CN-121299143-B - ELISA (enzyme-Linked immuno sorbent assay) reagent composition and kit for detecting brain-derived neurotrophic factor

Abstract

The invention provides an ELISA (enzyme-Linked immuno sorbent assay) reagent composition and a kit for detecting brain-derived neurotrophic factors, and relates to the technical field of biological detection. The invention provides an ELISA reagent composition for detecting brain-derived neurotrophic factors, which is a sample diluent, wherein the sample diluent consists of buffer solution, bovine serum albumin, trehalose, polyethylene glycol 6000, propamidine dihydrochloride, glutathione and AEBSF. The ELISA reagent composition and the kit containing the same have good specificity and accuracy, and the variation coefficient of the repeatability experiment is less than 10%, which indicates that the kit has good repeatability and can be applied to detecting the BDNF content in human serum.

Inventors

• WANG LIANG
• JIANG YUTAO
• DAI MENGXUAN
• Lin Zhuoang
• TANG JIAJUN
• ZHANG JIE
• YANG CHAO
• AO XIANG
• ZHANG WENSHENG
• HE MINYE
• Wei Zhengnong
• YUE SHIYONG

Assignees

• 南方医科大学第三附属医院（广东省骨科研究院）

Dates

Publication Date

20260508

Application Date

20251210

Claims (7)

1. The application of the ELISA reagent composition in preparing a product for detecting brain-derived neurotrophic factors is characterized in that the ELISA reagent composition is a sample diluent, and the sample diluent consists of 0.01-1M PBS buffer solution, 1-3% (w/v) bovine serum albumin, 1-3% (w/v) trehalose, 0.1-0.5% (w/v) polyethylene glycol 6000, 0.01-0.05% (w/v) propamidine dihydrochloride, 0.1-0.5mM glutathione and 0.05-1g/L AEBSF.
2. 2. The use according to claim 1, wherein the sample dilution consists of 0.05M PBS buffer, 2% (w/v) bovine serum albumin, 3% (w/v) trehalose, 0.3% (w/v) polyethylene glycol 6000, 0.02% (w/v) propamidine dihydrochloride, 0.5mM glutathione and 0.02g/L AEBSF.
3. 3. The use according to claim 1, wherein the test sample of the product is selected from any one of serum, cerebrospinal fluid, and tissue samples.
4. 4. An ELISA kit for detecting brain-derived neurotrophic factor, comprising the ELISA kit of any of claims 1-2.
5. 5. The ELISA kit of claim 4, wherein the detection sample of the kit is selected from any one of serum, cerebrospinal fluid and tissue sample.
6. 6. The kit according to claim 4, further comprising a blocking solution, a washing solution, a standard substance, an enzyme binding working solution, a color development solution and a stop solution.
7. 7. The kit according to claim 6, wherein the color developing solution contains TMB at a concentration of 0.5-2 mg/ml.

Description

ELISA (enzyme-Linked immuno sorbent assay) reagent composition and kit for detecting brain-derived neurotrophic factor Technical Field The invention belongs to the technical field of biological detection, and particularly relates to an ELISA (enzyme-Linked immuno sorbent assay) reagent composition and a kit for detecting brain-derived neurotrophic factors. Background Brain-derived neurotrophic factor (BDNF) is a core member of the neurotrophic factor family, and is widely involved in key physiological processes such as development of central and peripheral nervous systems, neuron survival, synaptic plasticity regulation and nerve injury repair. A large amount of clinical and scientific evidences show that abnormal expression level of BDNF is closely related to occurrence and development of various diseases, particularly in patients with lumbago, the BDNF plays roles in regulating nerve sensitization, regulating central pain processing, promoting memory formation and the like, the rise degree is positively related to disease progress, serum BDNF concentration is confirmed to be used as a potential biomarker for disease diagnosis and efficacy evaluation, in addition, BDNF is also involved in pathological regulation of cardiovascular diseases and metabolic syndrome, and the circulating level of BDNF is closely related to prognosis of patients. Therefore, the method accurately quantifies the content of BDNF in human serum, and has important practical value for early screening, mechanism research and clinical intervention of related diseases. The ELISA technique has simple operation, controllable cost and suitability for batch detection, but the existing ELISA kit is faced with a plurality of technical bottlenecks in practical application, has insufficient specificity, and is characterized in that impurities such as TrkB receptor fragments, neurotrophic factors (such as NGF and NT-3) with similar structures and the like contained in serum samples are easy to cause non-specific combination, so that the detection result is higher, and the actual BDNF content cannot be accurately reflected. In addition, the human serum sample has complex components, contains hetero protein, lipid, protease, polyphenol substances and the like, can compete for an antibody binding site, increase a background signal, and can degrade BDNF target protein, so that the detection accuracy is further reduced. In addition, pretreatment conditions such as coagulation time, storage temperature fluctuation and the like in the serum preparation process also can obviously influence the stability of BDNF detection results, so that comparability of detection data of different laboratories and different batches is poor. The sample diluent is used as a core component of the ELISA kit, and the formula of the sample diluent directly determines the specificity, accuracy and repeatability of detection. At present, the conventional diluent has single functional component, only focuses on basic pH stability or simple protein protection, and the protective agent added into part of the diluent cannot resist the double threat of protease degradation and oxidative damage. These defects make it difficult for the existing kit to balance the specificity, stability and sensitivity, and cannot meet the requirements of clinical detection and scientific research on BDNF accurate quantification. Therefore, developing a sample diluent aiming at BDNF and capable of effectively solving the problems of interference of serum matrix, poor target stability, insufficient repeatability and the like, and further constructing a BDNF ELISA kit with high specificity, high accuracy and high repeatability becomes a technical problem to be solved urgently in the field. Disclosure of Invention In order to solve the above problems, the present invention provides an ELISA reagent composition and a kit for detecting brain-derived neurotrophic factor. In a first aspect, the invention provides an ELISA reagent composition for detecting brain-derived neurotrophic factors, wherein the ELISA reagent composition is a sample diluent, and the sample diluent consists of buffer solution, bovine serum albumin, trehalose, polyethylene glycol 6000, propamidine dihydrochloride, glutathione and AEBSF. Preferably, the sample diluent consists of 0.01-1M buffer, 1-3% (w/v) bovine serum albumin, 1-3% (w/v) trehalose, 0.1-0.5% (w/v) polyethylene glycol 6000, 0.01-0.05% (w/v) propamidine dihydrochloride, 0.1-0.5mM glutathione and 0.05-1g/L AEBSF. Specifically, the buffer solution is selected from any one of PBS buffer solution, tris-HCl buffer solution, carbonate buffer solution, citric acid-sodium citrate buffer solution and HEPES buffer solution. Further, the buffer solution is PBS buffer solution. According to some embodiments of the invention, the concentration of the buffer solution included in the sample dilution may be 0.01M、0.02M、0.03M、0.04M、0.05M、0.06M、0.07M、0.08M、0.09M、0.1M、0.2M、0.3M、0.4M、0.5M、0.6M、