Anti-human beta-ngf monoclonal antibodies and uses thereof

Abstract

The application belongs to the field of immunological detection, and discloses an anti-human beta-NGF monoclonal antibody and application thereof.The amino acid sequences of the light chain and heavy chain CDR1, CDR2 and CDR3 of the monoclonal antibody are KSVSTSGYSY, LAS, QHSRELPLT and GFTFSSHW, VRLKSNNYAT, TLNGGAMDH respectively.The enzyme-linked immunosorbent kit constructed by the application is based on a double antibody sandwich method, and simultaneously combines a biotin-streptavidin signal amplification system and an enzyme color development amplification system, greatly improving the detection sensitivity and specificity, and accurately and rapidly detecting low content samples.The kit exhibits excellent stability and specificity, and can be used for quantitatively detecting the content of beta-NGF in human serum, cell supernatant or tissue homogenate and the like, thereby providing an objective detection basis for the diagnosis, efficacy evaluation and pathogenesis research of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection, specifically, it relates to an anti-human β-NGF monoclonal antibody and its application. Background Technology

[0002] β-Nerve Growth Factor (β-NGF) is an important member of the neurotrophic factor family. This protein has the activity to stimulate nerve growth and participates in regulating the growth and differentiation of sympathetic and sensory neurons. β-NGF plays a crucial role in the development, maintenance, and repair of the nervous system. Abnormally elevated or decreased levels can lead to the development and progression of various diseases (e.g., reduced β-NGF is commonly seen in type 1 and type 2 diabetes, glaucoma, and neurological diseases such as Alzheimer's disease and Parkinson's disease; decreased β-NGF levels have also been observed in hypertension, atherosclerosis, bronchial asthma, chronic obstructive pulmonary disease, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease). Rapid and accurate detection of β-NGF levels is of great significance in neuroscience research, clinical disease diagnosis, and treatment monitoring. The β-NGF protein is a dimer composed of two identical subunits, each consisting of 118 amino acid residues with a molecular weight of approximately 13.2 kDa. Mature β-NGF is produced from β-NGF precursor protein through protease cleavage. It possesses a highly conserved three-dimensional structure containing six antiparallel β-sheet chains, forming a typical β-sheet barrel structure. This structure endows β-NGF with the ability to bind to both the high-affinity receptor TrkA (tyrosine kinase receptor A) and the low-affinity receptor p75NTR (nerve growth factor receptor). Binding to TrkA activates signaling pathways such as PI3K-Akt and Ras-MAPK, promoting neuronal survival, differentiation, and synapse formation; while binding to p75NTR mediates apoptosis, participating in the programmed cell death process of neurons. Clinically, detecting β-NGF levels in cerebrospinal fluid or blood helps in the early detection of neurological diseases such as Alzheimer's and Parkinson's, and in assessing disease progression. In addition, intervention strategies targeting β-NGF and its signaling pathways, such as exogenous β-NGF supplementation or the design of small molecule inhibitors targeting TrkA, have become a research hotspot for the treatment of neurodegenerative diseases, tumors and other conditions, and are expected to bring new breakthroughs in the treatment of related diseases.

[0003] Currently, the commonly used method for β-NGF determination is mass spectrometry (LC-MS / MS). Mass spectrometry utilizes the fact that different molecules, after ionization, produce ions with varying mass-to-charge ratios. These ions are separated using an electric or magnetic field, and the mass-to-charge ratio and intensity information are recorded by a detector. The type and content of substances in the sample are inferred based on the characteristic mass spectral peaks of the ions. Its disadvantages include: the need for cumbersome pretreatment steps such as immunoprecipitation, enzymatic digestion, and chromatographic separation before sample detection, which may lead to β-NGF loss and affect detection accuracy; the high cost of mass spectrometry instruments and maintenance, requiring professional personnel for sample analysis and data interpretation; furthermore, the limited number of samples that can be analyzed at one time cannot meet the needs of rapid detection of large-scale samples.

[0004] Other common detection methods include PC12 cell proliferation assays and Western blotting. However, PC12 cell proliferation assays for β-NGF are susceptible to interference from other neurotrophic factors and have low sensitivity (low concentrations of β-NGF cannot proliferate PC12 cells). Furthermore, cell culture requires specialized personnel and can take several days, making it unsuitable for rapid results or urgent testing scenarios. Western blotting primarily relies on semi-quantitative analysis, heavily depending on internal control proteins for relative quantification. It has poor sensitivity, typically requiring protein concentrations above μg / mL. The procedure is also cumbersome, involving multiple steps such as electrophoresis, transfer, blocking, and antibody incubation, which are prone to errors, affecting the reproducibility and consistency of results. Summary of the Invention

[0005] The purpose of this invention is to provide a double-antibody sandwich ELISA kit for detecting the content of human β-nerve growth factor (β-NGF).

[0006] To achieve the objective of this invention, in a first aspect, this invention provides an anti-human β-NGF monoclonal antibody 13E2, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of antibody 13E2 are KSVSTSGYSY (SEQ ID NO:14), LAS, and QHSRELPLT (SEQ ID NO:15), respectively, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFTFSSHW (SEQ ID NO:16), VRLKSNNYAT (SEQ ID NO:17), and TLNGGAMDH (SEQ ID NO:18), respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of antibody 13E2 is as shown in SEQ ID NO:7, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:7;

[0008] The amino acid sequence of the heavy chain variable region of antibody 13E2 is shown in SEQ ID NO:8, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:8.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody 11F1.

[0010] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or host cells.

[0011] Fourthly, the present invention provides an antibody conjugate, which is obtained by conjugating the antibody 11F1 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, colloidal gold labeling, etc.

[0012] Fifthly, the present invention provides an antibody composition against human β-NGF, the antibody composition comprising said antibody 13E2 and anti-human β-NGF monoclonal antibody 11F1;

[0013] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of antibody 11F1 are QTLVLSNGNTY (SEQ ID NO:9), KVS, and SQSAHFPYT (SEQ ID NO:10), respectively, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are AFNVKDKY (SEQ ID NO:11), IEPGNGNT (SEQ ID NO:12), and NAPDYDFDY (SEQ ID NO:13), respectively.

[0014] Furthermore, the amino acid sequence of the light chain variable region of antibody 11F1 is as shown in SEQ ID NO:3, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:3;

[0015] The amino acid sequence of the heavy chain variable region of the antibody 11F1 is shown in SEQ ID NO:4, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:4.

[0016] In a sixth aspect, the present invention provides any of the following applications of the antibody 13E2 or the nucleic acid molecule encoding the antibody or the biological material containing the nucleic acid molecule or the antibody conjugate or the antibody composition:

[0017] (1) Used for qualitative or quantitative detection of human β-NGF (including non-disease diagnosis and treatment purposes);

[0018] (2) Prepare reagents or kits for the qualitative or quantitative detection of human β-NGF;

[0019] (3) Used for quality control of products containing human β-NGF.

[0020] In a seventh aspect, the present invention provides a human β-NGF detection reagent or kit prepared from the antibody 13E2, such as an enzyme-linked immunosorbent assay (ELISA) kit, a fluorescence immunoassay kit, or a chemiluminescent immunoassay kit.

[0021] Eighthly, the present invention provides a double-antibody sandwich ELISA kit for detecting human β-NGF levels, using antibody 11F1 as the first antibody and antibody 13E2 as the second antibody; or,

[0022] Antibody 13E2 was used as the first antibody, and antibody 11F1 was used as the second antibody.

[0023] Furthermore, the antibody 11F1 is immobilized on a solid-phase support, which is selected from enzyme-labeled plates, microspheres, nitrocellulose membranes, glass cellulose membranes, or nylon membranes.

[0024] Furthermore, the enzyme used for enzyme labeling is selected from any of the following: horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc.

[0025] Furthermore, the kit also includes at least one of a substrate chromogenic solution, a blocking solution, or a stop solution.

[0026] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0027] (i) The double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit for detecting human β-NGF content provided by the present invention can be used to quantitatively detect the content of β-NGF in human serum, cell supernatant or tissue homogenate, thereby providing objective detection basis for the diagnosis, efficacy evaluation and pathogenesis research of related diseases.

[0028] (ii) This kit uses a double antibody sandwich method, combined with a biotin-streptavidin signal amplification system and an enzyme colorimetric amplification system, which greatly improves the detection sensitivity and specificity, and can accurately and quickly detect low-content samples.

[0029] (III) Compared with other methods such as mass spectrometry detection (ng level), PC12 cell proliferation detection (μg level), and Western blotting detection (μg level), the enzyme-linked immunosorbent assay kit of the present invention has high sensitivity (pg level), a minimum detection limit of 1.452 pg / mL, and good linearity in the detection range of 31.25-2000 pg / mL.

[0030] (iv) Compared with other methods such as mass spectrometry detection (about 8-12h), PC12 cell proliferation detection (about 48h), and Western Blot detection (about 8-10h), the enzyme-linked immunosorbent assay kit of the present invention has a shorter detection time (4h), and the independent detachable well design of the ELISA plate can realize the detection of dozens to hundreds of samples in a single test (e.g., a 96-well plate can process more than 80 samples in a single test), which greatly improves the detection efficiency compared with traditional methods, and is especially suitable for large-scale screening scenarios.

[0031] (V) The enzyme-linked immunosorbent assay (ELISA) kit of the present invention exhibits excellent stability and specificity: Accelerated stability was assessed at 37°C for 12 days, with no significant fluctuations in the linearity of the standard curve and detection sensitivity; cross-tabulation results showed that it does not react with human GDNF, CNTF, BDNF, ENA-78, FGF4, IL-1α, IL-1β, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-19, IL-10, IL-17A, IL-12p40, IL-12p70, IL-13, IgG, IgA, IgM, or IgE. Simultaneously, the standard curve exhibits good linearity, with good spiking and linearity across different sample types. Its sensitivity is better than similar kits from abroad, making it a viable alternative to imported products. Attached Figure Description

[0032] Figure 1 The SDS identification results of the purified antibody are shown in the preferred embodiment of the present invention.

[0033] Figure 2 This is the standard curve corresponding to the human β-NGF double antibody sandwich ELISA method established in a preferred embodiment of the present invention. Detailed Implementation

[0034] The present invention aims to provide an enzyme-linked immunosorbent assay (ELISA) kit for accurate, sensitive, rapid, and high-volume detection of human β-NGF content in samples. This kit is an ELISA detection kit prepared based on the principle of double antibody sandwich method.

[0035] The present invention adopts the following technical solution:

[0036] A specific mouse anti-human β-NGF monoclonal antibody was coated onto an ELISA plate. Then, serially diluted β-NGF standards and the sample to be tested were added. The β-NGF contained in the standards and the sample was captured by the coating antibody on the ELISA plate. After washing the plate, a biotin-labeled β-NGF detection antibody was added, which specifically binds to the coating antibody-β-NGF complex. Then, horseradish peroxidase (HRP) labeled with streptavidin (SA) was added. The streptavidin in HRP binds with high affinity to the biotin in the detection antibody. After washing to remove free HRP, the chromogenic substrate TMB (tetramethylbenzidine) was added. This substrate reacts with different concentrations of HRP in the complex to produce different shades of blue (positively correlated). After stopping the color development with a stop solution, the wells become detection wells that can be measured at λmax = 450 nm (OD = 450 nm). The concentration of human β-NGF protein in the sample is directly proportional to the OD value in this detection method. A standard curve constructed using serially diluted β-NGF standards allows for accurate quantitative calculation of the β-NGF content in the sample. This kit significantly improves detection sensitivity and specificity through a double-antibody sandwich method, a biotin-streptavidin signal amplification system, and an enzyme colorimetric amplification system, enabling accurate and rapid detection of low-concentration samples.

[0037] An ELISA plate for capturing mouse anti-human β-NGF monoclonal antibody; the DNA sequences of its light chain and heavy chain variable regions are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The amino acid sequences of its light chain and heavy chain variable regions are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0038] A biotin-labeled mouse anti-human β-NGF monoclonal antibody; its light chain and heavy chain variable region DNA sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. Its light chain and heavy chain variable region amino acid sequences are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0040] Example 1: Preparation of anti-human β-NGF monoclonal antibody

[0041] 1. Animal immunization

[0042] Female Balb / c mice aged 6-8 weeks were selected. The eukaryotically expressed human β-NGF recombinant protein antigen was emulsified with an equal volume of complete Freund's adjuvant for the first immunization. Two weeks after the first immunization, an equal volume of recombinant protein antigen was emulsified with an equal volume of incomplete Freund's adjuvant for a second immunization. Subsequent immunizations were performed every two weeks using the same method, for a total of three immunizations. Tail tip blood was collected after immunization for titer testing. A booster immunization was administered three days before fusion to enhance the fusion effect.

[0043] 2. Cell fusion

[0044] (1) Activation of myeloma cells (SP2 / 0):

[0045] After thawing and resuscitating commercially available SP2 / 0 cells, they were resuspended in RPMI-1640 medium supplemented with fetal bovine serum and cultured at 37°C in a 5% CO2 incubator. Once the cell density reached 70%–80%, they were passaged. Cells were collected, resuspended in RPMI-1640 medium, counted, and 0.5 × 10⁶ cells were collected. 6 ~1×10 6 One cell was injected subcutaneously into the back of Balb / c mice and cultured for 9-10 days. When the tumor diameter grew to approximately 0.5-0.8 cm, the mice were euthanized by cervical dislocation, and the tumor was aseptically removed after soaking in 75% alcohol. The tumor mass was placed in a sterile homogenizer, and RPMI-1640 basal solution was added and homogenized thoroughly. 10 mL of RPMI-1640 medium was added, and the mixture was allowed to stand for 2 minutes. The supernatant cell suspension was aspirated into a centrifuge tube, and 10 mL of RPMI-1640 medium was added again, and homogenization was repeated twice. The obtained cell suspension was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the suspension was then resuspended in 10 mL of RPMI-1640 medium.

[0046] Add 10 mL of lymphocyte separation medium to another centrifuge tube, carefully spread the cell suspension on top of the separation medium, centrifuge at 8000×g for 20 minutes, aspirate the dense white cell layer at the interface, wash the cells twice with RPMI-1640 medium, resuspend them in 10 mL of RPMI-1640 medium, and count them for later use.

[0047] (2) Preparation of immune spleen cells:

[0048] One Balb / c mouse that had completed the immunization program was euthanized by exorbitant blood collection from the orbital cavity (the collected serum was positive). After euthanasia, the mouse was immersed in 75% alcohol for 10 minutes for disinfection. The mouse was then fixed on a dissecting board for dissection. The spleen was removed, shredded, and placed in a sterile homogenizer. After processing the cells according to the previously described method for grinding and preparing the cell suspension, the cells were counted and stored for later use.

[0049] (3) Preparation of feeder cells:

[0050] A standard Balb / c female mouse that had not undergone an immunization program was euthanized by orbital bleeding (the collected serum was negative). After euthanasia, the mouse was immersed in 75% alcohol for 5 minutes for disinfection. The mouse was then fixed on a dissecting board, and 2-3 mL of RPMI 1640 basal medium was injected intraperitoneally. After repeated pipetting, the medium was aspirated and placed in a clean centrifuge tube for later use (this step should not puncture organs to prevent contamination). The cells were centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the feeder cells were resuspended in HAT medium and incubated at 37°C with 5% CO2 for later use.

[0051] (4) Hybridoma cell fusion:

[0052] Add SP2 / 0 and immune spleen cells at a ratio of 1:5 to 1:10 to a 50 mL centrifuge tube and mix well (to ensure fusion effect, the number of immune spleen cells used should be 1×10). 8 (Several cells were collected), and then centrifuged at 1000 rpm for 8 min. After discarding the supernatant, the centrifuge tube containing the cell mixture was transferred to a 37°C water bath, and 0.8 mL of preheated PEG (Sigma) at 37°C was added. The mixture was gently stirred and allowed to stand for 30 s. After standing, 10 mL of preheated RPMI 1640 basal medium at 37°C was added, and the mixture was mixed by pipetting. The mixture was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the mixture was incubated at 37°C for 5-8 min to promote the fusion of SP2 / 0 cells with immune spleen cells. The treated cells were then mixed with the resuspended feeder cells by pipetting and seeded into 96-well plates at a rate of 250 μL / well. The plates were then incubated at 37°C in a 5% CO2 incubator. On day 4 of fusion, the HAT medium was replaced with HT medium and cultured. When the fusion cell colonies grew to 1 / 4 of the bottom area of ​​the well and the medium turned slightly yellow, antibody detection was performed.

[0053] 3. Clonal screening and subcloning of positive hybridoma cells

[0054] In the HAT medium screening system, unfused myeloma cells (SP2 / 0) die because they lack hypoxanthine-guanine-phosphoribotransferase and cannot synthesize DNA through the salvage pathway. Unfused immunosplenic cells can survive briefly in HAT medium, but gradually undergo apoptosis due to their lack of unlimited in vitro proliferation. Only successfully fused hybridoma cells, which acquire hypoxanthine-guanine-phosphoribotransferase from spleen cells, enabling DNA synthesis via the salvage pathway, and retain the unlimited proliferation characteristic of myeloma cells, can survive and proliferate in HAT medium. Based on this screening principle, specific antibodies were screened and identified using an indirect ELISA method on the cell culture supernatant in 96-well plates.

[0055] The steps for indirect ELISA screening of positive hybridoma cells are as follows:

[0056] (1) Antigen coating and blocking: Dilute the human β-NGF recombinant protein antigen to 2 μg / ml using coating buffer; add 100 μL to each well of a 96-well microplate and incubate overnight at 4°C or for 1 h at 37°C to allow the microplate to adsorb the antigen; discard the solution and wash 3 times with washing buffer and pat dry. Then add 250 μL of blocking buffer (2% BSA) to each well, shake gently, and incubate at 37°C for 1 h to block nonspecific sites; discard the solution and wash 3 times with washing buffer and pat dry.

[0057] (2) Sample incubation with enzyme-labeled secondary antibody: Add 50 μL of the supernatant of the hybridoma cells to be tested to each well in sequence to the enzyme-labeled wells. Incubate at 37℃ for 1 h to promote antibody-antigen binding. Discard the solution, wash 3 times and pat dry. Add 100 μL of enzyme-labeled secondary antibody working solution to each well, incubate at 37℃ for 1 h, discard the solution, wash 4 times and pat dry to completely remove free enzyme-labeled secondary antibody.

[0058] (3) Color development and result determination: Add 100 μL of TMB color development solution to the enzyme-labeled wells, shake gently, and develop color at 37°C in the dark for 10 min. After adding 50 μL of stop solution, immediately read the OD value at a wavelength of 450 nm using an enzyme-labeled reader. When the OD value of the positive well is greater than 3 times the OD value of the negative well, it can be determined as positive.

[0059] (4) Subcloning of hybridoma cells (limiting dilution method):

[0060] Cell dilution and culture observation: Hybridoma cells to be cloned were gently pipetted into single cells and collected. Viable cells were counted using a hemocytometer. After counting, the cells were diluted with complete culture medium to 5, 10, and 30 cells / mL. The cell suspensions at the above three concentrations were added to 96-well culture plates containing prepared mouse peritoneal macrophage feeder layers (100 μL / well), so that each well theoretically contained 0.5, 1, and 3 cells, respectively. On day 4 of culture, 50 μL of fresh culture medium was added to each well of the 96-well culture plate. On days 5-6, the single-clone growth wells were observed and labeled under a microscope.

[0061] Positive clone expansion culture: On days 7-9 after cloning, when the cell clones have grown to fill 1 / 3 to 1 / 2 of the field of view, the cell supernatant can be collected for ELISA detection; positive clones are transferred to 24-well plates for expansion culture. Once the cell density in the 24-well plates reaches the target, Balb / c mice can be intraperitoneally inoculated to prepare ascites fluid.

[0062] 4. RNA extraction and sequencing of monoclonal antibody 11F1 and 13E2 positive hybridoma cells

[0063] (1) Cell collection and RNA extraction: Two hybridoma cell lines (11F1 and 13E2) were collected, ensuring a cell count ≥10. 6 The samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0064] (2) Sequencing results:

[0065] Sequencing data of 11F1 cell line: Light chain variable region (VL): DNA sequence length 336 bp (SEQ ID NO:1), encoding 112 amino acids (SEQ ID NO:3). Heavy chain variable region (VH): DNA sequence length 348 bp (SEQ ID NO:2), encoding 116 amino acids (SEQ ID NO:4).

[0066] Sequencing data of 13E2 cell line: Light chain variable region (VL): DNA sequence length 333bp (SEQ ID NO:5), encoding 111 amino acids (SEQ ID NO:7). Heavy chain variable region (VH): DNA sequence length 354bp (SEQ ID NO:6), encoding 118 amino acids (SEQ ID NO:8).

[0067] 5. Large-scale preparation of monoclonal antibodies 11F1 and 13E2

[0068] The expanded hybridoma cell line was intraperitoneally injected into Balb / c mice. Ascites fluid was collected approximately 7 days post-injection, and antibodies were purified using Protein G affinity chromatography (SDS identification results of the purified antibodies are shown in [link to relevant documentation]). Figure 1 ).

[0069] Example 2: Construction of a double-antibody sandwich ELISA kit for human β-NGF content

[0070] 1. Biotin labeling of monoclonal antibody 13E2

[0071] Transfer the purified 13E2 antibody solution to a dialysis bag (preferably, the selected dialysis bag has a molecular weight cutoff of 10-15 kDa), place the dialysis bag in 0.1 M pH 9.5 CB buffer for overnight dialysis, and replace the 13E2 antibody buffer (using a high pH value helps with antibody activation and labeling).

[0072] Weigh 2.2 mg of NHS-D-Biotin and dissolve it in 1 mL of DMSO. Take 1 mL of the antibody solution after buffer replacement and add 1 / 10 of the total volume of NHS-D-Biotin to it. Stir at room temperature in the dark for 4 hours.

[0073] After the reaction, the labeled antibody solution was transferred to a dialysis bag (10-15 kDa) and dialyzed overnight in 0.01 M PBS, changing the medium 3-4 times during the process (to remove free biotin). After dialysis, the antibody solution was removed, and an equal volume of sterile glycerol (final concentration 50%, v / v) was added sequentially. The mixture was then mixed and stored at -20°C.

[0074] 2. Preparation of monoclonal antibody 11F1 ELISA plate

[0075] Take the monoclonal antibody 11F1 stock solution and dilute it to 2 μg / ml with 0.05M pH 9.6 carbonate coating buffer. Add 100 μL of the diluted antibody solution to each well of a 96-well polystyrene plate, ensuring the liquid level completely covers the bottom of the well. Seal the microplate with sealing film and incubate overnight at 4°C. The next day, remove the microplate, peel off the sealing film, discard the antibody coating solution in the wells, and wash three times with 250 μL of PBST washing buffer for 3 minutes each time to thoroughly remove unadsorbed antibody and impurities.

[0076] After the above steps, seal each well of the reaction plate with 300 μL of 2% BSA solution and let it stand at room temperature for 2 hours. Discard the solution in the wells, dry in a desiccant chamber for 16-18 hours, put the microplate into an aluminum foil bag, add desiccant, vacuum seal, and store at 4°C.

[0077] 3. Establishment of a human β-NGF double-antibody sandwich ELISA method

[0078] Thirty minutes before the experiment, remove the pre-dried and packaged ELISA plate (coated with monoclonal antibody 11F1) and allow it to equilibrate to room temperature to avoid condensation in the wells due to temperature differences. Wash the plate three times with PBST wash buffer and pat dry. Add 100 μL of human β-NGF standard at different dilutions: 2000, 1000, 500, 250, 125, 62.5, and 31.25 pg / mL, and set up a blank control. After sealing with the sealing membrane, incubate at room temperature with shaking at 300–400 rpm for 2 hours. Remove the sealing membrane, wash the plate four times, and pat dry. Then add 100 μL of biotinylated antibody 13E2 to the reaction wells, seal with the sealing membrane, and incubate at room temperature with shaking at 300–400 rpm for 1 hour. Remove the sealing membrane, wash the plate four times, and pat dry. Add 100 μL of horseradish peroxidase-labeled streptavidin (SA-HRP) to each reaction well, seal with a sealing film, and incubate at room temperature with shaking at 300-400 rpm for 30 min. Remove the sealing film, wash the plate 5 times, and blot dry. Add 100 μL of chromogenic substrate TMB to each reaction well, seal the plate, and incubate at room temperature in the dark for 15 min. Add 50 μL of 2M sulfuric acid solution as a stop solution to each reaction well, and then measure the OD value at 450 nm using a microplate reader (within 5 minutes). Plot a standard curve with different concentrations of human β-NGF standards on the x-axis and the corresponding OD values ​​on the y-axis to establish a regression equation. The results show that the detection range is 31.25-2000 pg / ml, and the R² is 0.99990 (…). Figure 2 ).

[0079] 4. Specific detection of human β-NGF double antibody sandwich ELISA

[0080] This embodiment verifies the specific recognition ability of the above-mentioned double antibody sandwich ELISA method for human β-NGF by detecting the cross-reactivity rate, and excludes non-specific binding with homologous proteins or interfering substances. Substances that may share homology with human β-NGF (such as members of the neurotrophic factor family) or are commonly found in biological samples (such as serum and tissue fluid) were selected as interference sources, including: human glial cell-derived neurotrophic factor (GDNF), human ciliary neurotrophic factor (CNTF), human brain-derived neurotrophic factor (BDNF), human epithelial neutrophil activating peptide-78 (ENA-78), human fibroblast growth factor-4 (FGF-4), members of the human interleukin family (IL-1α, IL-1β, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-19, IL-10, IL-17A, IL-12p40, IL-12p70, IL-13) and human immunoglobulins (IgG, IgA, IgM, IgE). High concentrations of interfering substances were set up for validation to ensure that non-specific binding could be avoided even when the concentration of interfering substances in the sample was high. Human β-NGF standards with different dilution ratios (such as the aforementioned dilution gradient, 31.25-2000 pg / mL) were selected to determine the cross-reactivity concentration results and to serve as positive controls to verify the effectiveness of the detection system.

[0081] The detection was then performed according to the previously described method. The results showed that the OD values ​​of all high-concentration interfering substances were not significantly different from the blank control, and the cross-reactivity rates were all <0.1%. Furthermore, the OD values ​​of human β-NGF at different dilutions were positively correlated with the β-NGF concentration and were significantly higher than those of the interfering groups (Table 1). This demonstrates that the human β-NGF double-antibody sandwich ELISA detection method established in this invention can specifically recognize the target antigen and has no cross-reactivity with the aforementioned homologous proteins, cytokines, and immunoglobulin-like substances.

[0082] Table 1 Cross-reactivity test

[0083]

[0084] Example 3: Stability Study of Human β-NGF Double Antibody Sandwich ELISA Kit

[0085] This embodiment verifies the stability of key components of the aforementioned double-antibody sandwich ELISA kit through accelerated stability testing to simulate performance changes under long-term storage conditions. The ELISA plate (coated with monoclonal antibody 11F1), biotinylated antibody 13E2, and human β-NGF lyophilized standard were stored in a 37°C incubator for 12 days (approximately 18 months at 4°C). Stability experiments were conducted at five time points: 0 days (untreated components under original conditions), 3 days, 6 days, 9 days, and 12 days. The experiments were performed according to the previously described methodology. Standard curves were plotted with different concentrations of human β-NGF standard on the x-axis and the corresponding OD values ​​on the y-axis, and regression equations were established. The results showed that the standard curves at 37°C for 0, 3, 6, 9, and 12 days exhibited good linearity, with R0... 2 All values ​​were greater than 0.9995, indicating good stability of the kit (Table 2).

[0086] Table 2 Accelerated stability test

[0087]

[0088] Example 4: Detection of recovery rate of human β-NGF double antibody sandwich ELISA

[0089] This embodiment verifies the quantitative accuracy of the aforementioned double-antibody sandwich ELISA kit for human β-NGF and its matrix adaptability through spiked recovery experiments and linear dilution tests. Commonly used biological samples in the detection process, matrix cell supernatant and serum, were selected to simulate the actual sample detection environment and assess the impact of matrix interference on the recovery results. In the spiked experiment, three concentration levels of human β-NGF standard were added to the two matrices: high (1000 pg / mL), medium (250 pg / mL), and low (62.5 pg / mL) (covering the kit's detection range of 31.25-2000 pg / mL), with a matrix blank control (unspecified). In the linear dilution experiment, a high-value human β-NGF standard (1000 pg / mL) was added to both matrices, thoroughly mixed, and then serially diluted 2-, 4-, and 8-fold with the corresponding matrices (the theoretical concentrations after dilution were 500 pg / mL, 250 pg / mL, and 125 pg / mL, respectively). In addition, the concentrations of human β-NGF standards at different dilutions (as described above, dilution gradient 31.25-2000 pg / mL) were quantified. Following the previously established methodological procedures, the results showed that the recoveries of spiked samples in both matrices (cell supernatant and serum) ranged from 70% to 100% for high (1000 pg / mL), medium (250 pg / mL), and low (62.5 pg / mL) values. Simultaneously, the linear recoveries of the two matrices ranged from 70% to 120% (Tables 3 and 4).

[0090] Table 3 Spike Recovery Rate

[0091]

[0092] Table 4. Linear dilution recovery rate

[0093]

[0094] Example 5: Comparison of detection results with similar reagent kits from abroad

[0095] A comparative test was conducted using a foreign R&D brand reagent kit (Human beta-NGF DuoSet ELISA kit, catalog number DY256), and experiments were performed according to the optimal methods for each brand (following their instructions exactly). The performance of the three kits was compared in the following two aspects.

[0096] a. Standard curve comparison: The linear correlation coefficient (R²) of the standard curve of the kit of this invention. 2 =0.99999) is significantly higher than that of foreign brands (R 2 =0.99985), indicating that the fitting accuracy of the present invention is higher and the quantitative accuracy is better. At the same time, the OD value of well 0 of the kit of the present invention is 0.016, which is significantly lower than that of foreign brands (0.086), proving that it has a better detection capability for low concentration samples (Table 5).

[0097] Table 5 Standard Curve

[0098]

[0099] b. Comparison of Sample Measurement Results: To verify the accuracy and sensitivity of the kit for detecting actual samples, PC12 cells were divided into four groups: a 24h blank group, a 24h treatment group with 100 ng / mL human IL-1β, a 24h treatment group with 500 ng / mL human IL-1β, a 48h blank group, a 48h treatment group with 100 ng / mL human IL-1β, and a 48h treatment group with 500 ng / mL human IL-1β. The experimental results showed that the concentration detection results of both the kit from this invention and foreign kits exhibited IL-1β concentration and stimulation time dependence; moreover, the concentration measured by the kit from this invention was higher than that of foreign brands, indicating that the kit from this invention has a better detection effect on β-NGF (Table 6).

[0100] Table 6 Sample Concentration Measurement

[0101]

[0102] Example 6 Antibody Affinity Constant Detection

[0103] Human β-NGF antigen was coated onto an ELISA plate and blocked. After washing with PBST, monoclonal antibodies 11F1 and 13E2 diluted to saturated concentration were added to the wells at 100 μL / well and incubated at room temperature for 2 h. After washing with PBST, 60 μL of different concentration gradients of NaSCN (sodium thiocyanate) solutions (0M, 1M, 2M, 3M) were added to the wells sequentially, ensuring the liquid surface completely covered the bottom of the wells to ensure sufficient contact between the antigen and antibody conjugates. The reaction was carried out at room temperature for 15 min. After washing with PBST, 100 μL of enzyme-labeled secondary antibody working solution was added and incubated at room temperature for 45 min. After washing with PBST, TMB chromogenic solution and stop solution were added sequentially before detection. Using the 0M pore OD value as a reference for the maximum OD value of antibody-antigen binding, when the OD value of the antibody after treatment with a certain concentration of sodium thiocyanate is still higher than 50% of the 0M pore OD value, it indicates that the sodium thiocyanate at that concentration did not destroy more than half of the antigen-antibody bindings. The corresponding sodium thiocyanate concentration is the antibody affinity level. As shown in Table 7, the affinity of both 11F1 and 13E2 antibodies is >3M, indicating that they both possess excellent affinity.

[0104] Table 7 Results of monoclonal antibody affinity assay

[0105]

[0106] Example 7: Monoclonal antibody screening and optimized combination for human β-NGF double antibody sandwich ELISA method

[0107] In this embodiment, the checkerboard method was used to screen for monoclonal antibody pairings (a total of 31 monoclonal antibodies) through a double-antibody sandwich pattern.

[0108] Monoclonal antibodies were diluted to 2 μg / mL using 0.05 M pH 9.6 carbonate coating buffer. 100 μL of the diluted antibody solution was added to each well of a 96-well polystyrene plate. The plate was sealed and incubated overnight at 4°C. The solution was discarded and the plate washed the next day. 250 μL of blocking buffer containing 2% (w / v) BSA was added to each well, and the plate was blocked at room temperature for 2 h. After discarding the blocking buffer and washing the plate, 100 μL of 2000 pg / mL standard and PBST blank control were added to the corresponding wells, and the plate was incubated at room temperature for 2 h. Diluted biotin-labeled monoclonal antibodies were added using a checkerboard pattern, and the plate was incubated at room temperature for 1 h. Streptavidin-labeled horseradish peroxidase (SA-HRP) was added, and the plate was incubated at room temperature for 30 min. After discarding the solution and washing the plate, TMB chromogenic buffer and stop solution were added sequentially, and the results were detected. Antibody pairs with relatively high peak values ​​and low blank well values ​​were selected for further optimization. The test results showed that using 11F1 as the coating antibody and biotinylated 13E2 as the detection antibody, the OD value of the positive well was >2.5, the background OD value was <0.1, the background was low, and the standard curve gradient was good.

[0109] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Anti-human β-NGF monoclonal antibody 13E2, characterized in that, The amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain variable region of antibody 13E2 are KSVSTSGYSY, LAS, and QHSRELPLT, respectively, and the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region are GFTFSSHW, VRLKSNNYAT, and TLNGGAMDH, respectively.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of antibody 13E2 is shown in SEQ ID NO:7, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:

7. The amino acid sequence of the heavy chain variable region of antibody 13E2 is shown in SEQ ID NO:8, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:

8.

3. A nucleic acid molecule encoding the antibody of claim 1 or 2.

4. A biomaterial containing the nucleic acid molecule of claim 3, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or host cell.

5. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the antibody of claim 1 or 2 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, and colloidal gold labeling.

6. Any of the following applications of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the biomaterial of claim 4, or the antibody conjugate of claim 5: (1) Qualitative or quantitative detection of human β-NGF for non-disease diagnosis and treatment purposes; (2) Prepare reagents or kits for the qualitative or quantitative detection of human β-NGF; (3) Used for quality control of products containing human β-NGF.

7. A human β-NGF detection reagent or kit prepared from antibody 13E2 as described in claim 1 or 2.

8. The reagent kit according to claim 7, characterized in that, This includes enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescence immunoassay kits.

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