Phosphorylated α-synuclein antigen epitope peptide, antibody and kit for measuring phosphorylated α-synuclein in saliva and their application in the diagnosis of Parkinson's disease

Abstract

The present invention provides a P-α-Syn specific antigen epitope peptide, wherein the amino acid sequence of the P-α-Syn specific antigen epitope peptide is one of the following two sequences: Tyr-Arg-Glu-Met-Pro-Ser(PO3H2)-Glu-Glu-Gly(1); or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala(2). The present invention also discloses a P-α-Syn specific antibody. The present invention further discloses an α-synuclein assay kit, comprising: a solid phase carrier, a P-α-Syn specific antibody, a marker and a P-α-Syn calibrator. The present invention also discloses the use of the kit provided by the present invention in the preparation of a product for diagnosing Parkinson's disease. The kit prepared by the present invention needs to have good diagnostic sensitivity, specificity and repeatability, and meet clinical application requirements.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a phosphorylated α-synuclein antigen epitope peptide, a specific antibody prepared using the antigen epitope peptide, a kit for measuring phosphorylated α-synuclein in saliva prepared using the antibody, and application of the kit in diagnosing Parkinson's disease. Background Art

[0002] Parkinson's disease is a common neurodegenerative disorder affecting middle-aged and elderly people. It is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. Biochemical changes in the striatum, including decreased dopamine production and an imbalance between dopamine and acetylcholine, are associated with motor symptoms such as tremor, rigidity, bradykinesia, and postural balance disorders, as well as non-motor symptoms such as sleep disturbances, olfactory impairment, autonomic dysfunction, and cognitive and psychiatric impairments. Epidemiological studies have shown that the prevalence of Parkinson's disease in Europe and the United States reaches 1% in those aged 60 and over, exceeding 4% in those aged 80 and over, and 1.7% in those aged 65 and over in my country. As the disease progresses, the motor and non-motor symptoms of Parkinson's disease gradually worsen, impairing daily activities and imposing a significant social and medical burden.

[0003] Current diagnostic criteria for Parkinson's disease include the "Chinese Parkinson's Disease Diagnostic Criteria (2016 Edition)" and the "Movement Disorder Society's Clinical Diagnostic Criteria for Parkinson's Disease (MDS-2015)." Both are based on the "Clinical Diagnostic Criteria for Parkinson's Disease" established by the UK Parkinson's Disease Research Institute (UK PD Brain Bank) in 1997 and have been refined. These diagnostic criteria primarily follow a three-step approach to diagnosing Parkinson's disease: inclusion, exclusion / alert, and support. A clinically confirmed diagnosis of Parkinson's disease (PD) requires first determining whether the patient presents with either of the two cardinal signs of bradykinesia (slow initiation of voluntary movement and progressive decrease in the speed and amplitude of repetitive movements) or resting tremor (4-6 Hz) / muscle rigidity, in addition to meeting the absolute exclusion criteria, at least two supportive criteria, and the absence of red flags. Exclusion criteria and red flags include the efficacy of dopamine therapy, functional status, and imaging test results. Supportive criteria include a clear and significant response to dopaminergic therapy, the presence of levodopa-induced dyskinesias, single-limb resting tremor documented on clinical examination (either previously or during the current examination), and the presence of anosmia or cardiac sympathetic denervation on cardiac MIBG scintigraphy. All core cardinal signs must be assessed 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 long-term observation of clinical symptoms and the effectiveness of dopaminergic drugs. In addition, PD symptoms overlap a lot with multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration in terms of clinical symptoms and pathology. Clinical symptom assessment as a key diagnostic indicator is unstable, and the rates of missed diagnosis and misdiagnosis are high, which brings great inconvenience to the diagnosis and treatment of PD.

[0005] Given the current status of Parkinson's disease diagnosis, clinical researchers have been trying to find a stable and reliable biological marker that can objectively diagnose Parkinson's disease and evaluate its efficacy. Currently, brain imaging and body fluid biomarkers are the most studied.

[0006] Parkinson's disease is a neurodegenerative disease whose occurrence is closely related to changes in brain structure and function. Brain imaging is an important tool for the study of neurodegenerative diseases. Among them, transcranial ultrasound, magnetic resonance imaging, and positron emission tomography can all detect 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 abnormal substantia nigra, namely the strong echo (SN+) phenomenon, but this is only for people with a higher possibility of PD, and the above clinical diagnosis still needs to be prioritized. The difference in excitability between Parkinson's disease (PD) patients and healthy controls revealed by blood oxygen level-dependent functional magnetic resonance imaging (fMRI) lacks specific quantitative criteria and can be influenced by the diagnostician's subjective perception. Current MRI technology is limited by numerous factors, including disease heterogeneity, the lack of imaging standards for the normal brain, the high sensitivity of MRI to physiological or exogenous chemical changes in the subject (such as agitation, coffee, smoking, and temperature), limitations in statistical analysis methods, and limitations in measured variables. Furthermore, individual variability in excitability in the cerebellar-thalamic-cortical and striatal-thalamic-cortical circuits is influenced by numerous external factors, making PD diagnosis difficult and its performance far from meeting the requirements for clinical application. PET-based presynaptic dopamine transporters are currently considered the most sensitive marker of PD. With their high sensitivity, they can be used as an indicator of striatal functional status, significantly reducing the misdiagnosis rate of Parkinson's disease and playing a key role in the differential diagnosis of Parkinson's disease. However, due to the long scanning time, high cost, and high requirements for operator skills and equipment, and the fact that PET tracers may increase the genotoxicity of ionizing radiation, imaging cannot currently be used as a diagnostic indicator for Parkinson's disease in clinical practice.

[0007] The pathological hallmark of Parkinson's disease is currently believed to be Lewy bodies, which are eosinophilic inclusions within the cytoplasm of the remaining neurons following the degeneration and loss of a large number of dopaminergic neurons in the substantia nigra. Lewy bodies found in the neurons of patients with Parkinson's disease (PD) contain numerous α-synuclein phosphorylated at Ser129. Comparative studies have shown that less than 4% of α-synuclein in the normal human brain is phosphorylated at Ser129, but at least 90% of Lewy bodies formed in the brains of patients with Parkinson's disease (PD) are phosphorylated at this site. This suggests that α-synuclein phosphorylated at Ser129 is prone to aggregation and is crucial in the pathogenesis of PD. Therefore, researchers have sought to investigate the potential of phosphorylated α-synuclein (P-α-Syn) as a biomarker for Parkinson's disease. However, due to the difficulty in obtaining cerebrospinal fluid (CSF) samples, these studies are often based on small clinical sample sizes. Furthermore, CSF P-α-Syn levels and structural imaging have not been shown to correlate well with the overall course of Parkinson's disease, and further longitudinal studies are needed to validate their potential. Blood Pa-Syn levels are affected by α-syn in red blood cells and exosomal α-syn, and lack the ability to distinguish, making it unable to be used as an individual biomarker for the diagnosis of Parkinson's disease. The diagnostic accuracy is low and it cannot be considered as a single diagnostic or prognostic biomarker for PD.

[0008] As research deepens, it has been discovered that P-α-Syn is not limited to the central nervous system and blood; phosphorylated α-synuclein has also been detected in saliva. Saliva specimens are not only noninvasive and easy to collect, but recent studies have also shown that the submandibular gland, which secretes saliva, is a target organ affected in the early stages of Parkinson's disease (PD), suggesting that salivary phosphorylated α-synuclein may serve as a potential biomarker for early PD screening.

[0009] Currently, there is no clinical research kit for measuring phosphorylated α-synuclein in saliva for Parkinson's disease diagnosis. This area plans to develop a kit for measuring the concentration of phosphorylated α-synuclein in human saliva, providing a more practical and objective indicator for auxiliary diagnosis and early screening of Parkinson's disease. Summary of the Invention

[0010] In order to solve the problems existing in the prior art, the present invention screened and obtained a phosphorylated α-synuclein-specific antigen epitope peptide, characterized in that the amino sequence of the antigen epitope peptide is one of the following:

[0011] Tyr-Arg-Glu-Met-Pro-Ser(PO3H2)-Glu-Glu-Gly (1) or

[0012] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala(2).

[0013] The present invention thus provides a specific antibody, which is a monoclonal antibody prepared by coupling the above-mentioned P-α-Syn specific antigen epitope peptide (1) to a carrier protein and then immunizing an animal.

[0014] The present invention thus provides a specific antibody, which is a monoclonal antibody or a polyclonal antibody prepared by coupling the above-mentioned P-α-Syn specific antigen epitope peptide (2) to a carrier protein and then immunizing an animal.

[0015] The present invention further provides a kit for determining salivary phosphorylated α-synuclein, which comprises: a solid phase carrier, a specific antibody, a marker and a P-α-syn calibrator, wherein the specific antibody is the specific antibody mentioned above.

[0016] According to the present invention, the P-α-syn assay kit is developed using an immunoassay method, and any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), fluorescent immunochromatography, or colloidal gold assay can be used.

[0017] According to the present invention, the solid phase carrier can be one of a microporous reaction plate, magnetic microspheres or a nitrocellulose membrane (NC membrane).

[0018] 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 chemiluminescent immunoassay (CLIA) kit, the label is preferably alkaline phosphatase AP; when used in a fluorescent immunochromatography kit, the label is preferably fluorescent microspheres; and when used in a colloidal gold assay kit, the label is colloidal gold.

[0019] According to the present invention, the phosphorylated α-synuclein calibrator / quality control is diluted extracted phosphorylated α-synuclein.

[0020] The specific antibodies are prepared by respectively coupling the antigen epitope peptide (1) and the antigen epitope peptide (2) with a carrier protein and then immunizing animals (mouse, rat, rabbit, sheep, etc.).

[0021] In the present invention, the specific antibody prepared from the antigenic epitope peptide (1) is the P-α-Syn specific antibody (1), which is a monoclonal antibody; the specific antibody prepared from the antigenic epitope peptide (2) is the P-α-Syn specific antibody ((2), which can be a monoclonal antibody or a polyclonal antibody, 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).

[0022] In a preferred embodiment, the kit of the present invention is a chemiluminescent immunoassay kit, which is developed using the principles of chemiluminescent immunoassay (CLIA) combined with double antibody sandwich method.

[0023] In a specific embodiment of the chemiluminescent immunoassay kit, the solid phase carrier is magnetic microbeads, and the magnetic microbeads are directly or indirectly connected to the P-α-Syn specific antibody (1) to form a magnetic separation reagent; the magnetic separation reagent is preferably an immunomagnetic bead coated with the P-α-Syn specific antibody (1) or a magnetic bead in which streptavidin is connected to the P-α-Syn specific antibody (1).

[0024] In a specific embodiment of the chemiluminescent immunoassay kit, the kit further comprises a luminescent substrate. Examples of luminescent substrates include, but are not limited to, luminol, isoluminol and its derivatives or (adamantane)-1,2-dioxyethane and its derivatives.

[0025] In a specific embodiment, the kit comprises: a magnetic separation reagent, an enzyme-labeled α-synuclein monoclonal antibody [P-α-Syn specific antibody (2)], a phosphorylated α-synuclein calibrator / quality control, a washing solution, and a chemiluminescent substrate. The magnetic separation reagent is preferably an immunomagnetic bead coated with the P-α-Syn specific antibody (1) or a magnetic bead in which streptavidin is linked to the P-α-Syn specific antibody (1); and the α-synuclein monoclonal antibody is prepared by immunizing an animal with an α-synuclein specific antigen epitope peptide (2) coupled to a carrier protein.

[0026] The present invention further provides use of the kit of the present invention in preparing products for detecting and diagnosing Parkinson's disease.

[0027] When the kit of the present invention is used to prepare a product for diagnosing Parkinson's disease, the concentration of phosphorylated α-synuclein in the subject sample measured by the kit is compared with the control reference interval (i.e., the concentration level of healthy subjects). If the concentration of phosphorylated α-synuclein in the subject sample is higher than the concentration in the control reference interval, the subject is at risk of developing Parkinson's disease.

[0028] Preferably, the test is a saliva sample test. Further preferably, the test is any one of an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence assay (CLIA), a fluorescent immunochromatographic assay kit, and a colloidal gold immunoassay; and further preferably, the chemiluminescence assay (CLIA) is used.

[0029] Chemiluminescence detection offers high sensitivity (minimum detection limit of 7.8 pg / mL) and a wide linear range, allowing direct measurement of samples up to 2000 pg / mL without dilution. It also boasts high accuracy (93.45% recovery in the recovery test) and high precision (CV less than 10%). The short reaction time, with results available in 15 minutes, significantly improves detection efficiency. The high degree of automation eliminates the need for manual operation, reducing human error.

[0030] Compared with the prior art, the present invention has the following advantages and positive effects:

[0031] 1. The present invention applies phosphorylated α-synuclein as a marker to prepare a Parkinson's disease auxiliary diagnosis kit, overcoming the defects of the indicators currently used in the clinical diagnosis of Parkinson's disease being too subjective and lacking objectivity. In addition, sampling is convenient and non-invasive, providing a more accurate, reliable and convenient objective indicator for auxiliary diagnosis and screening of Parkinson's disease.

[0032] 2. The kit prepared by the present invention must have good diagnostic sensitivity, specificity and repeatability, and meet the requirements of clinical application.

[0033] 3. The specific antibodies (1) and (2) prepared by the present invention can bind to phosphorylated α-synuclein in the sample with high specificity.

[0034] 4. The two-segment phosphorylated α-synuclein specific antigen epitope peptide screened by the present invention is hydrophilic, highly antigenic and easy to synthesize. The antigen (immunogen) prepared therefrom can produce highly specific monoclonal antibodies or polyclonal antibodies when used to immunize animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shows the level of salivary phosphorylated α-synuclein in controls and Parkinson's disease patients (test results using the kit prepared in Example 4)

[0036] Figure 2 The ROC curve of salivary phosphorylated α-synuclein for the diagnosis of Parkinson's disease is shown (test results using the kit prepared in Example 4) DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0038] Example 1: Screening of phosphorylated α-synuclein specific antigen epitope peptides

[0039] The amino acid sequence used is known in the art and can be found in professional databases such as NCBI (GenBank: AAL15443.1).

[0040] After extensive theoretical research and experimental exploration, the inventors considered factors such as hydrophilicity, antigenicity, and ease of synthesis, and ultimately screened out the following two epitope peptides with good antigenicity, whose amino acid sequences are:

[0041] Tyr-Arg-Glu-Met-Pro-Ser(PO3H2)-Glu-Glu-Gly (1) or

[0042] Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

[0043] Subsequent experiments showed that the above-mentioned epitope peptide (1) and epitope peptide (2) have the following characteristics:

[0044] a. antigenicity;

[0045] b. After being linked to a carrier protein, it can serve as an immunogen to stimulate animals to produce specific antibodies;

[0046] c. The prepared antibody can specifically bind to phosphorylated α-synuclein in the sample.

[0047] Example 2: Synthesis of antigenic epitope peptide (1) and antigenic epitope peptide (2)

[0048] 1. Raw materials: HMP resin (P-hydroxymethylphenoxymethyl polyvinyl resin, purchased from Sigma); Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acid, purchased from Merck); Fmoc-Ser(PO(OBzl)OH)-OH (phosphorylated amino acid with side chain monobenzyl protection, purchased from Merck); NMP (nitromethylpyrrolidone, purchased from Sigma); DCM (dichloromethane, purchased from Zhongyuan Chemical Company); MeoH (methanol, purchased from Zhongyuan Chemical Company); Piperidin e (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 Company, 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, and place 0.1 mmol of HMP resin in the reaction chamber of an American ABI431A automatic peptide synthesizer. The synthesizer automatically connects specific amino acids in different sequences, with a coupling rate of 99%.

[0052] 4. Purification of Synthetic Epitope Peptide (1) and Epitope Peptide (2):

[0053] Separation and purification by high performance liquid chromatography:

[0054] Conditions: Chromatographic column: C8 10×100 mm, purchased from Waters, USA

[0055] Chromatograph: Waters 600, Waters, USA

[0056] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0057] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0058] Detection wavelength: 214nm

[0059] Flow rate: 4ml / min

[0060] Elution gradient: 20-60% B, 30 min

[0061] HPLC (high performance liquid chromatography) analysis

[0062] Chromatographic column: C18 4.6×150 mm, purchased from Waters, USA

[0063] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0064] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0065] Detection wavelength: 214nm

[0066] Flow rate: 1ml / min

[0067] Elution gradient: 0-60% B, 30 min

[0068] The results of peptide analysis showed that the purity of the purified antigen epitope peptide (1) and antigen epitope peptide (2) were both above 95%.

[0069] Example 3: Preparation of specific antibodies

[0070] 1. Preparation of antigens:

[0071] Antigen (1) or antigen (2) is prepared by linking antigen epitope peptide (1) or antigen epitope peptide (2) with carrier protein KLH (keyhole limpet hemocyanin) using the bis-diazotizedbenzidine dichloride (BDB) method.

[0072] 20.0 mg of the antigenic epitope peptide (1) or antigenic epitope peptide (2) prepared in Example 1 was dissolved in 0.2 M borate buffer (pH 9.0); 7.36 ml of 6.25 mg / ml KLH was cooled to 0°C, and 1 ml of BDB was added. The mixture was placed in an ice-water mixture and mixed in the dark. The mixture was 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 packaged and stored at -20°C.

[0073] The formula of borate buffer is: 80 ml of 0.05 mol / L borax and 20 ml of 0.2 mol / L boric acid.

[0074] 2. Immunize animals to prepare monoclonal antibodies:

[0075] 2.1. The antigen (1) or antigen (2) prepared above was mixed with an equal volume of Freund's complete adjuvant (purchased from Shanghai Yuanju Biotechnology Co., Ltd.) and then immunized with Balb / c mice. 50 μg of antigen was injected subcutaneously at multiple sites. 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 per mouse. The mice were injected subcutaneously at multiple sites. The number of booster immunizations was 6 times. The booster immunizations were repeated twice before fusion. After that, spleen cells were fused with Sp2 / 0 myeloma cells using 50% PEG (MW4000) (purchased from Zhongyuan Chemical Co., Ltd.) according to conventional methods 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 9-11 days. Large cell clones appeared in the wells. Screening was performed using indirect ELISA starting 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 screened cells were divided and multiplied in large quantities), and then the cells were expanded, frozen, and ascites was prepared.

[0076] 2.2. Balb / c mice were treated with 0.5 ml of pristane (purchased from Sigma) and 2×10 hybridoma cells were inoculated into the abdominal cavity one week later. 6 The ascites was collected after 10 days.

[0077] 2.3. Determination of Antibody Titer: The titer of the prepared monoclonal antibody was determined by indirect ELISA method. The results showed that the titer of the monoclonal antibody reached 1:32000.

[0078] 3. Immunize animals to prepare polyclonal antibodies:

[0079] 3.1. Three-month-old New Zealand white rabbits weighing approximately 2 kg were selected as immunized animals. For the primary immunization, 2 mg of the above-prepared antigen (2) was mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and then subcutaneously injected at multiple points on the rabbit's back. For booster immunization every two weeks, the antigen was fully emulsified with incomplete Freund's adjuvant and then subcutaneously injected at multiple points on the back at a rate of 1 ml per rabbit. Ten days after the final booster immunization, the carotid artery was bled and the serum was separated.

[0080] 3.2. Determination of Antibody Titer: The titer of the polyclonal antibody was determined by indirect ELISA. The results showed that the antibody titer reached 1:32000.

[0081] 3.3. Blood collection and serum separation: Blood was collected through carotid artery cannulation and serum was separated.

[0082] 4. Separation and purification of antibodies:

[0083] The specific antibody to be loaded was first dialyzed against equilibration buffer (0.02 M PB, pH 8.0) to a consistent pH. The column was then loaded and connected to a protein chromatography system, and washed with equilibration buffer until pH 8.0. The purified phosphorylated α-synuclein antibody was added to the column. After the entire sample was injected, the column was washed with elution buffer (0.05 M PB, pH 8.0). The protein solution was collected when the protein nucleic acid detector indicated a peak. The collection process was stopped when the peak gradually decreased. The antibody concentration was measured and calculated using an L5S UV-Vis spectrophotometer (manufactured by Broadtech). The purified specific antibody was aliquoted and stored at -20°C.

[0084] 5. Identification of Antibody Specificity

[0085] ELISA was used to detect the specific reactivity of the prepared monoclonal or polyclonal antibodies with the phosphorylated α-synuclein. Normal BALB / c mouse serum or rabbit serum was used as a negative control, and PBS was used as a blank control.

[0086] Results: Monoclonal antibodies (1) and (2) prepared from antigen (1) and (2) as well as polyclonal antibody (2) prepared from antigen (2) all reacted positively only with phosphorylated α-synuclein (P / N>2.1), but reacted negatively with GFAP protein and S-100B protein, indicating that the monoclonal antibodies (1), (2) and polyclonal antibody (2) of the present invention are respectively specific.

[0087] Example 4: Preparation of a chemiluminescent kit for phosphorylated α-synuclein

[0088] 1. Preparation of magnetic separation reagent (working solution of immunomagnetic beads coated with monoclonal antibody (1))

[0089] (1) Washing of magnetic beads

[0090] Transfer 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube, add 2 mg of JSR magnetic bead stock solution (Shenzhen Ruisi Biotechnology Co., Ltd.), and vortex mix for 1 minute. Place the coated tube on a magnetic separation rack for 1 minute and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex mix for 1 minute. Repeat this process twice.

[0091] (2) Activation of magnetic beads

[0092] After adding 800 μL of 0.1M MES (pH 6.0) buffer to the coating tube, add 100 μL of NHS (10 mg / L) solution and vortex mix for 1 minute. Then, add 100 μL of EDC (10 mg / L) solution to the coating tube. Place the coating tube on a rotary mixer at a constant temperature of 25 ± 1°C, set the rotary mixer speed to 50 ± 1 RPM, and react for 30 minutes.

[0093] (3) Coating monoclonal antibodies with activated magnetic beads (1)

[0094] Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex mix for 1 minute. Add 12-16 μL of monoclonal antibody (1) 10 mg / mL to the coated tube and vortex mix for 1 minute. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex mix for 1 minute. Place the coated tube on a rotary mixer at a constant temperature of 25 ± 1°C, set the rotary mixer speed to 50 ± 1 rpm, and react for 2 hours.

[0095] (4) Magnetic bead sealing

[0096] Place the coated tube on a magnetic separation rack for 1 minute, discard the supernatant, add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube, and vortex to mix thoroughly. Repeat this procedure twice. Place the coated tube on a rotary mixer at a constant temperature of 25 ± 1°C, set the rotary mixer speed to 50 ± 1 RPM, and incubate for 30 minutes.

[0097] (5) Cleaning

[0098] After blocking, place the coated tube on a magnetic separation rack for 1 minute, discard the supernatant, add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube, and vortex mix for 1 minute. Repeat this procedure twice. Add 1 mL of 0.05 M TRIS (pH 7.4) to the coated tube and vortex mix for 1 minute.

[0099] (6) Preparation of magnetic separation reagent

[0100] The magnetic beads coated with the monoclonal antibody (1) were diluted with the final washing buffer and the enzyme label diluent 0.05M TRIS (pH 7.4) to obtain a final concentration of 0.2 mg / mL.

[0101] 2. Preparation of enzyme-labeled antibodies (monoclonal antibodies labeled with alkaline phosphatase (2))

[0102] Soak an ultrafiltration centrifuge tube (30KD) in 250µl of 0.1M MES (pH 4.5) for 2 minutes. Add 250µl of 0.1M MES (pH 4.5) to a centrifuge column to a constant volume of 500µl. Add 12.5µl of AP enzyme to the ultrafiltration centrifuge tube, gently mix by inverting, and centrifuge at 13,000rpm for 15 minutes. After centrifugation stops, 100µl of liquid will be observed in the centrifuge column. Discard the waste liquid and add 200µl of 0.1M MES (pH 4.5) to the ultrafiltration centrifuge tube. Centrifuge at 13,000rpm for 20 minutes. After the centrifugation stops, 100ul of liquid can be observed in the centrifuge column. After discarding the waste liquid, add 50ul of EDC (10mg / l) and 6ul of NHS (10mg / l) to the ultrafiltration centrifuge tube, then add 100ul of 0.1M MES (PH4.5) to make the volume 250ul, mix well, and place on a shaker for activation for 1.5 hours. Take 10ul of monoclonal antibody (2) 10mg / l, mix well, and centrifuge at 13000rpm for 20min, and discard the centrifuged liquid. Add 250ul of 0.1MPB (PH9.0), mix well, and centrifuge at 13000rpm for 20min. Repeat the previous step and centrifuge once. Then add 0.1MPB (PH9.0) to make the volume 250ul and place on a shaker at 120r for coupling for 2h. Add 0.05M TRIS PH8.02, add 250ul, mix well, and place on a shaker at 120rpm for 30min. Remove 500 μl of the coupled solution from the centrifuge column, add an equal amount of glycerol, mix well, and store at -20°C. Measure and calculate the concentration of the alkaline phosphatase-labeled antibody using an L5S UV-visible spectrophotometer (Boda Jingke). The enzyme-labeled antibody was diluted 1:1000 with 0.05 M TRIS (pH 7.4) enzyme diluent to obtain the enzyme-labeled antibody.

[0103] 3. Preparation of washing solution

[0104] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0105] 4. Preparation of phosphorylated α-synuclein calibrators / quality controls

[0106] Phosphorylated α-synuclein was serially diluted with a calibrator diluent (containing 10 mM phosphate buffer (PBS) (pH 7.2), 1% BSA, and 0.03% biological preservative Proclin-300) to prepare high and low concentration quality control products.

[0107] 5. Preparation of Luminescent Substrate

[0108] The chemiluminescent substrate is (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt (APS-5) and 0.3 M Tris buffer containing 0.0003% lucigenin, 0.001% sodium sulfite, 0.1% sodium dodecyl sulfate (SDS), and 0.03% Tween 20.

[0109] 6. Composition of the kit

[0110] The kit mainly consists of reagent strips (24 strips), phosphorylated α-synuclein calibration and quality control products. Each reagent strip is for one test. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (immunomagnetic bead working solution coated with monoclonal antibody (1)), 130 μL of enzyme-labeled antibody, 2 mL of washing solution and 250 μL of luminescent substrate.

[0111] Example 5: Preparation of a chemiluminescent kit for phosphorylated α-synuclein

[0112] 1. Preparation of magnetic separation reagent

[0113] (1) Preparation of Streptavidin Magnetic Bead Working Solution

[0114] JSR magnetic bead solution (100 mg / mL) was purchased from Shenzhen Ruisi Biotechnology Co., Ltd. with a particle size of 1.5 μm. It was diluted to a working concentration of 0.2 mg / l with a final wash buffer 0.05 M TRIS (pH 7.4) to obtain a working solution.

[0115] (2) Preparation of biotin-labeled monoclonal antibody (1)

[0116] Take 20ul (0.2mg) of monoclonal antibody (1) and add it to a centrifuge tube. Purify it once by ultracentrifugation with 400ul 1xPBS solution and then purify it twice with 300ul 1xPBS solution, each time for 6-7 minutes.

[0117] Take 3.4mg APE-biotin (Shenzhen Ruisi Biotechnology Co., Ltd.) and 1ml DMSO and shake to dissolve in a cryopreservation tube. Keep it away from light for later use. Dilute the purified antibody to 100ul with PBS and store it in a cryopreservation tube. Take 100ul APE-biotin solution and shake to mix. Place it at 4°C to react for 2h. Dilute the biotin-labeled antibody to 400ul with 1xPBS solution and add it to a centrifuge tube. Purify it once by ultracentrifugation, and then purify it a second time with 300ul PBS solution, each for 6-7 minutes. Dilute the cleaned antibody to 50ul with 1xPBS solution and store it in a cryopreservation tube. Take 50ul glycerol and shake to mix, and freeze it.

[0118] 2. Preparation of enzyme-labeled antibodies (labeled with alkaline phosphatase monoclonal antibody (2))

[0119] Same as Example 4.

[0120] 3. Preparation of phosphorylated α-synuclein calibrators / quality controls, wash buffer, and luminescent substrate

[0121] Same as Example 4.

[0122] 4. Composition of the kit

[0123] The kit mainly consists of reagent strips (24 strips), phosphorylated α-synuclein calibrators and quality control products. Each reagent strip is for one test. Each reagent strip is pre-installed with magnetic separation reagent (80 μL each of streptavidin magnetic bead working solution and biotin-labeled antibody), 130 μL of enzyme-labeled antibody, 2 mL of washing solution and 250 μL of luminescent substrate.

[0124] Example 6: Preparation of a chemiluminescent kit for phosphorylated α-synuclein

[0125] 1. Preparation of immunomagnetic beads coated with monoclonal antibodies (1)

[0126] Same as Example 4.

[0127] 2. Preparation of horseradish peroxidase-labeled monoclonal antibody (2)

[0128] 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 incubate at 4°C in the dark for 30 minutes. Add 0.5 ml of 160 mM ethylene glycol and incubate at room temperature for 30 minutes. Add 2 mg of monoclonal antibody (2) to the supernatant and mix well. Pour the above solution into a dialysis bag and dialyze against 2000 ml of 0.05 mM CB buffer and stir overnight at 4°C. (0.05 MCB buffer: 3.18 g of Na2CO3 + 5.88 g of NaHCO3, up to 2 L of distilled water) The dialyzed solution is aspirated into a 15 ml centrifuge tube, 0.2 ml of freshly prepared 5 mg / ml NaBH4 solution is added, mixed well, and incubated at 4°C for 2 hours. Add an equal amount of saturated ammonium sulfate solution and incubate at 4°C for 30 minutes. Centrifuge at 4000 rpm for 20 minutes at 4°C. Discard the supernatant and drain. Dissolve the precipitate in a small amount of PBS (0.02M, pH 7.4), place it in a dialysis bag, and dialyze against 0.02M pH 7.4 PBS at 4°C overnight (change PBS once during the process). Pipette the dialysate into an EP tube, centrifuge, remove the supernatant, add an equal amount of glycerol, mix well, and store at -20°C.

[0129] 3. Preparation of washing solution

[0130] Same as Example 4.

[0131] 4. Preparation of phosphorylated α-synuclein calibrators / quality controls

[0132] Same as Example 4.

[0133] 5. Preparation of Luminescent Substrate

[0134] 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 pH 8.6 borate buffer; 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 pH 8.6 borate buffer.

[0135] 6. Composition of the kit

[0136] The kit mainly consists of reagent strips (24 strips), phosphorylated α-synuclein calibrators and quality control products. Each reagent strip is for one test. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (immunomagnetic bead working solution coated with monoclonal antibody (1)), 130 μL of horseradish peroxidase-labeled monoclonal antibody (2), 2 mL of washing solution and 250 μL each of luminescent substrate A and B solution.

[0137] Example 7: Preparation of ELISA assay kit

[0138] 1. Preparation of various buffers and reagents:

[0139] 1.1 Coating buffer: 0.05M, pH 9.6, CB (carbonate buffer)

[0140] Na2CO3: 16.0 g

[0141] NaHCO3: 29.0 g

[0142] Add deionized water to 1000 ml.

[0143] 1.2, pH7.2, 10×PBS-Tween 20

[0144] Na2HPO4·12H2O: 58 g

[0145] KH2PO4: 4 g

[0146] NaCl: 100 g

[0147] KCl: 4 g

[0148] Tween 20: 20ml

[0149] Add deionized water to 1000 ml.

[0150] 1.3. Blocking solution / antibody diluent:

[0151] 10×PBS-Tween 20: 100ml

[0152] BSA (bovine serum albumin): 10 g

[0153] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0154] Add deionized water to 1000 ml.

[0155] 1.4. Enzyme marker diluent:

[0156] 10×PBS-Tween 20: 10ml

[0157] FCS (fetal calf serum): 20ml

[0158] Enzyme stabilizer (purchased from Shanghai Xibao Company, model ACE0070A): 1 gram Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0159] Add deionized water to 1000 ml.

[0160] 1.5. Color developer A:

[0161] Citric acid: 35.5 g

[0162] Carbamide peroxide: 10 g

[0163] Tween 20: 10ml

[0164] Add deionized water to 1000 ml.

[0165] 1.6. Chromogen B:

[0166] Citric acid: 120 g

[0167] EDTA-2Na: 1 g

[0168] TMB·2HCl: 2 g

[0169] Add deionized water to 1000 ml.

[0170] 1.7. Concentrated washing solution (pH 7.2, 25× PBS-Tween 20)

[0171] Na2HPO4·12H2O: 145 g

[0172] KH2PO4: 10 g

[0173] NaCl: 250 g

[0174] KCl: 10 g

[0175] Tween 20: 50ml

[0176] Add deionized water to 1000 ml.

[0177] 1.8. Stop solution: 2M H2SO4

[0178] Concentrated sulfuric acid (95-98%): 22.2 ml

[0179] Deionized water: 177.3 ml

[0180] While mixing, slowly add concentrated sulfuric acid to the deionized water until the volume is 5, and shake while adding.

[0181] 2. Preparation of pre-coated plates

[0182] The monoclonal antibody (1) was dissolved in coating buffer to prepare a pre-coating solution. 100 μl of the solution was added to each well of an enzyme-labeled coated plate (purchased from Shenzhen Jincanhua Company) at a concentration of 0.1 μg / well. The plate was placed at 4°C for 18-24 hours, removed, the coating solution was discarded, and the plate was washed. 100 μl of blocking solution was added to each well and the plate was blocked at 4°C for 16 hours. The blocking solution was discarded, and the plate was dried and placed in an aluminum foil bag, vacuum-sealed, and stored at 4°C.

[0183] 3. Preparation of combined antibodies and enzyme markers

[0184] The binding antibody (polyclonal antibody (2)) and enzyme marker (horseradish peroxidase-labeled goat anti-rabbit IgG antibody, purchased from Beijing Zhongshan Jinqiao Company) were diluted with antibody diluent to the working concentration, which was determined by matrix titration experiment.

[0185] 4. Preparation of phosphorylated α-synuclein calibrators and quality control products

[0186] Recombinant phosphorylated α-synuclein was diluted with sample diluent to prepare phosphorylated α-synuclein calibrators (25-1000 pg / ml) and high and low concentration quality control products.

[0187] 5. ELISA kit composition

[0188] The kit mainly consists of pre-coated plates (48 or 96 servings), one set of phosphorylated α-synuclein calibrators, phosphorylated α-synuclein controls (high and low concentrations), binding antibodies (10 mL), enzyme markers (10 mL), color development solution A (5 mL), color development solution B (5 mL), concentrated washing solution (20 mL) and stop solution (5 mL).

[0189] Example 8: Preparation of a phosphorylated α-synuclein fluorescence chromatography assay kit

[0190] 1. Coated bonding pad

[0191] 1.1. Fluorescent microsphere-labeled monoclonal antibodies (1)

[0192] 1.1.1 Activation of fluorescent microspheres:

[0193] (1) Take 500 μl of a 1 (w / v)% aqueous dispersion of fluorescent microspheres (purchased from Bangs Laboratories, Inc.) and add a primary wash buffer (50 mM MES aqueous solution, pH 6.5) to 1 ml. Centrifuge at 16,000 rpm for 20 min at 4°C. Remove the supernatant and disperse the precipitate in 1 ml of the primary wash buffer. Ultrasonicate (240 W) for 2 min.

[0194] (2) Repeat the above process twice;

[0195] (3) Add 375 μl (3 / 4 of the volume of microspheres) of a mixture of 10 mg / ml carbodiimide solution and 10 mg / ml N-hydroxysulfosuccinimide solution in a ratio of 1:3 and shake for 15 minutes to activate the fluorescent microspheres.

[0196] 1.1.2. Labeling monoclonal antibodies with activated fluorescent microspheres (1):

[0197] (1) The precipitate was dispersed in 1 ml of coupling buffer (50 mM MES aqueous solution, pH 6.0) and treated with ultrasound (240 W) for 2 min;

[0198] (2) Repeat the above process twice;

[0199] (3) Obtain 500 μl of buffer solution containing dispersed fluorescent microspheres;

[0200] (4) adding monoclonal antibody (1) to the activated fluorescent microspheres at a ratio of 15 mg antibody / g, and shaking at room temperature for 2 hours;

[0201] (5) Add 1 ml of blocking buffer (0.5 (w / v)% BSA-0.05 M ethanolamine), continue shaking for 1 hour, then centrifuge at 16,000 rpm for 20 minutes, repeat the centrifugation three 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), treat with ultrasound (240W) for 2 minutes, and adjust the volume to 500 μl with the above-mentioned final wash buffer.

[0202] 1.2 Coated bonding pad

[0203] The monoclonal antibody (1) labeled with fluorescent microspheres prepared above was diluted with microsphere diluent (0.5 (w / v)% BSA-2 (w / v)% S9-15% sucrose-0.5% PVP-40000-0.5% PEG20000-20mM Tris solution) at a ratio of 1:240 of microsphere volume to diluent to obtain a working solution, which was then evenly sprayed onto the conjugate pad using a micropipette (purchased from DRAGON) at a volume of 1200 μl / 30 cm, then dried in a 37°C oven and stored at 45% humidity for future use.

[0204] 2. Preparation of reaction membrane

[0205] Monoclonal antibody (2) and goat anti-mouse IgG monoclonal antibody (purchased from Arista) were diluted to 0.5 mg / ml with 1% (w / v) PEG20000-5% (v / v) methanol-3% (w / v) sucrose 10 mM PBS (pH 8.4) buffer, respectively. The detection line and quality control line interval parameters of the gold spraying machine (purchased from Hangzhou Fenghang Company) were set to 8 mm, and the coating volume was set to 1.0 μl / cm. Monoclonal antibody (2) and goat anti-mouse IgG monoclonal antibody were streaked on nitrocellulose membrane using the gold spraying machine, dried in a 37°C oven, and stored at 45% humidity for future use.

[0206] 3. Assembly and cutting of test strips

[0207] The sample pad, conjugate pad, NC reaction membrane and absorbent filter paper were overlapped and pasted on the bottom plate in sequence to obtain a test paper board, which was then cut into test strips with a width of 4 mm.

[0208] 4. Preparation of phosphorylated α-synuclein fluorescent immunoassay card

[0209] Secure the cut test strips to the plastic base card and press the surface of the test strip with the top card. The top card has sample injection holes and an observation window located above the sample pad and reaction membrane of the test strip. Once assembled, place the test card in an aluminum foil bag, add desiccant, and seal for storage. It can be stored in dry conditions at room temperature for over a year.

[0210] 5. Composition of the Phosphorylated α-Synuclein-Fluorescence Chromatography Assay Kit (20T)

[0211] Phosphorylated α-synuclein fluorescent immunoassay card 20

[0212] Example 9: Determination and performance verification of phosphorylated α-synuclein using the chemiluminescent immunoassay (CLIA) kit prepared in Example 4

[0213] 1. Saliva sample collection:

[0214] (1) The subjects were asked to remain calm for 10 minutes before collection and to rinse their mouths with clean water for more than 1 minute 5 minutes before collection.

[0215] (2) The subject lowered his head, opened his mouth, and allowed saliva to flow naturally into a sterile tube, collecting 2 ml of saliva;

[0216] (3) The collected saliva was transferred to a 2 ml centrifuge tube, placed on ice, and various protease inhibitors (0.1 μl / ml saliva) were added to prevent protein degradation;

[0217] (4) Centrifuge the saliva at 2600 g for 15 minutes at 4°C, then at 15,000 g for 15 minutes at 4°C. Collect the supernatant from both centrifugations.

[0218] (5) Store in a refrigerator at -80°C.

[0219] 2. Determine the concentration of phosphorylated α-synuclein using a chemiluminescent immunoassay (CLIA) kit. The specific steps are as follows:

[0220] (1) All reagents must be brought to room temperature before the test, and the automatic luminometer must be preheated for at least 30 minutes;

[0221] (2) Scan the reagent kit barcode, and the automatic luminometer automatically selects different built-in measurement programs based on the reagent kit information;

[0222] (3) Loading reagents and samples: Follow the operating instructions of the fully automatic luminometer assay program to complete the loading of magnetic separation reagents, enzyme-labeled antibodies, and luminescent substrate solution. Mix the calibrator / quality control product / sample and transfer it to the sample well of each reagent strip using a quantitative pipette.

[0223] (4) Start the automatic measurement program: The measurement process and related parameters have been pre-defined in the instrument operation software, and the reaction and test time is set to 15 minutes.

[0224] (5) Result output: The fully automatic luminometer automatically calculates the concentration of phosphorylated α-synuclein in each sample using the working curve obtained by adjusting the built-in master curve by two-point calibration of the calibrator.

[0225] 3. Performance Verification of the Kit in Example 4

[0226] 3.1 Linear range verification

[0227] Take a sample near the upper limit of the kit's linear range and serially dilute it with the calibrator diluent. Measure each diluted sample twice and calculate the mean concentration (yi). Perform a linear fit using the dilution ratio (xi) as the independent variable and the corresponding mean concentration (yi) as the dependent variable. Calculate the linear correlation coefficient (r).

[0228] The test results are shown in Table 1 below:

[0229] Table 1 Linear range test results and calculation unit: pg / mL

[0230]

[0231]

[0232] The test results show that the linear correlation coefficient of the kit of Example 4 can reach 0.992 in the range of 10 to 2000 pg / mL.

[0233] 3.2 Minimum detection limit

[0234] The zero-concentration reference substance was tested 20 times using the kit prepared in Example 4. The mean (M) and standard deviation (SD) of the 20 test concentrations were calculated, and M+2SD was calculated as the minimum detection limit.

[0235] The test results showed that the minimum detection limit was 7.8 pg / mL, indicating that the kit has high sensitivity.

[0236] 3.3 Precision

[0237] Precision was verified on samples at high and low concentrations, with each concentration tested 10 times using the kit of Example 4. The two concentrations were 80 pg / mL and 1000 pg / mL, respectively.

[0238] Calculate the average value of 10 measurement results for each concentration sample and standard deviation SD, according to The coefficient of variation (CV) was obtained to evaluate the precision of the kit.

[0239] The test results are shown in Table 2 below:

[0240] Table 2 Precision test results

[0241]

[0242] The test results show that the CV% of the test kit prepared in Example 4 for samples with high and low concentrations is less than 10%, indicating that the kit has high precision.

[0243] 3.4 Accuracy

[0244] The accuracy of the kit was evaluated using a recovery test.

[0245] High-concentration sample A was added to low-concentration sample B, with the volume ratio of sample A to sample B being 1:9. The concentrations of sample B and the mixed sample were tested, and the recovery rate was calculated according to the following formula.

[0246]

[0247] Where: R—recovery rate;

[0248] V—volume of sample A added;

[0249] V0 - volume of sample B;

[0250] C - the detection concentration of sample B after adding sample A;

[0251] C0—detection concentration of sample B;

[0252] CS—concentration of sample A.

[0253] After calculation, the recovery rate was 93.45%, indicating that the kit had high accuracy.

[0254] Verification conclusion:

[0255] The validation results of the kit prepared in Example 4 showed that the kit had a wide linear range and could directly measure samples with a concentration of up to 2000 pg / mL without dilution. It had high sensitivity (the minimum detection limit was 7.8 pg / mL), high accuracy (the recovery rate was 93.45%), and good precision (the high and low concentration CV% were 5.17% and 5.39%, respectively). In addition, the entire reaction time was short, with results available in 15 minutes, greatly improving the detection efficiency.

[0256] Example 10: Determination of phosphorylated α-synuclein using the enzyme-linked immunosorbent assay (ELISA) kit prepared in Example 7 and verification of the kit's performance

[0257] 1. Sample collection: same as Example 9.

[0258] 2. Determine the concentration of phosphorylated α-synuclein using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0259] (1) Preparation of washing liquid:

[0260] Dilute the 25x concentrated washing solution with deionized water at a ratio of 1:25;

[0261] (2) Take out the kit prepared in Example 7 and equilibrate it to room temperature, prepare samples, calibrators, and quality control products, and dilute the clinical samples with sample diluent at a ratio of 1:20;

[0262] (3) Add 50 μl of the diluted sample / calibrator / quality control to each well of the microplate, then add 50 μl of the binding antibody to the corresponding well, tap to mix, seal the microplate with sealing film, and incubate at 37°C for 30 minutes;

[0263] (4) Remove the reaction plate, discard the liquid in the plate, add 200-300 μl of washing solution to each well, wash the plate 5 times, and pat dry;

[0264] (5) Add two drops or 100 μl of enzyme marker to each well, seal the plate with sealing film, and incubate at 37°C for 30 minutes;

[0265] (6) Remove the reaction plate, discard the liquid in the plate, add 200-300 μl of washing solution to each well, wash the plate 5 times, and pat dry;

[0266] (7) Add 50 μl of color developer A and B solution to each well, mix thoroughly, and incubate at 37°C for 15 minutes;

[0267] (8) Add one drop (50 μl) of stop solution to each well as quickly as possible and gently tap to mix;

[0268] (9) Measure the OD value of each well using a microplate reader (using a dual wavelength of 450 / 630 nm);

[0269] (10) Draw a calibration curve and calculate the concentration of phosphorylated α-synuclein in the sample based on the calibration curve.

[0270] 3. Kit performance verification

[0271] 3.1 Linear range verification

[0272] The verification method is the same as Example 9.

[0273] The test results are shown in Table 3 below:

[0274] Table 3 Linear range test results and calculation unit: pg / mL

[0275]

[0276]

[0277] The test results show that the linear correlation coefficient of the kit prepared in Example 7 can reach 0.998 in the range of 25 to 1000 pg / mL.

[0278] 3.2 Minimum detection limit

[0279] The verification method is the same as Example 9.

[0280] The test results showed that the minimum detection limit was 16.3 pg / mL, indicating that the kit has high sensitivity.

[0281] 3.3 Precision

[0282] The verification method was the same as that in Application Example 9. The two concentrations used for precision verification were 80 pg / mL and 500 pg / mL, respectively.

[0283] The test results are shown in Table 4 below:

[0284] Table 4 Precision test results

[0285]

[0286] The test results show that the CV% of the test kit prepared in Example 7 for samples with high and low concentrations is less than 10%, indicating that the kit has high precision.

[0287] 3.4 Accuracy

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

[0289] After calculation, the recovery rate was 92.8%, indicating that the kit had high accuracy.

[0290] Verification conclusion:

[0291] The validation results of the kit prepared in Example 7 showed that the kit had good linearity in the range of 25 to 1000 pg / mL, high sensitivity (the minimum detection limit was 16.3 pg / mL), a recovery rate of 92.8%, high accuracy, and good precision (both high and low concentration CV% were less than 10%). It did not require complex equipment and could be used for batch testing. More than 80 test results could be produced simultaneously in 1.5 hours, with high detection efficiency.

[0292] Example 11: Determination of phosphorylated α-synuclein by the fluorescent immunochromatographic kit prepared in Example 8 and verification of the kit performance

[0293] 1. Sample collection: same as Example 9.

[0294] 2. Determine the concentration of phosphorylated α-synuclein using a fluorescent immunochromatographic kit. The specific steps are as follows:

[0295] (1) Before testing, the reagents were returned to room temperature (20 ± 5°C) and the test was performed at room temperature;

[0296] (2) Turn on the fluorescence analyzer and adjust the instrument to the waiting state;

[0297] (3) Add 100 μL of the 1:4 diluted calibrator / quality control / sample to each test card sample port and react for 15 minutes;

[0298] (4) After the reaction is completed, the test card is inserted into the fluorescence analyzer for scanning and testing. The concentration of phosphorylated α-synuclein in each sample is calculated based on the built-in calibration curve imported into the analyzer.

[0299] 3. Kit performance verification

[0300] 3.1 Linear range verification

[0301] The testing method is the same as that in Example 9.

[0302] The test results are shown in Table 5 below:

[0303] Table 5 Linear range test results and calculation unit: pg / mL

[0304]

[0305]

[0306] The test results show that the linear correlation coefficient of the kit of Example 8 can reach 0.997 in the range of 25 to 1000 pg / mL.

[0307] 3.2 Minimum detection limit

[0308] The testing method is the same as that in Example 9.

[0309] The test results showed that the minimum detection limit was 20.5pg / mL.

[0310] 3.2 Precision

[0311] The test method was the same as in Application Example 9. The two concentrations used for precision verification were 80 pg / mL and 500 pg / mL. The test results are shown in Table 6 below:

[0312] Table 6 Precision test results

[0313]

[0314]

[0315] The test results show that the CV% of the test kit prepared in Example 8 for samples with high and low concentrations is less than 10%, indicating that the kit has high precision.

[0316] 3.4 Accuracy

[0317] The verification method is the same as that in Application Example 9.

[0318] After calculation, the recovery rate is 91.5%, which meets the recovery rate requirement of [90% to 110%].

[0319] Verification conclusion:

[0320] The validation results of the kit prepared in Example 8 showed that the kit had good linearity in the range of 25 to 1000 pg / mL, a sensitivity of 20.5 pg / mL, a recovery rate of 91.5%, met the requirements for accuracy, and had good precision (both high and low concentration CV% were less than 10%). It can be used for bedside diagnosis, is easy to operate, can be tested on demand, and can produce results in 15 minutes, making the detection rapid.

[0321] Test comparison example:

[0322] A chemiluminescence kit (comparative kit) was prepared using α-synuclein (phospho-S129) antibody (ab51253) and α-synuclein antibody (ab138501) (purchased from abcam) according to the method of Example 4, and the analytical performance was compared with the kit of Example 4.

[0323] The performance comparison results are shown in Table 7 below:

[0324] Table 7 Comparison results of performance of the comparative kit and the kit of Example 4

[0325]

[0326] Comparing various analytical performances, the kit prepared in Example 4 has better indicators.

[0327] Example 12: Phosphorylated α-synuclein Assay Kit for Auxiliary Diagnosis of Parkinson's Disease

[0328] 1. Research subjects

[0329] The Parkinson's disease group included 32 patients with Parkinson's disease, including 15 males and 17 females, aged (65.4 ± 15.7) years. These patients had typical features and met the Chinese diagnostic criteria for Parkinson's disease (2016 edition), including bradykinesia and one of the following symptoms: resting tremor (4-6 Hz) / muscle rigidity.

[0330] Healthy control group: 30 healthy control group, including 15 males and 15 females, aged (64.6±16.8) years, without mental illness, neurological disease or physical disease.

[0331] There was no statistically significant difference in age and gender between the two groups. All subjects gave their informed consent to this study.

[0332] 2. Experimental methods

[0333] 2.1. The phosphorylated α-synuclein test kit prepared in Example 4 of the present invention was used to test samples from a control group and patients with Parkinson's disease.

[0334] 2.2. Receiver operating characteristic (ROC) curve: A ROC curve was constructed based on the levels of phosphorylated α-synuclein in patients and controls to analyze the value of salivary phosphorylated α-synuclein in the auxiliary diagnosis of Parkinson's disease.

[0335] 3. Results

[0336] like Figure 1 As shown in the results, the salivary phosphorylated α-synuclein level in the Parkinson's disease group (78.76±22.21 pg / mL) was significantly higher than that in the healthy control group (59.86±12.56 pg / mL).

[0337] Draw a receiver operating curve for Parkinson's disease diagnosis (eg Figure 2 The area under the receiver operating characteristic (ROC) curve (AUC) was 0.78 (95% CI = 0.66-0.90), with a P < 0.01. This suggests that phosphorylated α-synuclein has a good value in assisting the diagnosis of Parkinson's disease.

Claims

1. A P-α-Syn specific antigen epitope peptide, characterized in that: The amino sequence of the antigen epitope peptide is one of the following: Tyr-Arg-Glu-Met-Pro-Ser(PO3H2)-Glu-Glu-Gly (1) or Tyr-Glu-Gly-Ile-Leu-Glu-Asp-Met-Pro-Val-Asp-Pro-Asp-Asn-Glu-Ala (2).

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