A kit for determining brain-derived neurotrophic factor, its application, and a brain-derived neurotrophic factor-specific antibody

Abstract

The present invention discloses a kit for determining brain-derived neurotrophic factor (BDNF), comprising: a solid-phase carrier, a BDNF-specific antibody, a label, and a BDNF calibrator. The present invention also discloses the application of the kit provided by the present invention in the preparation of products for diagnosing depression. The present invention further provides a BDNF-specific antibody, which is prepared by immunizing an animal after conjugating a BDNF-specific antigen epitope peptide with a carrier protein, wherein the amino acid sequence of the BDNF-specific antigen epitope peptide is one of the following two sequences: Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser (1) or Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser (2). 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 biological field, and particularly to a kit for detecting brain-derived neurotrophic factor (BDNF), the application of the kit in the preparation of products for diagnosing depression, and a brain-derived neurotrophic factor-specific antibody. Background Art

[0002] Depression is an affective disorder mental illness that seriously affects physical and mental health, characterized by persistent low mood, loss of interest, slow thinking, etc. Its lifetime prevalence rate is about 17%, and about 350 million people worldwide are affected by it. The World Health Organization reported that by 2030, depression will rise to the first place in the global disease burden. The latest data in 2019 showed that the lifetime prevalence rate of depression in China has reached 3.4%. Based on this estimate, about 44 million people are suffering from this disease. More importantly, this disease often recurs, and the suicide rate is as high as 15% - 25%. It has become a major mental disorder that plagues human physical and mental health, bringing a heavy burden to patients, families and society.

[0003] The current diagnostic criteria for depression include the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) (DSM-5) in the United States, the International Classification of Diseases, Eleventh Revision (ICD-11), and the Classification and Diagnostic Criteria for Mental Disorders in China, Third Edition (CCMD-3). The above diagnostic criteria for depression mainly rely on symptomatology, that is, judging according to clinical manifestations, symptoms and scales. Its diagnostic effectiveness largely depends on a complete and true medical history and a comprehensive and effective mental status examination. The so-called mental status examination is for the doctor to talk to the patient face to face, and through the conversation and observing the patient's tone, expression, movement, look in the eyes and other manifestations, to find out whether the patient's mental activities are abnormal and what mental symptoms exist, providing a basis for the diagnosis of the disease. The doctor's evaluation of the patient's mental status mainly relies on subjective judgment, supplemented by the results of some scoring scales. This diagnostic method cannot well exclude the subjective factors of the patient, nor can it well avoid the influence of the doctor's experience, etc. on the examination results, lacking objectivity.

[0004] Due to the lack of objective examination means, the stability and consistency of the current diagnosis of depression are poor. Meta-analysis shows that the current correct diagnosis rate of depression is only 47%. The on-site test after the release of the DSM-5 diagnostic criteria found that the diagnostic consistency of depression is only 28%, indicating that the clinical symptom assessment as the key diagnostic index is unstable.

[0005] In view of the current situation of depression diagnosis, clinically, attempts have been made to find a stable, reliable, and objective biological marker for diagnosing depression and evaluating its curative effect. Currently, more research has been done on brain imaging and peripheral blood protein markers.

[0006] Depression belongs to mental 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 mental diseases. Among them, magnetic resonance examination is the most widely used in current brain function research and is an important means for studying mental disorders such as depression. Researchers measure the volume of brain regions in depression patients, introduce new variables such as cortical thickness, surface area, and cortical folding coefficient, and analyze the structural changes in the onset and development of depression. However, due to the current magnetic resonance technology being restricted by many influencing factors, such as disease heterogeneity; lack of imaging standards for normal brains; high sensitivity of magnetic resonance to changes in the physiology of subjects or exogenous chemical substances (such as excitement, coffee, smoking, temperature, etc.); limitations of statistical analysis methods; limitations of measured variables, etc., its performance is far from meeting the requirements of clinical applications and cannot be used as a diagnostic indicator for depression in clinical practice.

[0007] So far, the exact neuropathological basis of depression is still unclear. Over the years, various hypotheses have been proposed in the academic community to explain the pathogenesis of depression. Researchers have also studied the central nervous system-specific proteins in peripheral blood involved in different mechanisms, including monoamine metabolites (HVA, 5-HIAA, MHPG), cytokines (such as TNF-α, INF-γ, IL-6), etc. However, most of these studies come from small-sample clinics, with poor sensitivity, specificity, and / or reproducibility, and it is difficult to directly use them to guide clinical practice.

[0008] With the in-depth study, some scholars proposed the neurotrophic hypothesis in 2006. This hypothesis holds that the occurrence, development, and outcome of depression are closely related to neurotrophic factors represented by brain-derived neurotrophic factor. This hypothesis believes that the occurrence of depression is due to the reduction of neurotrophic factors represented by BDNF in the brain and the decline of related neural plasticity.

[0009] BDNF is widely distributed in the brain and regulates important physiological processes in the body such as nerve growth and development and synaptic plasticity. There have been many research reports confirming that the expression levels of BDNF protein in the serum and hippocampus of depressed patients and depression animal models are significantly lower than those of normal individuals, and it is a central nervous system-specific protein in peripheral blood related to the pathogenesis of depression. BDNF protein in peripheral blood can reflect the function of the central system to a certain extent and can be used as an objective and reliable examination method to assist in the diagnosis and guide the treatment of depression.

[0010] Although there are currently preliminary studies on the use of BDNF for the auxiliary diagnosis and treatment judgment of depression, since there is still no objective standard for the clinical diagnosis of depression, the clinical trials are difficult, and there is no kit for measuring brain-derived neurotrophic factor for the diagnosis of depression. Therefore, there is a need in this field to develop a kit that can measure the concentration level of BDNF in human serum to overcome the deficiency of subjective diagnosis of depression clinically and provide a more accurate and reliable objective indicator for the auxiliary diagnosis, condition monitoring and evaluation of the efficacy of antidepressants of depression. Summary of the Invention

[0011] To solve the problems existing in the prior art, the present invention provides a kit for measuring brain-derived neurotrophic factor (BDNF), which includes: a solid-phase carrier, a BDNF-specific antibody, a labeling agent and a BDNF calibrator.

[0012] According to the present invention, the BDNF measurement kit is developed by an immunoassay method and can use any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), fluorescence immunoassay chromatography or colloidal gold assay.

[0013] According to the present invention, the solid-phase carrier can be one of a microplate, magnetic microspheres or a nitrocellulose membrane (NC membrane).

[0014] According to the present invention, the labeling agent 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 labeling agent is preferably horseradish peroxidase HRP; when used in a chemiluminescent immunoassay (CLIA) kit, the labeling agent is preferably alkaline phosphatase AP; when used in a fluorescence immunoassay chromatography kit, the labeling agent is preferably fluorescent microspheres; when used in a colloidal gold assay kit, the labeling agent is colloidal gold.

[0015] According to the present invention, the BDNF calibrator / quality control product is a diluted recombinant or extracted BDNF.

[0016] The BDNF-specific antibody can be prepared by immunizing animals (such as mice, rats, rabbits, sheep, etc.) after conjugating a BDNF-specific antigen epitope peptide with a carrier protein. The BDNF-specific antibody can be a BDNF 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.

[0017] Preferably, the amino acid sequence of the BDNF-specific antigen epitope peptide is one of the following two sequences:

[0018] Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser(1) or

[0019] Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser(2).

[0020] In a specific embodiment, the BDNF specific antigen epitope peptide is prepared by a method comprising the following steps:

[0021] (1) Solid-phase synthesis of antigen epitope peptide resin

[0022] The resin is placed in the reaction chamber of a polypeptide automatic synthesizer, and the synthesizer automatically connects specific amino acids in different orders according to the amino acid sequences of antigen epitope peptide (1) or (2).

[0023] Examples of the resin include, but are not limited to, HMP resin.

[0024] (2) Cleavage of peptide resin to prepare crude epitope peptide

[0025] The peptide chain is cleaved with a cleavage reagent, then the cleavage reagent is removed with a scavenger, and then extraction is carried out with a solvent to obtain crude products of BDNF antigen epitope peptides (1) and (2) respectively.

[0026] Examples of the cleavage reagent include, but are not limited to, trifluoroacetic acid (TFA); examples of the scavenging reagent include, but are not limited to, EDT and thioanisole.

[0027] (3) Purification and analysis of crude antigen epitope peptide

[0028] The two crude antigen epitope peptides are further separated, purified and their purity is detected.

[0029] The methods of separation and purification include, but are not limited to, high performance liquid chromatography separation.

[0030] 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 double antibody sandwich method.

[0031] In a specific embodiment of the chemiluminescent immunoassay kit, the solid phase carrier is magnetic microspheres, and the magnetic microspheres are directly or indirectly connected with the BDNF specific antibody to form a magnetic separation reagent; the magnetic separation reagent is preferably an immunomagnetic bead coated with DNF monoclonal antibody or a streptavidin magnetic bead connected with BDNF antibody.

[0032] In a specific embodiment of the chemiluminescence 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-dioxoethane and its derivatives.

[0033] In one specific embodiment, the kit includes: a magnetic separation reagent, an enzyme-labeled BDNF monoclonal antibody, a BDNF calibrator / quality control product, a washing solution, and a chemiluminescent substrate. Among them, the BDNF monoclonal antibody is prepared by immunizing an animal after coupling a BDNF-specific antigen epitope peptide to a carrier protein, and the amino acid sequence of the BDNF-specific antigen epitope peptide is one of the following two sequences:

[0034] Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser(1) or

[0035] Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser(2).

[0036] In the second aspect of the present invention, there is provided the use of the kit of the present invention in the preparation of a product for detecting depression.

[0037] When the kit of the present invention is used for the preparation of a product for diagnosing depression, the BDNF concentration in the subject sample measured by the kit is compared with a control reference interval (i.e., the concentration level of healthy subjects). If the BDNF concentration in the subject sample is lower than the concentration in the control reference interval, the subject is at risk of suffering from depression, and the severity of the disease is negatively correlated with the BDNF concentration level in the sample.

[0038] The BDNF concentrations in the samples of the patient before and after treatment measured by the kit are compared. If the BDNF concentration in the patient sample does not change or shows an increasing trend during the treatment, it indicates that the drug treatment is ineffective. If the BDNF concentration in the patient sample shows a decreasing trend during the treatment, it indicates that the drug treatment is effective.

[0039] Preferably, the detection is serum detection. More preferably, the detection is any one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), fluorescence immunochromatography assay kit, and colloidal gold immunoassay; even more preferably, it is chemiluminescent immunoassay (CLIA).

[0040] In the detection by chemiluminescence method, it has high detection sensitivity (the lowest detection limit is 8 pg / mL), a wide linear range of 3 - 6 orders of magnitude, can directly measure samples with a concentration as high as 50 ng / mL without dilution, high accuracy (the recovery rate of the recovery test is 95.61%), 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, without manual operation, reducing human operation errors.

[0041] In the third aspect of the present invention, a BDNF-specific antibody is prepared by immunizing an animal after conjugating a BDNF-specific antigenic epitope peptide with a carrier protein, wherein the amino acid sequence of the BDNF-specific antigenic epitope peptide is one of the following two sequences:

[0042] Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser(1) or

[0043] Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser(2).

[0044] The present invention has the following advantages and positive effects compared with the prior art:

[0045] 1. The present invention applies BDNF as a biomarker to the preparation of a kit for the auxiliary diagnosis or efficacy evaluation of depression, overcoming the defect that the indicators used in the current clinical diagnosis process of depression are too subjective and lack objectivity, and providing a more accurate and reliable objective indicator for the auxiliary diagnosis, condition monitoring and antidepressant efficacy evaluation of depression.

[0046] 2. The kit prepared by the present invention needs to have good diagnostic sensitivity, specificity and repeatability, meeting the requirements of clinical applications.

[0047] 3. The BDNF-specific monoclonal antibody and polyclonal antibody prepared by the present invention can highly specifically bind to BDNF in the sample.

[0048] 4. The two BDNF-specific antigenic epitope peptides screened by the present invention have the characteristics of hydrophilicity, strong antigenicity and easy synthesis. Immunizing animals with the antigens (immunogens) prepared therefrom can produce highly specific monoclonal antibodies and polyclonal antibodies. Description of the Drawings

[0049] Figure 1 Shows the serum BDNF levels of healthy controls and patients with depression (test results using the kit prepared in Example 1)

[0050] Figure 2 Shows the relationship between the severity of depression and serum BDNF levels (test results using the kit prepared in Example 1)

[0051] Figure 3 Shows the ROC curve of serum BDNF for the diagnosis of depression (test results using the kit prepared in Example 1)

[0052] Figure 4 Shows the comparison of serum BDNF levels before and after treatment in 67 patients with depression (test results using the kit prepared in Example 1)

[0053] Figure 5 Shows the ROC curve before and after treatment in patients with depression (test results using the kit prepared in Example 1) Detailed implementation mode

[0054] 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 generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0055] Preparation Example 1: Synthesis of BDNF-specific antigenic epitope peptides (1) and (2)

[0056] Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser (1)

[0057] Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser (2)

[0058] 1. Raw materials used:

[0059] HMP resin (P-hydroxymethylphenoxymethyl polyethylene resin, purchased from sigma company)

[0060] Fmoc-AA (9-fluorenylmethoxycarbonyl-protected amino acid, purchased from Merck company)

[0061] NMP (N-methylpyrrolidone, purchased from sigma company)

[0062] DCM (dichloromethane, purchased from Zhongyuan Chemical Industry Company)

[0063] MeoH (methanol, purchased from Zhongyuan Chemical Industry Company)

[0064] Piperidine (purchased from sigma company)

[0065] DMAP (Dimethylaminopyridine, purchased from Sigma)

[0066] HOBT (Hydroxybenzotriazole, purchased from Sigma)

[0067] DCC (Dicyclohexylcarbodiimide, purchased from Sigma)

[0068] TFA (Trifluoroacetic acid, purchased from Sigma)

[0069] EDT (1,2-Ethanedithiol, purchased from Sigma)

[0070] Thioanisole, purchased from Guangzhou Weibo Chemical Co., Ltd.

[0071] Crystalline phenol, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0072] Acetonitrile, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0073] 2. Instruments used:

[0074] Automatic peptide synthesizer, model 431A, purchased from ABI

[0075] Rotary evaporator, model R-201, purchased from Shanghai Shenshun Co., Ltd.

[0076] High performance liquid chromatograph, Waters 600, purchased from Waters Corporation, USA

[0077] Freeze dryer, model VFD-2000, purchased from Beijing Boyikang Co., Ltd.

[0078] 3. Synthesis method and process:

[0079] 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 orders with a coupling rate of 99%. The reaction is as follows:

[0080] (1) Activation of amino acids (HOBt / DCC method)

[0081]

[0082] Fmoc-protected amino acid

[0083]

[0084] (2) Connect the amino acid to the resin

[0085]

[0086] (3) Deprotect the Fmoc group of the amino acid

[0087]

[0088] (4) Activation of another amino acid (HOBt / DCC method)

[0089]

[0090] (5) Coupling

[0091]

[0092] (6) Repeat steps (3) to (5) until the synthesis is completed.

[0093] Peptide resins of BDNF epitope peptide (1) and BDNF epitope peptide (2) are obtained respectively.

[0094] (7) Cleavage of the peptide resin:

[0095] 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 BDNF epitope peptide (1) and (2) respectively.

[0096] 4. Purification of the crude products of BDNF epitope peptide (1) and (2):

[0097] Separation and purification are carried out by high performance liquid chromatography:

[0098] Conditions: Chromatographic column: C8 10×100mm, purchased from Waters, USA

[0099] Chromatograph: Waters 600, Waters, USA

[0100] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0101] B: 0.1% TFA (trifluoroacetic acid) in 60% acetonitrile

[0102] Detection wavelength: 214nm

[0103] Flow rate: 4 ml / min

[0104] Elution gradient: 20 - 60% B, 30 minutes

[0105] HPLC (High Performance Liquid Chromatography) analysis

[0106] Chromatographic column: C18 4.6×150mm, purchased from Waters Company, USA

[0107] Mobile phase: A: 0.1% TFA (trifluoroacetic acid) aqueous solution

[0108] B: 0.1% TFA (trifluoroacetic acid) in acetonitrile

[0109] Detection wavelength: 214nm

[0110] Flow rate: 1ml / minute

[0111] Elution gradient: 0 - 60% B, 30 minutes

[0112] The analysis results of the peptide segments show that the purities of the BDNF antigenic epitope peptides (1) and (2) of the present invention are both above 95%.

[0113] Preparation Example 2: Preparation of BDNF-specific antibody

[0114] 1. Preparation of antigen:

[0115] The BDNF-specific antigenic epitope peptide was linked with the carrier protein KLH (keyhole limpet hemocyanin) by the Bis-diazotizedbenzidine dichloride (BDB) method to prepare the BDNF antigen.

[0116] Take 20.0mg of the BDNF-specific antigenic epitope peptide (1) or (2) prepared in Preparation Example 1 and dissolve it with 0.2M borate buffer (pH 9.0); take 7.36ml of KLH at 6.25mg / ml and cool it to 0°C. Take 1ml of BDB and put it into a mixture of the two in an ice-water bath and mix in the dark. React on a shaker for 1 - 1.5h. After the reaction is completed, adjust the pH to 9.0 with 0.2NaOH, dialyze overnight, dispense, and store at -20°C.

[0117] The formula of the borate buffer is: 80ml of 0.05mol / L borax plus 20ml of 0.2mol / L boric acid mixed together.

[0118] 2. Immunize animals to prepare monoclonal antibodies:

[0119] 2.1. The prepared BDNF antigen (immunogen) was respectively mixed thoroughly with an equal volume of Freund's complete adjuvant (purchased from Shanghai Yuanju Biotech Co., Ltd.), and then used to immunize Balb / c mice at a dose of 50 μg antigen per mouse by multiple subcutaneous injections. After 4 weeks, the serum titer was measured, and mice with good immunoreactivity were selected for booster immunization: the antigen was mixed thoroughly with an equal volume of Freund's incomplete adjuvant, and the antigen dose was 25 μg per mouse for multiple subcutaneous injections. The number of booster immunizations was 6 times, with two consecutive booster immunizations before fusion. Then, splenocytes and Sp2 / 0 myeloma cells were fused by 50% PEG (MW4000) (purchased from Zhongyuan Chemical Industry Co., Ltd.) according to the conventional method and cultured in HAT conditioned medium (purchased from sigma company). After fusion, the cells were cultured in a CO2 incubator at 37°C for 9 - 11 days, and large cell clones appeared in the wells. Screening started on the 11th day using indirect ELISA. The wells with positive primary screening results were subjected to 4 rounds of cloning culture by the limiting dilution method (even if the screened cells divided and propagated in large numbers), and then the cells were amplified, cryopreserved, and ascites were prepared.

[0120] 2.2. Balb / c mice were treated with 0.5 ml of pristane (purchased from sigma company) per mouse. One week later, 2×10 6 hybridoma cells per mouse were inoculated intraperitoneally, and ascites were collected 10 days later.

[0121] 2.3. Determination of antibody titer: The titer of the prepared monoclonal antibody was measured by the indirect ELISA method, and the results showed that the titer of the monoclonal antibody reached 1:32000 or higher.

[0122] 3. Preparation of polyclonal antibody by immunizing animals:

[0123] 3.1. New Zealand white rabbits at the age of three months and weighing about 2 kg were selected as immunized animals. In the primary immunization, 2 mg of the above-prepared BDNF antigen (immunogen) was respectively mixed with an equal volume of Freund's complete adjuvant, thoroughly emulsified, and then injected subcutaneously at multiple points on the back of the rabbits. Booster immunization was performed once every 2 weeks. After thorough emulsification of the antigen and incomplete Freund's adjuvant, 1 ml per rabbit was injected subcutaneously at multiple points on the back. Ten days after the last booster immunization, blood was collected from the carotid artery and serum was separated.

[0124] 3.2. Determination of antibody titer: The titer of the polyclonal antibody was measured by the indirect ELISA method, and the results showed that the antibody titer reached 1:32000 or higher.

[0125] 3.3. Blood collection and serum separation: Blood was collected through carotid artery intubation and serum was separated.

[0126] 4. Isolation and purification of antibodies:

[0127] The BDNF antibody to be loaded is first dialyzed to a consistent pH using 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, the BDNF antibody to be purified is 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 detector shows the start of a peak, and then the protein solution is collected. Collection is stopped when the peak drops smoothly. The concentration of the antibody is measured and calculated using an L5S ultraviolet-visible spectrophotometer (produced by Boda Jingke), and the purified BDNF antibody is aliquoted and stored at -20°C.

[0128] Example 1: Preparation of a chemiluminescent kit for BDNF

[0129] 1. Preparation of the magnetic separation reagent (working solution of immunomagnetic beads coated with BDNF monoclonal antibody 1)

[0130] (1) Initial washing and activation of magnetic beads

[0131] Take 200 μl of magnetic beads, a 1 (w / v)% aqueous dispersion of magnetic beads (purchased from Wuxi Baimaige Biotechnology Co., Ltd.), and precipitate them with a magnetic stand; discard the supernatant, add 0.8 ml of initial washing buffer (50 mM 2-(N-morpholino)ethanesulfonic acid (MES aqueous solution, pH 6.5)), mix well by sonication (240 W) for 4 - 5 s, precipitate by adsorption, discard the supernatant, repeat the above process 3 times, add 750 μl of a mixed solution of 10 mg / ml carbodiimide solution and 10 mg / ml N-hydroxysulfosuccinimide solution, and place it on a thermostatic shaker for 15 minutes to activate the carboxyl magnetic beads. After activation is completed, mix the microspheres by sonication; discard the supernatant (initial washing buffer and activator).

[0132] (2) Coating of the activated magnetic beads with BDNF monoclonal antibody 1

[0133] Add 1000 ul of coupling buffer (50 mM MES aqueous solution, pH 6.0), mix well by sonication; adsorb the precipitate with a magnetic stand, and discard the supernatant coupling buffer; repeat the washing 3 times, add 1000 ul of coupling buffer, and mix the carboxyl magnetic beads by sonication to obtain a buffer solution dispersed with carboxyl magnetic beads. Add 37.5 ul of BDNF monoclonal antibody for coupling, mix well by sonication for 4 - 5 s, place it on a constant temperature shaker for 2 hours of coupling, and sonicate the microspheres for 4 - 5 s every 30 min. Add 1 ml of blocking buffer (0.5 (w / v)% BSA - 0.05 M ethanolamine), place it on a shaker for 1 hour, after blocking is completed, adsorb the precipitate and discard the supernatant; add 1 ml of final washing buffer (0.5 (w / v)% BSA - 2 (w / v)% S9 - 20 mM Tris solution); mix well by sonication for 4 - 5 s, adsorb the precipitate, repeat the washing 3 times, disperse the precipitate into 1 ml of final washing buffer, sonicate for 4 - 5 s, and make up the volume to 1 ml with the above final washing buffer.

[0134] (3) Preparation of magnetic separation reagent

[0135] Dilute the magnetic beads coated with BDNF monoclonal antibody 1 with the final washing buffer (0.5 (w / v)% BSA - 2 (w / v)% S9 - 20 mM Tris solution) at a ratio of 1:100 to obtain the magnetic separation reagent.

[0136] 2. Preparation of enzyme - labeled antibody (BDNF monoclonal antibody 2 labeled with alkaline phosphatase)

[0137] Take 1 ml of purified BDNF monoclonal antibody 2, add 2 mg of alkaline phosphatase (AKP, purchased from sigma), mix well in an ice bath, then add 8 ul of 25% glutaraldehyde (Aladdin), and couple at room temperature for 2 h. Activate the dialysis bag (Shanghai Yuanju Biotechnology Co., Ltd.), take a 20 - cm - long dialysis bag, boil it in boiling water for 5 min, after cooling, seal one end of the dialysis bag with cotton thread, then add the coupled antibody into the dialysis bag, seal the other end with cotton thread, then prepare 0.01 M PBS (pH 7.2), perform dialysis at room temperature, change the dialysis solution every 1.5 h, repeat five times, and finally place it in a 4°C refrigerator for overnight dialysis, and collect about 1 ml the next morning. Measure and calculate the concentration of the alkaline phosphatase - labeled antibody with an L5S ultraviolet - visible spectrophotometer (Boda Jingke), and dilute the enzyme - labeled antibody 1:1000 with an enzyme - labeled diluent (0.01 M phosphate - buffered saline (PBS) containing 20% calf serum, 0.1% enzyme stabilizer (purchased from Shanghai Xibao Company, model ACE0070A), 0.03% biocide Proclin - 300).

[0138] 3. Preparation of washing solution

[0139] It consists of 10 mM PBS (pH 7.2), 0.08% Tween-20 and 0.03% Proclin-300.

[0140] 4. Preparation of BDNF Calibrator / Quality Control

[0141] A series of calibrators with concentrations of 0.02 ng / mL, 0.10 ng / mL, 0.50 ng / mL, 2.0 ng / mL, 10.0 ng / mL, and 50.0 ng / mL were prepared by diluting BDNF recombinant protein (purchased from abcam) with calibrator diluent (containing 10 mM phosphate buffer (PBS) (pH 7.2), 1% BSA, 0.03% biopreservative Proclin-300). The calibrator diluent consists of 10 mM phosphate buffer (PBS) (pH 7.2), 1% BSA, and 0.03% biopreservative Proclin-300.

[0142] The BDNF recombinant protein was diluted with calibrator diluent to 1.0 ng / mL and 40 ng / mL, which were used as low-value and high-value quality controls, respectively.

[0143] 5. Preparation of Luminescent Substrate

[0144] It consists of chemiluminescent substrate (4-chlorophenylthio)(10-methyl-9,10-dihydroacridiniumyl) disodium phosphate (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.

[0145] 6. Composition of the Kit

[0146] The kit mainly consists of 24 reagent strips, 1 set of BDNF calibrator, and 1 vial of quality control. Each reagent strip is for one-person testing and pre-loaded with 80 μL of magnetic separation reagent (working solution of BD NF monoclonal antibody 1-coated immunomagnetic beads), 130 μL of enzyme-labeled antibody, 2 mL of washing solution, and 250 μL of luminescent substrate.

[0147] Example 2: Preparation of Chemiluminescent Kit for BDNF

[0148] 1. Preparation of Magnetic Separation Reagent

[0149] (1) Preparation of Streptavidin Magnetic Bead Working Solution

[0150] The streptavidin magnetic bead solution (10 mg / mL) was purchased from Wuxi Baimaige Company, with a particle size of 0.01 - 3 μm, and diluted 1:5 with final wash buffer (0.5 (w / v)% BSA - 2 (w / v)% S9 - 20 mM Tris solution) to obtain the working solution.

[0151] (2) Preparation of biotin-labeled BDNF monoclonal antibody

[0152] After biotin-labeling BDNF monoclonal antibody 1 with a biotin conjugation kit (purchased from abcam), it was diluted 1:2000 with an antibody diluent (0.01M PBS buffer containing 20% calf serum, 0.1% enzyme stabilizer (purchased from Shanghai Sibao Co., Ltd., model ACE0070A), and 0.03% biopreservative) to obtain the biotin-labeled antibody.

[0153] 2. Preparation of enzyme-labeled antibody (BDNF monoclonal antibody 2 labeled with alkaline phosphatase)

[0154] Same as Example 1.

[0155] 3. Preparation of BDNF calibrator / quality control product, washing solution, and luminescent substrate

[0156] Same as Example 1.

[0157] 4. Composition of the kit

[0158] The kit mainly consists of a reagent strip (24 strips), 1 set of BDNF calibrator, and 1 vial of quality control product. Each reagent strip is for 1 person's test. 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.

[0159] Example 3: Preparation of chemiluminescent kit for BDNF

[0160] 1. Preparation of immunomagnetic beads coated with BDNF monoclonal antibody 1

[0161] Same as Example 1.

[0162] 2. Preparation of BDNF monoclonal antibody 2 labeled with horseradish peroxidase

[0163] 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. Then add 2 mg of BDNF 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 (0.05 M CB buffer: 3.18 g of Na2CO3 + 5.88 g of NaHCO3, up to 2 L of distilled water) at 4 °C with stirring overnight. 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 for 20 minutes at 4 °C. Discard the supernatant and drain well. 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 at 4 °C overnight (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.

[0164] 3. Preparation of washing solution

[0165] Same as Example 1.

[0166] 4. Preparation of BDNF calibrator / quality control

[0167] Same as Example 1.

[0168] 5. Preparation of luminescent substrate

[0169] The luminescent substrate is divided into solution A and solution B. Solution A is prepared from 0.4 g / L urea peroxide and 0.1 mol / L borate buffer at pH 8.6; Solution 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 at pH 8.6.

[0170] 6. Composition of the kit

[0171] The kit mainly consists of reagent strips (24 strips), 1 set of BDNF calibrator, and 1 vial of quality control. Each reagent strip is for one-person testing. Each reagent strip is pre-loaded with 80 μL of magnetic separation reagent (working solution of immunomagnetic beads coated with BDNF monoclonal antibody 1), 130 μL of BDNF monoclonal antibody 2 labeled with horseradish peroxidase, 2 mL of washing solution, and 250 μL each of luminescent substrate solution A and solution B.

[0172] Example 4: Preparation of ELISA assay kit

[0173] 1. Preparation of various buffers and reagents:

[0174] 1.1 Coating buffer: 0.05M, pH9.6, CB (carbonate buffer)

[0175] Na2CO3: 16.0 g

[0176] NaHCO3: 29.0 g

[0177] Make up to 1000 ml with deionized water.

[0178] 1.2 pH7.2, 10×PBS-Tween 20

[0179] Na2HPO4·12H2O: 58 g

[0180] KH2PO4: 4 g

[0181] NaCl: 100 g

[0182] KCl: 4 g

[0183] Tween 20: 20 ml

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

[0185] 1.3 Blocking buffer / Sample diluent / Antibody diluent:

[0186] 10×PBS-Tween 20: 100 ml

[0187] BSA (bovine serum albumin): 10 g

[0188] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0189] Make up to 1000 ml with deionized water.

[0190] 1.4 Enzyme conjugate diluent:

[0191] 10×PBS-Tween 20: 10 ml

[0192] FCS (fetal calf serum): 20 ml

[0193] Enzyme stabilizer (purchased from Shanghai Xibao Company, model number ACE0070A): 1 g

[0194] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0195] Make up to 1000 ml with deionized water.

[0196] 1.5 Chromogen A:

[0197] Citric acid: 35.5 g

[0198] Urea peroxide: 10 g

[0199] Tween 20: 10 ml

[0200] Make up to 1000 ml with deionized water.

[0201] 1.6, Chromogenic reagent B:

[0202] Citric acid: 120 g

[0203] EDTA-2Na: 1 g

[0204] TMB·2HCl: 2 g

[0205] Make up to 1000 ml with deionized water.

[0206] 1.7, Concentrated washing solution (pH 7.2, 25×PBS-Tween 20)

[0207] Na2HPO4·12H2O: 145 g

[0208] KH2PO4: 10 g

[0209] NaCl: 250 g

[0210] KCl: 10 g

[0211] Tween 20: 50 ml

[0212] Make up to 1000 ml with deionized water.

[0213] 1.8, Stop solution: 2M H2SO4

[0214] Concentrated sulfuric acid (95-98%): 22.2 ml

[0215] Deionized water: 177.3 ml

[0216] When preparing, slowly drip concentrated sulfuric acid into deionized water and make up to volume, shaking well while adding.

[0217] 2. Preparation of pre-coated plate

[0218] Dissolve the BDNF-specific monoclonal antibody in the coating buffer to prepare a pre-coating solution. Add 100 μl per well at 0.1 μg / well on the enzyme-linked immunosorbent assay (ELISA) 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, air dry and then put into an aluminum foil bag, evacuate and seal, and store at 4°C.

[0219] 3. Preparation of combined antibody and enzyme label

[0220] The combined antibody (BDNF-specific polyclonal antibody) and enzyme label (goat anti-rabbit IgG antibody labeled with horseradish peroxidase, purchased from Beijing Zhongshan Golden Bridge Co., Ltd.) were diluted to the working concentration with antibody diluent, and the working concentration was determined by checkerboard titration experiment.

[0221] 4. Preparation of BDNF calibrators and quality control products

[0222] Recombinant BDNF was diluted with sample diluent to prepare 6 concentrations of BDNF calibrators (concentrations were 50, 100, 200, 500, 1000, 2000 pg / ml respectively) and 2 concentrations of quality control products (concentrations were 300, 800 pg / ml respectively).

[0223] 5. Composition of ELISA kit

[0224]

[0225] Example 5: Preparation of BDNF-fluorescence chromatography assay kit

[0226] 1. Coating the conjugate pad

[0227] 1.1. Labeling of BDNF monoclonal antibody 1 with fluorescent microspheres

[0228] 1.1.1. Activation of fluorescent microspheres:

[0229] (1) Take 500 μl of 1% (w / v) aqueous dispersion of fluorescent microspheres (purchased from Bangs laboratories, lnc), 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 into 1 ml of primary wash buffer, and sonicate (240 W) for 2 minutes;

[0230] (2) Repeat the above process twice;

[0231] (3) Add 375 μl (3 / 4 of the amount of microspheres) 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.

[0232] 1.1.2. Labeling of activated fluorescent microspheres with BDNF monoclonal antibody (1):

[0233] (1) Disperse the precipitate into 1 ml of coupling buffer (50 mM MES aqueous solution, pH 6.0), and sonicate (240 W) for 2 minutes;

[0234] (2) Repeat the above process twice;

[0235] (3) Obtain 500 μl of buffer solution in which fluorescent microspheres are dispersed;

[0236] (4) Add BDNF monoclonal antibody (1) thereto at a ratio of 15 mg of antibody per gram of activated fluorescent microspheres, and oscillate at room temperature for 2 hours;

[0237] (5) Add 1 ml of blocking buffer (0.5 (w / v)% BSA - 0.05 M ethanolamine), continue to oscillate for 1 hour, then centrifuge at 16000 rpm for 20 minutes, repeat centrifugation 3 times, disperse the precipitate into 500 μl of final wash buffer (0.5 (w / v)% BSA - 0.1 (v / v)% Tween - 20 mM Tris solution), perform ultrasonic treatment (240 W) for 2 minutes, and make up the volume to 500 μl with the above - mentioned final wash buffer.

[0238] 1.2. Coating the conjugate pad

[0239] Dilute the above - prepared BDNF monoclonal antibody (1) labeled with fluorescent microspheres with microsphere diluent (0.5 (w / v)% BSA - 2 (w / v)% S9 - 15% sucrose - 0.5% PVP - 40000 - 0.5% PEG20000 - 20 mM Tris solution) at a ratio of microsphere amount: diluent of 1:240 to obtain a working solution, then evenly spray it on the conjugate pad with a micropipette (purchased from DRAGON company) at a rate of 1200 μl / 30 cm, then dry it in an oven at 37 °C and store it for standby at 45% humidity.

[0240] 2. Preparation of the reaction membrane

[0241] Dilute BDNF monoclonal antibody (2) and goat anti - mouse IgG monoclonal antibody (purchased from arista company) to 0.5 mg / ml respectively with 1% (w / v) PEG20000 - 5% (v / v) methanol - 3% (w / v) sucrose 10 mM PBS (pH 8.4) buffer solution. Set the interval parameters of the test line and the quality control line of the gold - spraying machine (purchased from Hangzhou Fenghang company) to 8 mm, and set the coating amount to 1.0 μl / cm respectively. Use the gold - spraying machine to draw BDNF monoclonal antibody (2) and goat anti - mouse IgG monoclonal antibody on the nitrocellulose membrane, dry it in an oven at 37 °C and store it for standby at 45% humidity.

[0242] 3. Assembly and cutting of the test strip

[0243] Sequentially and overlappingly paste the sample pad, the conjugate pad, the NC reaction membrane and the absorbent filter paper on the bottom plate to obtain a test strip board, and cut it into test strips with a width of 4 mm.

[0244] 4. Preparation of BDNF Fluorescence Immunoassay Card

[0245] Fix the cut test strip on the plastic bottom card, and press the surface of the test strip tightly with the surface card. The surface card has a sample addition hole and an observation window at the positions of the sample pad and the reaction membrane of the test strip. After the test card is assembled, it is put into an aluminum foil bag, and a desiccant is added and sealed for storage. It can be stored for more than one year under dry conditions at room temperature.

[0246] 5. Preparation of Sample Diluent

[0247] 10×PBS-Tween 20: 100 ml

[0248] BSA (Bovine Serum Albumin): 10 g

[0249] Biological preservative (Proclin-300, purchased from Shanghai Xibao Company): 1 ml

[0250] Make up to 1000 ml with deionized water.

[0251] 6. Composition of BDNF-Fluorescence Chromatography Assay Kit (20T)

[0252] BDNF Fluorescence Immunoassay Cards: 20 pieces

[0253] Sample Diluent: 20 vials

[0254] Application Example 1: Determination of BDNF by the Chemiluminescence Immunoassay (CLIA) Kit Prepared in Example 1 and Verification of the Performance of the Kit in Example 1

[0255] 1. Sample Collection:

[0256] Draw 3 - 5 ml of fasting venous blood from the subject, place it in an uncoagulated test tube at room temperature for 1 hour, centrifuge to separate the serum, and store it in a -20°C refrigerator.

[0257] 2. Determine the BDNF concentration using the Chemiluminescence Immunoassay (CLIA) Kit. The specific steps are as follows:

[0258] (1) Before the test, all reagents need to be placed at room temperature, and the fully automatic luminescence analyzer is turned on and preheated for at least 30 minutes;

[0259] (2) Scan the barcode of the kit, and the fully automatic luminescence analyzer automatically selects different built-in determination programs according to the kit information;

[0260] (3) Reagent and sample loading: According to the operation guide of the fully automatic luminescence analyzer determination program, complete the loading of the magnetic separation reagent, enzyme-labeled antibody, and luminescence substrate solution. After mixing the calibrator / quality control / sample, transfer it to the sample hole position of each reagent strip with a quantitative pipette;

[0261] (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.

[0262] (5) Result output: The fully automatic chemiluminescence analyzer automatically calculates the BDNF concentration of each sample by means of the working curve obtained by calibrating the built-in main curve with two-point calibration of the calibrator.

[0263] 3. Performance verification of the kit in Example 1

[0264] 3.1. Verification of linear range

[0265] 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 sample is measured 2 times, and the average value (yi) of its concentration is calculated. Taking the dilution ratio (xi) as the independent variable and the corresponding average concentration value (yi) as the dependent variable for linear fitting. Calculate the linear correlation coefficient (r).

[0266] The test results are as follows in Table 1:

[0267] Table 1 Linear range test results and calculation unit: ng / mL

[0268]

[0269] The test results show that for the kit prepared in Example 1, within the range of 0.02 - 50 ng / mL, the linear correlation coefficient can reach 0.9998, indicating that the kit has a wide linear range for testing.

[0270] 3.2. Minimum detection limit

[0271] Use the kit prepared in Example 1 to repeatedly detect the zero-concentration reference product 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.

[0272] The test results show that the minimum detection limit is 8 pg / mL, indicating that the kit has high sensitivity.

[0273] 3.3. Precision

[0274] The verification of precision is carried out on samples at two concentration levels, high and low. The kit in Example 1 is used to repeatedly detect each concentration sample 10 times. The two concentrations are 5 ng / mL and 40 ng / mL respectively.

[0275] Calculate the average value X and standard deviation SD of the 10 measurement results of each concentration sample respectively, and obtain the coefficient of variation CV according to CV = SD / X × 100% to evaluate the precision of the kit.

[0276] The test results are as follows in Table 2:

[0277] Table 2 Results of precision test

[0278]

[0279] The test results show that the CV% of the kit prepared in Example 1 for samples with high and low concentrations is less than 10%, indicating that the kit has high precision.

[0280] 3.4, Accuracy

[0281] The accuracy of the kit was evaluated by a recovery test.

[0282] High-concentration sample A was added to low-concentration sample B, and the volume ratio between the added sample A and sample B was 1:9. The concentrations of sample B and the mixed sample were detected, and the recovery rate was calculated according to the following formula.

[0283]

[0284] In the formula: R - recovery rate;

[0285] V - volume of added sample A;

[0286] V0 - volume of sample B;

[0287] C - detected concentration after adding sample A to sample B;

[0288] C0 - detected concentration of sample B;

[0289] CS - concentration of sample A.

[0290] After calculation, the recovery rate was 95.61%, indicating that the kit has high accuracy.

[0291] Verification conclusion:

[0292] The verification results of the kit prepared in Example 1 show that: the kit has a wide linear range, can directly measure samples with a concentration as high as 50 ng / mL without dilution, has high sensitivity (the lowest detection limit is 8 pg / mL), high accuracy (the recovery rate is 95.61%), good precision (the CV% of high and low concentrations are 5.51% and 5.76% respectively), and the whole reaction time is short, and the result can be obtained in 15 minutes, greatly improving the detection efficiency.

[0293] Application Example 2: Determination of BDNF by the enzyme-linked immunosorbent assay (ELISA) kit prepared in Example 4 and verification of the kit performance

[0294] 1. Sample collection: The same as Application Example 1.

[0295] 2. Determine the BDNF concentration using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0296] (1) Preparation of washing solution:

[0297] Dilute the 25-fold concentrated washing solution with deionized water at a ratio of 1:25.

[0298] (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.

[0299] (3) Add 50 μl of the diluted sample / calibrator / quality control product to each well of the microplate, then add 50 μl of the binding antibody to the corresponding wells. Tap gently to mix evenly, seal the microplate with a sealing film, and incubate at 37 °C for 30 minutes.

[0300] (4) Take out the reaction plate, discard the liquid in the plate, wash the plate 5 times with 200 - 300 μl of washing solution per well, and pat dry.

[0301] (5) Add two drops or 100 μl of the enzyme conjugate to each well, seal the plate with a sealing film, and incubate at 37 °C for 30 minutes.

[0302] (6) Take out the reaction plate, discard the liquid in the plate, wash the plate 5 times with 200 - 300 μl of washing solution per well, and pat dry.

[0303] (7) Add 50 μl of each of the chromogen solutions A and B to each well, mix well, and incubate at 37 °C for 15 minutes.

[0304] (8) As soon as possible, add 1 drop (50 μl) of the stop solution to each well and tap gently to mix evenly.

[0305] (9) Measure the OD value of each well using an enzyme-linked immunosorbent assay reader (measuring at a dual wavelength of 450 / 630 nm).

[0306] (10) Plot the calibration curve and calculate the BDNF concentration in the sample based on the calibration curve.

[0307] 3. Kit performance verification

[0308] 3.1. Linear range verification

[0309] The verification method is the same as that in Application Example 1.

[0310] The test results are shown in Table 3 below:

[0311] Table 3 Linear range test results and calculation unit: pg / mL

[0312]

[0313] The test results show that for the kit prepared in Example 4, in the range of 50 - 2000 pg / mL, the linear correlation coefficient can reach 0.992.

[0314] 3.2, Minimum detection limit

[0315] The verification method is the same as that in Application Example 1.

[0316] The test results show that the minimum detection limit is 10 pg / mL, indicating that the kit has high sensitivity.

[0317] 3.2, Precision

[0318] The verification method is the same as that in Application Example 1. The two concentrations used for precision verification are 500 pg / mL and 1500 pg / mL respectively.

[0319] The test results are as shown in Table 4 below:

[0320] Table 4 Precision test results

[0321]

[0322] The test results show that for the kit prepared in Example 4, the CV% for testing samples at both high and low concentrations is less than 10%, indicating that the kit has high precision.

[0323] 3.4, Accuracy

[0324] The verification method is the same as that in Application Example 1.

[0325] After calculation, the recovery rate is 95.4%, indicating that the kit has high accuracy.

[0326] Verification conclusion:

[0327] The verification results of the kit prepared in Example 4 show that: the kit has good linearity in the range of 50 - 2000 pg / mL, reaching 0.992, high sensitivity (minimum detection limit is 10 pg / mL), recovery rate of 95.4%, high accuracy, good precision (CV% at both high and low concentrations is less than 10%), 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.

[0328] Application Example 3: Determination of BDNF using the fluorescence immunochromatography kit prepared in Example 5 and verification of the kit performance

[0329] 1. Sample collection: The same as in Application Example 1.

[0330] 2. Determine the BDNF concentration using the fluorescence immunochromatography kit. The specific steps are as follows:

[0331] (1) Restore the reagent to room temperature (20±5°C) before testing, and conduct the test at room temperature;

[0332] (2) Turn on the fluorescence analyzer and adjust the instrument to the waiting state for detection;

[0333] (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 minutes;

[0334] (4) After the reaction is completed, insert the test card into the fluorescence analyzer for scanning test, and calculate the BDNF concentration of each sample according to the built-in calibration curve imported into the analyzer.

[0335] 3. Kit performance verification

[0336] 3.1. Linear range verification

[0337] The test method is the same as that in Application Example 1.

[0338] The test results are shown in Table 5 below:

[0339] Table 5 Linear range test results and calculation unit: pg / mL

[0340]

[0341]

[0342] The test results show that for the kit in Example 5, the linear correlation coefficient can reach 0.990 in the range of 50 - 2000 pg / mL.

[0343] 3.2. Minimum detection limit

[0344] The test method is the same as that in Application Example 1.

[0345] The test results show that the minimum detection limit is 20 pg / mL.

[0346] 3.2. Precision

[0347] The test method is the same as that in Application Example 1. The two concentrations used for precision verification are 500 pg / mL and 1500 pg / mL respectively.

[0348] The test results are shown in Table 6 below:

[0349] Table 6 Precision test results

[0350]

[0351] The test results show that for the kit prepared in Example 5, the CV% for testing samples at both high and low concentrations is less than 10%, indicating that the kit has high precision.

[0352] 3.4, Accuracy

[0353] The verification method is the same as that in Application Example 1.

[0354] After calculation, the recovery rate is 93.5%, meeting the requirement of the recovery rate [90% - 110%].

[0355] Verification conclusion:

[0356] The verification results of the kit prepared in Example 5 show that: the kit has good linearity in the range of 50 - 2000 pg / mL, up to 0.990, the sensitivity is 20 pg / mL, the recovery rate is 93.5%, the accuracy meets the requirements, and the precision is good (CV% of high and low concentrations are both 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 in 15 minutes, and the detection is fast.

[0357] Test Comparative Example 1:

[0358] The BDNF polypeptides of Orb374958 and orb72384 (purchased from Biorbyt) were used to prepare monoclonal antibody 1 and monoclonal antibody 2 respectively according to the method of Preparation Example 2, and a chemiluminescence kit (comparative kit) was prepared according to the method of Example 1, and the analytical performance comparison test was carried out with the kit of Example 1.

[0359] The comparison results of each performance are shown in Table 7 below:

[0360] Table 7 Comparison of the performance of the comparative kit and the kit of Example 1

[0361]

[0362] For the comparison of each analytical performance, the kit prepared in Example 1 has a wider linear detection range and better other indicators.

[0363] Application Example 5: The BDNF assay kit is used for the auxiliary diagnosis and condition monitoring of depression

[0364] 1. Research subjects

[0365] Depression group: 119 depression patients who have never taken antidepressants or have stopped taking them for ≥6 weeks, including 52 males and 67 females, with an age of (35.4 ± 14.9) years, an education duration of (11.8 ± 5.6), and a BMI index of (22.5 ± 3.4). All meet the depression diagnostic criteria in the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) (DSM-5) and have no serious physical diseases. The severity of the selected patients was scored according to the 24-item Hamilton Depression Scale (HAMD). There were 52 cases of mild depression (7 - 20 points), 42 cases of moderate depression (20 - 35 points), and 25 cases of severe depression (>35 points).

[0366] Healthy control group: 128 healthy subjects, including 56 males and 72 females, with an age of (34.3 ± 15.2) years, years of education (12.3 ± 6.2), and BMI index (23.1 ± 3.8). There was no history of depressive episodes, family psychiatric history, other mental illnesses, or physical diseases.

[0367] There was no significant difference in the general conditions between the two groups, and all the research subjects gave informed consent to this study.

[0368] 2. Experimental methods

[0369] 2.1. Use the BDNF kit prepared in Example 1 of the present invention to test the serum samples of the enrolled healthy control group and patients with various types of depression.

[0370] 2.2. Receiver operating characteristic curve (ROC curve): Based on the BDNF levels of patients and healthy control populations, a ROC curve was made to analyze the value of serum BDNF in the auxiliary diagnosis of depression.

[0371] 3. Results

[0372] As Figure 1 shown, compared with the healthy control group, the serum BDNF level in the depression group was significantly decreased ( Figure 1 ), and was correlated with the severity of depression ( Figure 2 ).

[0373] The receiver operating characteristic curve for the diagnosis of depression was drawn. As Figure 3 , the area under the ROC curve (AUC) for the diagnosis was 0.82 (95% CI = 0.76 - 0.87), P < 0.01. This indicates that serum BDNF has good value in the auxiliary diagnosis of depression.

[0374] Application Example 6: Serum BDNF assay kit for evaluating the efficacy of antidepressant drugs

[0375] 1. Test method

[0376] Sixty-seven patients with depression with a score of ≥ 20 on the Hamilton Depression Scale (HAMD) in Application Example 5 were selected, and were given the SSRI drug escitalopram for 8 weeks. The dosage was according to the drug instruction manual. After treatment, blood was drawn to collect serum samples and tested with the BDNF kit prepared in Example 1. The serum BDNF levels before and after treatment were compared, and a ROC curve was made to analyze the value of serum BDNF in the efficacy evaluation of depression.

[0377] 2. Results

[0378] As Figure 4, after treatment with antidepressants, the serum BDNF level of patients with depression increased significantly ( Figure 4 ).

[0379] The ROC curves of patients with depression before and after treatment are as Figure 5 shown. The area under the curve AUC is 0.88 (95% CI = 0.83 - 0.94), P < 0.01. This indicates that serum BDNF has good value for evaluating the curative effect of depression. Sequence Listing <110> Shenzhen Anqun Bioengineering Co., Ltd. <120> A kit for determining brain-derived neurotrophic factor, its application, and a brain-derived neurotrophic factor-specific antibody <130> 2020122301 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <400> 1 Tyr Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met Ser 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <400> 2 Tyr Lys Lys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser 1 5 10 15

Claims

1. A kit for determining brain-derived neurotrophic factor (BDNF), comprising: A solid-phase carrier, a BDNF-specific antibody, a label, and a BDNF calibrator, wherein the BDNF-specific antibody is prepared by immunizing an animal after conjugating a BDNF-specific antigenic epitope peptide with a carrier protein, and the amino acid sequence of the BDNF-specific antigenic epitope peptide is one of the following two sequences: Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser(1) or Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser(2), The BDNF-specific antibody is a polyclonal antibody.

2. The kit according to claim 1, 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.

3. The kit according to claim 1, wherein the solid-phase carrier is selected from any one of a microplate, magnetic microspheres, or a nitrocellulose membrane.

4. The kit according to claim 1, wherein the label is selected from horseradish peroxidase HRP, alkaline phosphatase AP, a fluorescent substance, a dye, or colloidal gold.

5. The kit according to claim 1, wherein the kit is a chemiluminescence assay kit and the solid-phase carrier is magnetic microbeads.

6. The kit according to claim 5, wherein the magnetic microbeads are directly or indirectly connected to the BDNF-specific antibody to form a magnetic separation reagent.

7. The kit according to claim 6, wherein the magnetic separation reagent is an immunomagnetic bead coated with the BDNF-specific antibody or a magnetic bead connecting streptavidin with the BDNF-specific antibody.

8. The kit according to claim 6, wherein the label is selected from horseradish peroxidase HRP and / or alkaline phosphatase AP.

9. Use of the kit according to any one of claims 1-8 in the preparation of a product for diagnosing depression.

10. A BDNF-specific antibody prepared by immunizing an animal after conjugating a BDNF-specific antigenic epitope peptide with a carrier protein, wherein the amino acid sequence of the BDNF-specific antigenic epitope peptide is one of the following two sequences: Tyr-Glu-Trp-Val-Thr-Ala-Ala-Asp-Lys-Lys-Thr-Ala-Val-Asp-Met-Ser(1) or Tyr-Lys-Lys-Arg-Ile-Gly-Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr-Ser(2), The BDNF-specific antibody is a polyclonal antibody.

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