal antibody or a monoclonal antibody prepared by immunizing an animal with the above-mentioned SNCA-specific antigenic epitope peptide. Specifically, it is prepared by immunizing rabbits and mice after coupling the SNCA-specific antigenic epitope peptide with a carrier protein.

[0015] The present invention further provides a kit for detecting salivary SNCA (α-synuclein), which comprises: a solid-phase carrier, an SNCA-specific antibody, a label, and an SNCA calibrator. The SNCA-specific antibody is a monoclonal antibody or a polyclonal antibody prepared from an SNCA antigen, and the SNCA antigen is prepared by coupling a human SNCA antigenic epitope peptide (1) or (2) with a carrier protein. The amino acid sequence of the SNCA antigenic epitope peptide (1) or (2) is one of the following two sequences:

[0016] Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu(1) or

[0017] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala(2).

[0018] According to the present invention, the kit for detecting SNCA is developed by immunoassay methods and can use any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), fluorescence immunochromatography, or colloidal gold assay.

[0019] According to the present invention, the solid-phase carrier can be one of a microplate, magnetic microspheres, or a nitrocellulose membrane (NC membrane).

[0020] According to the present invention, the label can be horseradish peroxidase HRP, alkaline phosphatase AP, biotin, a luminescent substance, fluorescent microspheres, colloidal gold, etc. For example, when used in an enzyme-linked immunosorbent assay (ELISA) kit, the label is preferably horseradish peroxidase HRP; when used in a chemiluminescence immunoassay (CLIA) kit, the label is preferably alkaline phosphatase AP; when used in a fluorescence immunochromatography kit, the label is preferably fluorescent microspheres; and when used in a colloidal gold assay kit, the label is colloidal gold.

[0021] According to the present invention, the SNCA calibrator / quality control product is a diluted recombinant or extracted SNCA.

[0022] The SNCA-specific antibody is prepared by immunizing animals (such as mice, rats, rabbits, sheep, etc.) with the SNCA-specific antigen epitope peptide (1) or (2) conjugated with a carrier protein. The SNCA-specific antibody can be a monoclonal antibody or a polyclonal antibody against SNCA, preferably a monoclonal antibody; the carrier protein can be bovine serum albumin BSA, ovalbumin OVA or keyhole limpet hemocyanin KLH, preferably keyhole limpet hemocyanin KLH.

[0023] In a preferred embodiment, the kit of the present invention is a chemiluminescent immunoassay kit, which is developed by combining chemiluminescent immunoassay (CLIA) with the principle of sandwich immunoassay.

[0024] In a specific embodiment of the chemiluminescent immunoassay kit, the solid-phase carrier is magnetic beads, and the magnetic beads are directly or indirectly connected to the SNCA-specific antibody to form a magnetic separation reagent; the magnetic separation reagent is preferably an immunomagnetic bead coated with a monoclonal antibody against SNCA or a magnetic bead connecting streptavidin and an SNCA antibody.

[0025] In a specific embodiment of the chemiluminescent immunoassay kit, the kit further comprises a luminescent substrate. Examples of the luminescent substrate include, but are not limited to, luminol, isoluminol and its derivatives, or (adamantane)-1,2-dioxetane and its derivatives.

[0026] In a specific embodiment, the kit includes: a magnetic separation reagent, an enzyme-labeled monoclonal antibody against SNCA, an SNCA calibrator / quality control product, a washing solution, and a chemiluminescent substrate. Among them, the monoclonal antibody against SNCA is prepared by immunizing animals with an SNCA-specific antigen epitope peptide conjugated with a carrier protein, and the amino acid sequence of the SNCA-specific antigen epitope peptide is one of the following two sequences:

[0027] Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu (1) or

[0028] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

[0029] The present invention further provides the application of the kit of the present invention in the preparation of products for detecting and diagnosing Parkinson's disease.

[0030] When the kit of the present invention is used for preparing a product for diagnosing Parkinson's disease, the SNCA concentration of the subject sample measured by the kit is compared with the control reference range (i.e., the concentration level of healthy subjects). If the SNCA concentration of the subject sample is lower than the concentration in the control reference range, the subject has a risk of suffering from Parkinson's disease.

[0031] Preferably, the detection is saliva sample detection. Further preferably, the detection is any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), fluorescence immunochromatography assay kit, and colloidal gold immunoassay; more preferably, it is chemiluminescence immunoassay (CLIA).

[0032] In the detection by chemiluminescence method, it has high detection sensitivity (the lowest detection limit is 1.41 pg / mL), a wide linear range with 3 - 6 orders of magnitude, can directly measure samples with a concentration as high as 5000 pg / mL without dilution, high accuracy (the recovery rate of the recovery test is 92.64%), high precision (CV is less than 10%), short reaction time, and the result can be obtained in 15 minutes, greatly improving the detection efficiency. It has a high degree of automation, does not require manual operation, and reduces human operation errors.

[0033] The present invention has the following advantages and positive effects compared with the prior art:

[0034] 1. The present invention applies saliva SNCA as a biomarker to the preparation of an auxiliary diagnostic kit for Parkinson's disease, overcomes the defect that the indicators used in the current clinical diagnosis process of Parkinson's disease are too subjective and lack objectivity, and the sampling is convenient and non-invasive, providing a more accurate, reliable and convenient objective indicator for the auxiliary diagnosis and screening of Parkinson's disease.

[0035] 2. The kit prepared by the present invention needs to have good diagnostic sensitivity, specificity and repeatability, meeting the requirements of clinical application.

[0036] 3. The SNCA-specific monoclonal antibody and polyclonal antibody prepared by the present invention can highly specifically bind to SNCA in the sample.

[0037] 4. The two SNCA-specific antigenic epitope peptides screened by the present invention have the characteristics of hydrophilicity, strong antigenicity and easy synthesis. Immunizing animals with the antigen (immunogen) prepared therefrom can produce highly specific monoclonal antibodies and polyclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shows the saliva SNCA levels of the control group and Parkinson's disease patients (test results using the kit prepared in Example 1).

[0039] Figure 2Shows the ROC curve of SNCA in saliva samples for the diagnosis of Parkinson's disease (test results using the kit prepared in Example 1). Detailed implementation mode

[0040] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0041] Example 1: Screening of SNCA-specific antigenic epitope peptides

[0042] The SNCA (α-synuclein) used has an amino acid sequence known in the art and can be found in professional databases such as NCBI (GenBank: AAL15443.1).

[0043] After a large amount of theoretical research and experimental exploration, the inventors considered factors such as hydrophilicity, antigenicity, and ease of synthesis, and finally screened the following two antigenic epitope peptides with good antigenicity, and their amino acid sequences are:

[0044] Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu (1) or

[0045] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2)

[0046] Subsequent experiments showed that these two antigenic epitope peptides a. have antigenicity; b. can stimulate animals to produce specific antibodies as an immunogen after being linked to a carrier protein; c. the antibodies prepared with the antigenic epitope peptides can specifically bind to human SNCA.

[0047] Example 2: Synthesis of SNCA-specific antigenic epitope peptides (1) and (2)

[0048] 1. Raw materials used: HMP resin (P-hydroxymethyl phenoxymethyl polyvinyl resin, purchased from Sigma); Fmoc-AA (9-fluorenylmethoxycarbonyl-protected amino acid, purchased from Merck); NMP (N-methylpyrrolidone, purchased from Sigma); DCM (dichloromethane, purchased from Zhongyuan Chemical Industry Company); MeOH (methanol, purchased from Zhongyuan Chemical Industry Company); Piperidine (purchased from Sigma); DMAP (dimethylaminopyridine, purchased from Sigma); HOBT (hydroxybenzotriazole, purchased from Sigma); DCC (dicyclohexylcarbodiimide, purchased from Sigma); TFA (trifluoroacetic acid, purchased from Sigma); EDT (1,2-ethanedithiol, purchased from Sigma); Thioanisole, purchased from Guangzhou Weibo Chemical Co., Ltd.; Crystalline phenol, purchased from Sinopharm Chemical Reagent Co., Ltd.; Acetonitrile, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0049] 2. Instruments used: Automatic peptide synthesizer, model 431A, purchased from ABI; Rotary evaporator, model R-201, purchased from Shanghai Shenshun Company; High performance liquid chromatograph, Waters 600, purchased from Waters, USA; Freeze dryer, model VFD-2000, purchased from Beijing Boyikang Company.

[0050] 3. Synthesis method and process:

[0051] Weigh 100 mg of HMP resin with a substitution equivalent of 1.0 meq, that is, place 0.1 mmol of HMP resin in the reaction chamber of the ABI 431A automatic peptide synthesizer in the United States. The synthesizer automatically connects specific amino acids in different sequences, and the coupling rate reaches 99%. The reaction is as follows:

[0052] (1) Activation of amino acids (HOBt / DCC method)

[0053]

[0054] Fmoc-protected amino acid

[0055]

[0056] (2) Connect the amino acid to the resin

[0057]

[0058] (3) Remove the Fmoc protecting group of the amino acid

[0059]

[0060] (4) Activation of another amino acid (HOBt / DCC method)

[0061]

[0062] (5) Coupling

[0063]

[0064] (6) Repeat steps (3) to (5) until the synthesis is completed. Peptide resins of SNCA antigen epitope peptide (1) and SNCA antigen epitope peptide (2) are obtained respectively.

[0065] (7) Cleavage of peptide resin:

[0066] Cut the peptide chain with TFA (trifluoroacetic acid), use EDT (2.5% by volume) and thioanisole (2.5% by volume) as scavengers, react at room temperature for 3.0 hours, remove the cleavage reagent, and then extract with diethyl ether to obtain the crude products of SNCA antigen epitope peptide (1) and (2) respectively.

[0067] 4. Purification of the crude products of SNCA antigen epitope peptide (1) and (2):

[0068] Purify by high performance liquid chromatography:

[0069] Conditions: Chromatographic column: C8 10×100mm, purchased from Waters, USA

[0070] Chromatograph: Waters 600, Waters, USA

[0071] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0072] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0073] Detection wavelength: 214nm

[0074] Flow rate: 4ml / min

[0075] Elution gradient: 20 - 60% B, 30 minutes

[0076] HPLC (High Performance Liquid Chromatography) analysis

[0077] Chromatographic column: C18 4.6×150mm, purchased from Waters, USA

[0078] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0079] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0080] Detection wavelength: 214nm

[0081] Flow rate: 1 ml / min

[0082] Elution gradient: 0 - 60% B, 30 minutes

[0083] The results of peptide segment analysis showed that the purity of the SNCA antigenic epitope peptides (1) and (2) of the present invention was both above 95%.

[0084] Example 3: Preparation of SNCA-specific antibody

[0085] 1. Preparation of antigen:

[0086] The SNCA-specific antigenic epitope peptide was linked with the carrier protein KLH (keyhole limpet hemocyanin) by the Bis-diazotizedbenzidine dichloride (BDB) method to prepare the SNCA antigen.

[0087] 20.0 mg of the SNCA-specific antigenic epitope peptides (1) and (2) prepared in Example 1 were respectively taken and dissolved in 0.2 M borate buffer (pH 9.0); 7.36 ml of KLH at 6.25 mg / ml was taken and cooled to 0°C, 1 ml of BDB was taken, and the two were placed in an ice-water mixture, mixed in the dark, and reacted on a shaker for 1 - 1.5 h. After the reaction was completed, the pH was adjusted to 9.0 with 0.2 NaOH, dialyzed overnight, and then aliquoted to prepare the corresponding SNCA antigen (1) or (2), and stored at -20°C.

[0088] The formulation of the borate buffer was: 80 ml of 0.05 mol / L borax plus 20 ml of 0.2 mol / L boric acid mixed together.

[0089] 2. Immunize animals to prepare monoclonal antibodies:

[0090] 2.1. The SNCA antigen (1) or (2) (immunogen) prepared above was mixed with an equal volume of Freund's complete adjuvant (purchased from Shanghai Yuanju Biotechnology Co., Ltd.) and then immunized Balb / c mice. 50 μg antigen / mouse was injected subcutaneously at multiple points. After 4 weeks, the serum titer was measured and mice with good immune reactivity were selected for booster immunization: the antigen was mixed with an equal volume of Freund's incomplete adjuvant and the antigen dose was 25 μg / mouse. The mice were injected subcutaneously at multiple points. The number of booster immunizations was 6 times. Before fusion, the booster immunizations were continued twice. After that, the spleen cells were fused with Sp2 / 0 myeloma cells using 50% PEG (weight average molecular weight (MW) = 4000) (purchased from Zhongyuan Chemical Co., Ltd.) according to the conventional method, and selected and cultured with HAT conditioned medium (purchased from Sigma). After fusion, the cells were placed in a CO2 incubator and cultured at 37°C for 9 to 11 days. Large cell clones appeared in the wells. Screening was started by indirect ELISA on the 11th day. The wells that were positive in the initial screening were cloned and cultured four times using the limiting dilution method (i.e., the cells after screening were divided and multiplied in large numbers), and then the cells were expanded, frozen, and ascites was prepared.

[0091] 2.2. Balb / c mice were treated with 0.5 ml of pristane (purchased from Sigma) and 2×10 hybridoma cells were inoculated intraperitoneally one week later. 6 The ascites was collected after 10 days.

[0092] 2.3. Determination of antibody titer: The titer of the prepared SNCA monoclonal antibodies (1) and (2) was determined by the indirect ELISA method. The results showed that the titer of the monoclonal antibodies reached 1:64,000.

[0093] 3. Immunize animals to prepare polyclonal antibodies:

[0094] 3.1. Three-month-old New Zealand white rabbits weighing about 2 kg were selected as immunized animals. In the primary immunization, 2 mg of the SNCA antigen (1) or (2) (immunogen) prepared above was mixed with an equal volume of Freund's complete adjuvant, and then fully emulsified and injected subcutaneously at multiple points on the back of the rabbit. For booster immunization every 2 weeks, the antigen was fully emulsified with incomplete Freund's adjuvant and then injected subcutaneously at multiple points on the back at a rate of 1 ml / rabbit. On the 10th day after the last booster immunization, the carotid artery was bled and the serum was separated.

[0095] 3.2. Determination of antibody titer: The titer of polyclonal antibody (1) or (2) was determined by indirect ELISA method. The result showed that the antibody titer reached 1:32000.

[0096] 3.3. Blood collection and serum separation: Blood was collected through carotid artery catheterization and serum was separated.

[0097] 4. Separation and purification of antibodies:

[0098] The SNCA antibody to be loaded is first dialyzed to a consistent pH with a equilibration buffer (0.02 M, PB, pH 8.0); packed into a column, connected to a protein chromatography system, and washed with the equilibration buffer until the pH reaches 8.0; loaded with the SNCA antibody to be purified added to the column. After all the sample has been injected, it is washed with an elution buffer (0.05 M PB pH 8.0) until the protein nucleic acid detector shows the start of a peak, at which point the protein solution is collected. Collection is stopped when the peak descends smoothly. The concentration of the antibody is measured and calculated using an L5S ultraviolet-visible spectrophotometer (produced by Boda Jingke), and the purified SNCA antibody is aliquoted and stored at -20 °C.

[0099] 5. Specific Identification of SNCA Antibody

[0100] Detection is performed by ELISA. ELISA plates are coated with GFAP protein, S-100B protein, and AD7C-NTP protein (all purchased from Shanghai Lianshuo Company) as detection antigens, and the specific reactions of the prepared SNCA monoclonal antibody or polyclonal antibody with the human SNCA protein are detected by ELISA, using normal BALB / c mouse serum or rabbit serum as negative controls and PBS solution as a blank control.

[0101] Results: SNCA monoclonal antibodies (1), (2) and polyclonal antibodies (1), (2) all reacted positively only with SNCA (P / N > 2.1), and reacted negatively with GFAP protein, S-100B protein, and AD7C-NTP protein, indicating that SNCA monoclonal antibodies (1), (2) and polyclonal antibodies (1), (2) of the present invention are specific respectively.

[0102] Example 4: Preparation of a Chemiluminescent Kit for SNCA

[0103] 1. Preparation of Magnetic Separation Reagent (Coated SNCA Monoclonal Antibody (1) Immunomagnetic Bead Working Solution)

[0104] (1) Washing of Magnetic Beads

[0105] Take 1 mL of 0.1 M MES (pH 6.0) buffer into a coated tube, add 2 mg of JSR magnetic bead mother liquor (Shenzhen Res Biotechnology Co., Ltd.), and vortex for 1 min. Place the coated tube on a magnetic separation rack for 1 min and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex for 1 min. Repeat the above process 2 times.

[0106] (2) Activation of Magnetic Beads

[0107] After adding 800 μL of 0.1 M MES (pH 6.0) buffer into the coated tube, add 100 μL of NHS (10 mg / l) solution and vortex for 1 min. Add 100 μL of EDC (10 mg / l) solution into the coated tube. Place the rotary mixer in a constant temperature environment at 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotation speed of the rotary mixer at 50 ± 1 RPM, and react for 30 minutes.

[0108] (3) Coat the phosphorylated α-synuclein monoclonal antibody (1) with the activated magnetic beads

[0109] Add 1 mL of 0.1 M MES (pH 6.0) buffer into the coated tube and vortex for 1 min. Add 14 μL of SNCA monoclonal antibody 1 (10 mg / mL) into the coated tube and vortex for 1 min. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer into the coated tube and vortex for 1 min. Place the rotary mixer in a constant temperature environment at 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotation speed of the rotary mixer at 50 ± 1 rpm, and react for 2 hours.

[0110] (4) Block the magnetic beads

[0111] Place the coated tube on the magnetic separation rack for 1 min and then discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer into the coated tube and vortex. Repeat the operation twice. Place the rotary mixer in a constant temperature environment at 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotation speed of the rotary mixer at 50 ± 1 RPM, and react for 30 minutes.

[0112] (5) Wash

[0113] After the blocking is completed, place the coated tube on the magnetic separation rack for 1 min and then discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer into the coated tube and vortex for 1 min. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) into the coated tube and vortex for 1 min.

[0114] (6) Preparation of magnetic separation reagent

[0115] Dilute the magnetic beads coated with SNCA monoclonal antibody (1) with the final wash buffer using 0.05 M TRIS (pH 7.4) enzyme-labeled diluent to obtain a final concentration of the enzyme-labeled antibody of 0.2 mg / mL.

[0116] 2. Preparation of enzyme-labeled antibody (labeled with alkaline phosphatase SNCA monoclonal antibody (2))

[0117] Soak the ultrafiltration centrifuge tube (30KD) with 250 μL of 0.1 M MES (pH 4.5) for 2 min. Add 250 μL of 0.1 M MES (pH 4.5) to the centrifuge column to make the volume up to 500 μL. Add 12.5 μL of AP enzyme to the ultrafiltration centrifuge tube, gently mix by inverting up and down, and then centrifuge at 13,000 rpm for 15 min on a centrifuge. After the centrifugation stops, it can be observed that there is 100 μL of liquid in the centrifuge column. After discarding the waste liquid, add 200 μL of 0.1 M MES (PH4.5) to the centrifuge column in the ultrafiltration centrifuge tube, and then centrifuge at 13,000 rpm for 20 min. After the centrifugation stops, it can be observed that there is 100 μL of liquid in the centrifuge column. After discarding the waste liquid, add 50 μL of EDC (10 mg / l) and 6 μL of NHS (10 mg / l) to the ultrafiltration centrifuge tube, and then add 100 μL of 0.1 M MES (PH4.5) to make the volume up to 250 μL and mix well. Place it on a shaker for activation for 1.5 hours. Take 10 μL of SNCA monoclonal antibody 2 (10 mg / l), mix well and centrifuge at 13,000 rpm for 20 min, and pour out the centrifuged liquid. Add 250 μL of 0.1 M PB (PH9.0), mix well and centrifuge at 13,000 rpm for 20 min. Repeat the above centrifugation step once. Then add 0.1 M PB (PH9.0) to make the volume up to 250 μL, place it on a shaker at 120 r for coupling for 2 h. Add 0.05 M TRIS PH8.02, add 250 μL and mix well, place it on a shaker at 120 rpm for 30 min. Take out the 500 μL solution with the coupling completed in the centrifuge column, then add an equal amount of glycerol, mix well and store at -20 °C. Measure and calculate the concentration of alkaline phosphatase-labeled antibody with an L5S ultraviolet-visible spectrophotometer, and dilute it 1:1000 with an enzyme-labeled diluent of 0.05 M TRIS (PH7.4) to obtain the enzyme-labeled antibody.

[0118] 3. Preparation of washing solution

[0119] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0120] 4. Preparation of SNCA calibrator / quality control

[0121] Gradiently dilute the SNCA recombinant protein (purchased from abcam company, concentration 2 mg / mL) with a calibrator diluent (containing 10 mM phosphate buffer (PBS) (pH 7.2), 1% BSA, 0.03% biopreservative Proclin-300) to prepare a series of calibrators (0 - 5000 pg / mL) and prepare high and low concentration quality controls.

[0122] 5. Preparation of luminescent substrate

[0123] It consists of a chemiluminescent substrate (4-chlorophenyl mercapto)(10-methyl-9,10-dihydroacridine methylene) disodium phosphate (APS-5) and a 0.3M Tris buffer solution containing 0.0003% lucigenin, 0.001% sodium sulfite, 0.1% sodium dodecyl sulfate (SDS), and 0.03% Tween 20.

[0124] 6. Composition of the kit

[0125] The kit mainly consists of reagent strips (24 strips), SNCA calibrator, and quality control products. Each reagent strip is for one-person testing. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (coated SNCA monoclonal antibody (1) immunomagnetic bead working solution), 130 μL of enzyme-labeled antibody, 2 mL of washing solution, and 250 μL of chemiluminescent substrate.

[0126] Example 5: Preparation of a chemiluminescent kit for SNCA

[0127] 1. Preparation of magnetic separation reagent

[0128] (1) Preparation of streptavidin magnetic bead working solution

[0129] The JSR magnetic bead solution (100 mg / mL) is purchased from Shenzhen Res Biotechnology Co., Ltd., with a particle size of 1.5 μm, and is diluted to a working concentration of 0.2 mg / l with a final wash buffer of 0.05M TRIS (PH7.4) to obtain the working solution.

[0130] (2) Preparation of biotin-labeled SNCA monoclonal antibody (1)

[0131] Take 20 μL (0.2 mg) of SNCA protein monoclonal antibody and add it to a centrifuge tube. Ultracentrifuge and purify it once with 400 μL of 1xPBS solution, and then purify it twice with 300 μL of 1xPBS solution, each time for 6 - 7 minutes. Take 3.4 mg of APE-biotin (Shenzhen Res Biotechnology Co., Ltd.) and 1 ml of DMSO and dissolve them by shaking in a cryotube, and keep it in the dark for later use. Dilute the purified antibody to 100 μL with PBS and store it in a cryotube. Take 100 μL of APE-biotin solution and mix it by shaking, and react it at 4°C for 2 h. Dilute the biotin-labeled antibody to 400 μL with 1xPBS solution and add it to a centrifuge tube. Ultracentrifuge and purify it once, and then purify it a second time with 300 μL of PBS solution, each time for 6 - 7 minutes. Dilute the washed antibody to 50 μL with 1xPBS solution and store it in a cryotube. Take 50 μL of glycerol, mix it by shaking, and freeze it.

[0132] 2. Preparation of enzyme-labeled antibody (labeled alkaline phosphatase SNCA monoclonal antibody (2))

[0133] Same as Example 4.

[0134] 3. Preparation of SNCA Calibrator / Control, Washing Solution, and Luminescent Substrate

[0135] Same as Example 4.

[0136] 4. Composition of the Kit

[0137] The kit mainly consists of reagent strips (24 strips), SNCA calibrator, and control. Each reagent strip is for one-person testing. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (streptavidin magnetic bead working solution and biotin-labeled antibody each), 130 μL of enzyme-labeled antibody, 2 mL of washing solution, and 250 μL of luminescent substrate.

[0138] Example 6: Preparation of Chemiluminescent Kit for SNCA

[0139] 1. Preparation of Immunomagnetic Beads Coated with SNCA Monoclonal Antibody (1)

[0140] Same as Example 4.

[0141] 2. Preparation of Horseradish Peroxidase-Labeled SNCA Monoclonal Antibody (2)

[0142] Weigh 2 mg of HRP and dissolve it in 0.5 ml of distilled water. Add 0.5 ml of freshly prepared 0.06 M NaIO4 solution and keep it in the dark at 4°C for 30 minutes. Add 0.5 ml of 160 mM ethylene glycol and keep it at room temperature for 30 minutes. Add 2 mg of SNCA to the supernatant and mix well. Put the above solution into a dialysis bag and dialyze it in 2000 ml of 0.05 mM CB buffer (where 0.05 M CB buffer: 3.18 g of Na2CO3 + 5.88 g of NaHCO3, up to 2 L of distilled water) with stirring overnight at 4°C. Transfer the dialysis solution to a 15 ml centrifuge tube, add 0.2 ml of freshly prepared 5 mg / ml NaBH4 solution, mix well, and then keep it at 4°C for 2 hours. Add an equal volume of saturated ammonium sulfate solution, keep it at 4°C for 30 minutes, and centrifuge at 4000 rpm at 4°C for 20 minutes. Discard the supernatant and drain. Dissolve the precipitate in a small amount of PBS (0.02 M, pH 7.4), put it into a dialysis bag, and dialyze it against 0.02 M pH 7.4 PBS overnight at 4°C (change PBS once in the middle). Transfer the liquid in the dialysis solution to an EP tube, centrifuge, suck out the supernatant, add an equal volume of glycerol, mix well, and store it at -20°C.

[0143] 3. Preparation of Washing Solution

[0144] Same as Example 4.

[0145] 4. Preparation of SNCA Calibrator / Control

[0146] Same as Example 4.

[0147] 5. Preparation of Luminescent Substrate

[0148] The luminescent substrate is divided into liquid A and liquid B. Liquid A is prepared from 0.4 g / L urea peroxide and 0.1 mol / L borate buffer solution with pH 8.6; liquid B is prepared from 0.2 g / L 4-iodophenol, 0.8 g / L luminol, 0.5 ml / L Tween-20, and 0.1 mol / L borate buffer solution with pH 8.6.

[0149] 6. Composition of the Kit

[0150] The kit mainly consists of reagent strips (24 strips), SNCA calibrator, and quality control product. Each reagent strip is for one-person test. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (working solution of immunomagnetic beads coated with SNCA monoclonal antibody (1)), 130 μL of SNCA monoclonal antibody (2) labeled with horseradish peroxidase, 2 mL of washing solution, and 250 μL each of liquid A and liquid B of the luminescent substrate.

[0151] Example 7: Preparation of ELISA Assay Kit

[0152] 1. Preparation of Various Buffer Solutions and Reagents:

[0153] 1.1 Coating Buffer: 0.05 M, pH 9.6, CB (Carbonate Buffer)

[0154] Na2CO3: 16.0 g

[0155] NaHCO3: 29.0 g

[0156] Make up to 1000 ml with deionized water.

[0157] 1.2 pH 7.2, 10×PBS-Tween 20

[0158] Na2HPO4·12H2O: 58 g

[0159] KH2PO4: 4 g

[0160] NaCl: 100 g

[0161] KCl: 4 g

[0162] Tween 20: 20 ml

[0163] Make up to 1000 ml with deionized water.

[0164] 1.3 Blocking Solution / Antibody Diluent:

[0165] 10×PBS-Tween 20: 100 ml

[0166] BSA (Bovine Serum Albumin): 10 g

[0167] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0168] Make up to 1000 ml with deionized water.

[0169] 1.4, Enzyme conjugate diluent:

[0170] 10×PBS-Tween 20: 10 ml

[0171] FCS (Fetal Calf Serum): 20 ml

[0172] Enzyme stabilizer (purchased from Shanghai Xibao Company, model number ACE0070A): 1 g

[0173] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0174] Make up to 1000 ml with deionized water.

[0175] 1.5, Chromogenic agent A:

[0176] Citric acid: 35.5 g

[0177] Urea peroxide: 10 g

[0178] Tween 20: 10 ml

[0179] Make up to 1000 ml with deionized water.

[0180] 1.6, Chromogenic agent B:

[0181] Citric acid: 120 g

[0182] EDTA-2Na: 1 g

[0183] TMB·2HCl: 2 g

[0184] Make up to 1000 ml with deionized water.

[0185] 1.7, Concentrated washing solution (pH 7.2, 25×PBS-Tween 20)

[0186] Na2HPO4·12H2O: 145 g

[0187] KH2PO4: 10 g

[0188] NaCl: 250 g

[0189] KCl: 10 g

[0190] Tween 20: 50 ml

[0191] Make up to 1000 ml with deionized water.

[0192] 1.8. Stop solution: 2M H2SO4

[0193] Concentrated sulfuric acid (95 - 98%): 22.2 ml

[0194] Deionized water: 177.3 ml

[0195] When preparing, slowly drip concentrated sulfuric acid into deionized water for volume making, and shake well while adding.

[0196] 2. Preparation of pre - coated plate

[0197] Dissolve the SNCA - specific monoclonal antibody (1) in the coating buffer to prepare a pre - coating solution. Add 100 μl per well at a concentration of 0.1 μg / well on the enzyme - labeled coating plate (purchased from Shenzhen Jin Canhua Company), place at 4°C for 18 - 24 hours, take out, discard the coating solution, wash, add 100 μl of blocking solution per well and block at 4°C for 16 hours, discard the blocking solution, dry, put into an aluminum foil bag, vacuum - seal, and store at 4°C.

[0198] 3. Preparation of conjugated antibody and enzyme label

[0199] Dilute the conjugated antibody (SNCA - specific polyclonal antibody (2)) and the enzyme label (horseradish peroxidase - labeled goat anti - rabbit IgG antibody, purchased from Beijing Zhongshan Jinqiao Company) to the working concentration, and the working concentration is determined by checkerboard titration experiment.

[0200] 4. Preparation of SNCA calibrator and quality control

[0201] Gradient - dilute the recombinant SNCA (purchased from abcam company, concentration 2 mg / mL) with the sample diluent to prepare SNCA calibrator (0 - 1000 pg / ml) and two quality controls with high and low concentrations.

[0202] 5. Composition of ELISA kit

[0203] The kit mainly consists of a pre - coated plate (for 48 or 96 people), one set of SNCA calibrator, SNCA quality controls (two concentrations of high and low), conjugated antibody (10 mL), enzyme label (10 mL), chromogenic solution A (5 mL), chromogenic solution B (5 mL), concentrated washing solution (20 mL) and stop solution (5 mL).

[0204] Example 8: Preparation of SNCA - fluorescence chromatography assay kit

[0205] 1. Coating the conjugate pad

[0206] 1.1. Fluorescent microsphere-labeled SNCA monoclonal antibody (1)

[0207] 1.1.1. Activation of fluorescent microspheres:

[0208] (1) Take 500 μl of a 1% (w / v) aqueous dispersion of fluorescent microspheres (purchased from Bangs laboratories, Inc.), add primary wash buffer (50 mM MES aqueous solution, pH 6.5) to 1 ml, centrifuge at 16000 rpm for 20 minutes at 4 °C, discard the supernatant, disperse the precipitate in 1 ml of primary wash buffer, and sonicate (240 W) for 2 minutes;

[0209] (2) Repeat the above process twice;

[0210] (3) Add 375 μl (3 / 4 of the microsphere amount) of a mixture of 10 mg / ml carbodiimide solution and 10 mg / ml N-hydroxysulfosuccinimide solution mixed in a ratio of 1:3, and shake for 15 minutes to activate the fluorescent microspheres.

[0211] 1.1.2. Label SNCA monoclonal antibody (1) with activated fluorescent microspheres:

[0212] (1) Disperse the precipitate in 1 ml of coupling buffer (50 mM MES aqueous solution, pH 6.0), and sonicate (240 W) for 2 minutes;

[0213] (2) Repeat the above process twice;

[0214] (3) Obtain 500 μl of buffer dispersed with fluorescent microspheres;

[0215] (4) Add SNCA monoclonal antibody (1) thereto at a ratio of 15 mg antibody / g of activated fluorescent microspheres, and shake at room temperature for 2 hours;

[0216] (5) Add 1 ml of blocking buffer (0.5% (w / v) BSA - 0.05 M ethanolamine), continue to shake for 1 hour, then centrifuge at 16000 rpm for 20 minutes, repeat centrifugation 3 times, disperse the precipitate in 500 μl of final wash buffer (0.5% (w / v) BSA - 0.1% (v / v) Tween - 20 mM Tris solution), sonicate (240 W) for 2 minutes, and make up the volume to 500 μl with the above final wash buffer.

[0217] 1.2. Coating the conjugate pad

[0218] The prepared SNCA monoclonal antibody (1) labeled with fluorescent microspheres was diluted 1:240 with a microsphere diluent (0.5 (w / v)% BSA - 2 (w / v)% S9 - 15% sucrose - 0.5% PVP - 40000 - 0.5% PEG20000 - 20 mM Tris solution) to obtain a working solution, and then evenly sprayed on the conjugate pad at a rate of 1200 μl / 30 cm using a micropipette (purchased from DRAGON company). After that, it was dried in an oven at 37°C and stored for later use at 45% humidity.

[0219] 2. Preparation of the reaction membrane

[0220] The SNCA monoclonal antibody (2) and the goat anti - mouse IgG monoclonal antibody (purchased from arista company) were respectively diluted to 0.5 mg / ml with a 1% (w / v) PEG20000 - 5% (v / v) methanol - 3% (w / v) sucrose 10 mM PBS (pH 8.4) buffer. The interval parameters of the test line and the control line of the gold - spraying machine (purchased from Hangzhou Fenghang company) were set to 8 mm, and the coating amounts were respectively set to 1.0 μl / cm. The SNCA monoclonal antibody (2) and the goat anti - mouse IgG monoclonal antibody were drawn on the nitrocellulose membrane using the gold - spraying machine, dried in an oven at 37°C, and stored for later use at 45% humidity.

[0221] 3. Assembly and cutting of the test strip

[0222] The sample pad, conjugate pad, NC reaction membrane, and absorbent filter paper were sequentially overlapped and pasted on the bottom plate to obtain a test strip board, which was cut into test strips with a width of 4 mm.

[0223] 4. Preparation of the SNCA fluorescence immunoassay card

[0224] The cut test strip was fixed on a plastic bottom card, and the surface of the test strip was pressed tightly with a surface card. The surface card was provided with a sample - adding hole and an observation window at the positions of the sample pad and the reaction membrane of the test strip. After the test card was assembled, it was put into an aluminum foil bag, added with a desiccant and sealed for storage, and could be stored for more than one year under room - temperature dry conditions.

[0225] 5. Composition of the SNCA - fluorescence chromatography assay kit (20T)

[0226] 20 SNCA fluorescence immunoassay cards

[0227] Example 9: Determination and performance verification of SNCA using the chemiluminescence immunoassay (CLIA) kit prepared in Example 4

[0228] 1. Collection of saliva samples:

[0229] (1) The subject was calmed for 10 minutes before collection. Five minutes before collection, the subject rinsed the mouth with clean water for more than 1 minute.

[0230] (2) The subject bows their head, opens their mouth, and allows saliva to flow naturally into a sterile tube, collecting 2 ml of saliva.

[0231] (3) Transfer the collected saliva to a 2-ml centrifuge tube, place it on ice, and add a variety of protease inhibitors (0.1 μl / ml of saliva) to prevent protein degradation.

[0232] (4) Centrifuge the saliva at 2600 g for 15 minutes at 4°C, and then centrifuge at 15000 g for 15 minutes at 4°C. Take the supernatant for both centrifugations.

[0233] (5) Store at -80°C in a refrigerator.

[0234] 2. Determine the concentration of SNCA using a chemiluminescent immunoassay (CLIA) kit. The specific steps are as follows:

[0235] (1) Before the experiment, all reagents need to be brought to room temperature, and the fully automatic luminometer is turned on and preheated for at least 30 minutes.

[0236] (2) Scan the barcode of the kit, and the fully automatic luminometer automatically selects different built-in measurement programs according to the kit information.

[0237] (3) Reagent and sample loading: According to the operation guide of the fully automatic luminometer measurement program, complete the loading of magnetic separation reagents, enzyme-labeled antibodies, and luminescent substrate solutions. After mixing the calibrator / quality control / sample, transfer it to the sample well positions of each reagent strip using a quantitative pipette.

[0238] (4) Start the automatic measurement program: The measurement process and related parameters have been predefined in the instrument operation software, and the reaction and test time are set to 15 minutes.

[0239] (5) Result output: The fully automatic luminometer automatically calculates the SNCA concentration of each sample by means of a working curve obtained by calibrating the built-in main curve with two-point calibration of the calibrator.

[0240] 3. Performance verification of the kit in Example 4

[0241] 3.1 Verification of the linear range

[0242] Take samples close to the upper limit of the linear range of the kit and perform serial dilutions with the calibrator diluent. Each diluted concentration of the sample is measured 2 times, and the average value of its concentration (yi) is calculated. Using the dilution ratio (xi) as the independent variable and the corresponding average concentration (yi) as the dependent variable for linear fitting. Calculate the linear correlation coefficient (r).

[0243] The test results are shown in Table 1 below:

[0244] Table 1 Test results and calculations of the linear range Unit: pg / mL

[0245]

[0246]

[0247] The test results show that for the kit prepared in Example 4, in the range of 2 - 5000 pg / mL, the linear correlation coefficient can reach 0.9998, indicating that the kit has a wide linear range for testing.

[0248] 3.2, Minimum detection limit

[0249] The kit prepared in Example 4 was used to repeatedly detect the zero - concentration reference sample 20 times. Calculate the average value (M) and standard deviation (SD) of the 20 detection concentrations, and then calculate M + 2SD, which is the minimum detection limit.

[0250] The test results show that the minimum detection limit is 1.41 pg / mL, indicating that the kit has high sensitivity.

[0251] 3.3, Precision

[0252] The verification of precision was carried out on samples at two concentration levels, high and low. The kit of Example 4 was used to repeatedly detect each concentration sample 10 times. The two concentrations were 57.8 pg / mL and 2420 pg / mL respectively.

[0253] Calculate the average value of the 10 measurement results of each concentration sample respectively and the standard deviation SD. According to calculate the coefficient of variation CV to evaluate the precision of the kit.

[0254] The test results are shown in Table 2 below:

[0255] Table 2 Precision test results

[0256]

[0257] The test results show that for the samples at high and low concentrations tested by the kit prepared in Example 4, the CV% is less than 10%, indicating that the kit has high precision.

[0258] 3.4, Accuracy

[0259] The accuracy of the kit was evaluated by a recovery test.

[0260] High - concentration sample A was added to low - concentration sample B. The volume ratio between the added sample A and sample B was 1:9. Detect the concentrations of sample B and the mixed sample, and calculate the recovery rate according to the following formula.

[0261]

[0262] In the formula: R — recovery rate; V — volume of sample A added; V0 — volume of sample B; C — detected concentration after adding sample A to sample B; C0 — detected concentration of sample B; CS — concentration of sample A.

[0263] After calculation, the recovery rate was 92.64%, indicating that the kit had high accuracy.

[0264] Verification conclusion:

[0265] The verification results of the kit prepared in Example 4 showed that: the kit had a wide linear range, could directly measure samples with a concentration up to 5000 pg / mL without dilution, had high sensitivity (the lowest detection limit was 1.41 pg / mL), high accuracy (the recovery rate was 92.64%), good precision (the CV% of high and low concentrations were 6.4% and 5.5% respectively), and the whole reaction time was short, and the result could be obtained in 15 minutes, greatly improving the detection efficiency.

[0266] Example 10: Determination of SNCA by the enzyme-linked immunosorbent assay (ELISA) kit prepared in Example 7 and verification of the kit performance

[0267] 1. Sample collection: The same as in Example 9.

[0268] 2. Determine the SNCA concentration by the enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0269] (1) Preparation of washing solution: Dilute the 25-fold concentrated washing solution with deionized water at a ratio of 1:25.

[0270] (2) Take out the kit prepared in Example 4 and equilibrate it to room temperature. Prepare the samples, calibrators, and quality control products. Dilute the clinical samples with the sample diluent at a ratio of 1:20.

[0271] (3) First add 50 μL of the diluted sample to be tested / calibrator / quality control product to each well of the microplate, then add 50 μL of the binding antibody to the corresponding wells, gently tap and mix well, seal the microplate with a sealing film, and incubate at 37°C for 30 minutes.

[0272] (4) Take out the reaction plate, discard the liquid in the plate, add 200 - 300 μL of washing solution to each well and wash the plate 5 times, and pat dry.

[0273] (5) Add two drops or 100 μL of the enzyme-labeled substance to each well, seal the plate with a sealing film, and incubate at 37°C for 30 minutes.

[0274] (6) Take out the reaction plate, discard the liquid in the plate, add 200 - 300 μL of washing solution to each well and wash the plate 5 times, and pat dry.

[0275] (7) Add 50 μl of chromogenic reagent A and B solution to each well, mix well, and incubate at 37 °C for 15 minutes;

[0276] (8) As soon as possible, add 1 drop (50 μl) of stop solution to each well and gently tap to mix;

[0277] (9) Measure the OD value of each well using an enzyme-labeled instrument (measured at a dual wavelength of 450 / 630 nm);

[0278] (10) Draw a calibration curve and calculate the SNCA concentration in the sample according to the calibration curve.

[0279] 3. Kit performance verification

[0280] 3.1. Linear range verification

[0281] The verification method is the same as that in Example 9.

[0282] The test results are shown in Table 3 below:

[0283] Table 3 Linear range test results and calculations Unit: pg / mL

[0284]

[0285]

[0286] The test results show that the kit prepared in Example 7 has a linear correlation coefficient of up to 0.993 in the range of 20 - 1000 pg / mL.

[0287] 3.2. Minimum detection limit

[0288] The verification method is the same as that in Example 9.

[0289] The test results show that the minimum detection limit is 15.2 pg / mL, indicating that the kit has high sensitivity.

[0290] 3.2. Precision

[0291] The verification method is the same as that in Example 9. The two concentrations used for precision verification are 57.8 pg / mL and 524 pg / mL respectively.

[0292] The test results are shown in Table 4 below:

[0293] Table 4 Precision test results

[0294]

[0295] The test results show that the kit prepared in Example 7 has a CV% less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0296] 3.4, Accuracy

[0297] The verification method is the same as that in Example 9.

[0298] After calculation, the recovery rate is 91.6%, indicating that the accuracy of the kit is relatively high.

[0299] Verification conclusion:

[0300] The verification results of the kit prepared in Example 7 show that: the kit has good linearity in the range of 20 - 1000 pg / mL, reaching 0.993, with high sensitivity (the lowest detection limit is 15.2 pg / mL), a recovery rate of 91.6%, high accuracy, and good precision (CV% of both high and low concentrations is less than 10%). It does not require complex equipment, can be used for batch detection, and can produce more than 80 test results simultaneously in 1.5 hours, with relatively high detection efficiency.

[0301] Example 11: Determination of SNCA by the fluorescence immunochromatographic kit prepared in Example 8 and verification of the kit performance

[0302] 1. Sample collection: The same as in Example 9.

[0303] 2. Determine the SNCA concentration with the fluorescence immunochromatographic kit. The specific steps are as follows:

[0304] (1) Restore the reagent to room temperature (20 ± 5°C) before testing, and the testing is carried out at room temperature;

[0305] (2) Turn on the fluorescence analyzer and adjust the instrument to the waiting detection state;

[0306] (3) Add 100 μL of each of the 1:4 diluted calibrator / quality control / sample to the sample addition port of each test card and react for 15 min;

[0307] (4) After the reaction is completed, insert the test card into the fluorescence analyzer for scanning and testing, and calculate the SNCA concentration of each sample according to the built-in calibration curve imported into the analyzer.

[0308] 3. Kit performance verification

[0309] 3.1. Verification of linear range

[0310] The verification method is the same as that in Example 9.

[0311] The test results are as follows in Table 5:

[0312] Table 5 Test results and calculation of linear range (unit: pg / mL)

[0313]

[0314] The test results show that for the kit of Example 8, in the range of 20 - 1000 pg / mL, the linear correlation coefficient can reach 0.998.

[0315] 3.2, Minimum detection limit

[0316] The verification method is the same as that of Example 9.

[0317] The test results show that the minimum detection limit is 16.5 pg / mL.

[0318] 3.2, Precision

[0319] The verification method is the same as that of Example 9. The two concentrations used for precision verification are 57.8 pg / mL and 524 pg / mL respectively.

[0320] The test results are as shown in Table 6 below:

[0321] Table 6 Precision test results

[0322]

[0323]

[0324] The test results show that for the kit prepared in Example 8, the CV% for testing samples at both high and low concentrations is less than 10%, indicating that the kit has high precision.

[0325] 3.4, Accuracy

[0326] The verification method is the same as that of Example 9.

[0327] After calculation, the recovery rate is 90.5%, meeting the requirement of the recovery rate [90% - 110%].

[0328] Verification conclusion:

[0329] The verification results of the kit prepared in Example 8 show that: the kit has good linearity in the range of 20 - 1000 pg / mL, reaching 0.998, the sensitivity is 16.5 pg / mL, the recovery rate is 90.5%, the accuracy meets the requirements, and the precision is good (CV% for both high and low concentrations is less than 10%). It can be used for bedside diagnosis, with simple operation, available for testing at any time, and the results can be obtained within 15 minutes, enabling rapid detection.

[0330] Test of comparative example:

[0331] The SNCA monoclonal antibodies of ab138501 (purchased from abcam) and LS - B2923 (purchased from lifespan) were used to prepare a chemiluminescence kit (comparative kit) according to the method of Example 4, and performance comparison tests were carried out with the kit of Example 4.

[0332] The comparison results of various performances are shown in Table 7 below:

[0333] Table 7 Comparison results of the performance of the comparison kit and the kit of Example 4

[0334]

[0335]

[0336] For the comparison of various analytical performances, the kit prepared in Example 4 has a wider linear detection range and higher sensitivity.

[0337] Example 12: The SNCA assay kit is used for the auxiliary diagnosis of Parkinson's disease

[0338] 1. Research objects

[0339] Parkinson's disease group: 55 patients with Parkinson's disease, including 32 males and 23 females, with an age of (64.8 ± 11.9) years. The patients had typical characteristics, met the diagnostic criteria for Parkinson's disease in China (2016 version), and had bradykinesia and one of the following symptoms: resting tremor (4 - 6 Hz) / muscle rigidity.

[0340] Healthy control group: 118 healthy controls, including 67 males and 51 females, with an age of (62 ± 13.2) years, without mental diseases, neurological diseases, and somatic diseases.

[0341] There was no significant difference in age and gender between the two groups. All research objects gave informed consent to this study.

[0342] 2. Experimental methods

[0343] 2.1. Use the SNCA kit prepared in Example 4 to test the samples of the control group and patients with Parkinson's disease.

[0344] 2.2. Receiver operating characteristic curve (ROC curve): Make a ROC curve based on the SNCA levels of patients and the control group to analyze the value of saliva SNCA in the auxiliary diagnosis of Parkinson's disease.

[0345] 3. Results

[0346] As Figure 1 shown, the saliva SNCA level (50.8 ± 30.1 pg / mL) in the Parkinson's disease group (PD) was significantly lower than that in the healthy control group (HC) (313.0 ± 223.8 pg / mL).

[0347] Draw the receiver operating characteristic curve for the diagnosis of Parkinson's disease (as Figure 2) The area under the ROC curve (AUC) for the diagnosis was 0.89 (95% CI = 0.84 - 0.94), P < 0.0001, indicating that salivary SNCA has good value for the auxiliary diagnosis of Parkinson's disease.

Claims

1. A SNCA-specific antigenic epitope peptide, characterized in that, The amino acid sequence is as follows: Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu (1) or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

2. An SNCA-specific antibody, which is a polyclonal antibody prepared by immunizing an animal with an SNCA-specific antigen epitope peptide, wherein the amino acid sequence of the SNCA-specific antigen epitope peptide is as follows: Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu (1) or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

3. The SNCA-specific antibody according to claim 2, wherein It is prepared by immunizing a rabbit or a mouse after coupling the SNCA-specific antigen epitope peptide with a carrier protein.

4. A kit for the determination of α-synuclein, detection or diagnosis of Parkinson's disease, comprising: SNCA-specific antibody; Wherein the SNCA-specific antibody is a polyclonal antibody prepared by immunizing an animal after coupling the SNCA-specific antigen epitope peptide with a carrier protein, and the amino acid sequence of the SNCA-specific antigen epitope peptide is as follows: Tyr-Lys-Thr-Lys-Gln-Gly-Val-Ala-Glu-Ala-Ala-Gly-Lys-Thr-Lys-Glu (1) or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

5. The kit according to claim 4, wherein the kit further comprises a solid-phase carrier, a label, and an SNCA calibrator.

6. The kit according to claim 5, wherein the kit is any one of an enzyme-linked immunosorbent assay kit, a chemiluminescence assay kit, a fluorescence immunochromatography assay kit, or a colloidal gold immunoassay kit.

7. The kit according to claim 5, wherein the solid-phase carrier is selected from any one of a microplate, magnetic microspheres, or a nitrocellulose membrane; the label is selected from horseradish peroxidase HRP, alkaline phosphatase AP, a luminescent substance, a fluorescent substance, a dye, or colloidal gold.

8. The kit according to claim 5, wherein the kit is a chemiluminescence assay kit and the solid-phase carrier is magnetic microspheres.

9. The kit according to claim 8, wherein the magnetic microspheres are directly or indirectly connected to the SNCA-specific antibody to form a magnetic separation reagent; the magnetic separation reagent is an immunomagnetic bead coated with an SNCA monoclonal antibody or a streptavidin magnetic bead formed by connecting streptavidin to an SNCA antibody.

10. The kit according to claim 7, wherein the label is selected from horseradish peroxidase HRP and / or alkaline phosphatase AP.

11. Use of the kit according to any one of claims 4 - 10 in the preparation of a product for detecting and diagnosing Parkinson's disease.

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