Preparation method of CGRP detection kit and application of CGRP detection kit in migraine diagnosis

Abstract

The invention discloses a preparation method of a CGRP detection kit and application of the CGRP detection kit in migraine diagnosis, and belongs to the technical field of biological medicine detection. According to the invention, alpha-CGRP polypeptide coupled KLH protein is subjected to efficient animal immunization, an antibody with good affinity is screened by using methods such as enzyme-linked immunosorbent assay, cross pairing analysis is carried out, and final antibody sequence information is obtained through sequencing. The paired monoclonal antibody screened by the invention has high sensitivity (KD value of 1nM), the limit of detection (LoD) can be as low as pg / mL level, a low-concentration CGRP sample can be effectively detected, the matrix effect of human serum / plasma can be effectively neutralized by an optimized sample diluent formula, and the repeatability is high (in-plate and inter-plate CV values of 1t and 10%). According to the invention, the kit prepared from the specific paired antibody is systematically and closely linked with the indication function of migraine for the first time, and a clinical diagnosis direction is provided.

Description

Technical Field

[0001] This invention relates to a method for preparing a CGRP detection kit and its application in the diagnosis of migraines, belonging to the field of biomedical detection technology. Background Technology

[0002] Migraine is a common neurological disorder and the seventh leading cause of disability worldwide, but its complexity poses a significant obstacle to its diagnosis and treatment. Genetic, epigenetic, and environmental factors all contribute to the pathogenesis of migraine, and this mechanism remains incompletely understood. The lack of specific biomarkers to date greatly impacts the accurate diagnosis, treatment selection, and prognostic assessment of migraine.

[0003] Numerous studies have found abnormal changes in neuropeptides in migraine patients, such as calcitonin gene-related peptide (CGRP), S100B, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), neurokinin A, substance P, matrix metalloproteinase-9 (MMP-9), and p-endorphin.

[0004] Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide composed of 37 amino acids, discovered in 1982. It is widely distributed throughout various systems of the human body, primarily in the dorsal root ganglia and the C and Aδ sensory fibers of the trigeminal nerve. Approximately 65%–80% of DRG neurons express CGRP, mostly belonging to small to medium-sized neurons. CGRP is mainly located at the terminals of primary afferent nerves; it is almost absent in the cell bodies of secondary neurons. Dorsal root ganglion neurons are the sole source of CGRP in the spinal cord. CGRP exists in two forms: α-CGRP and β-CGRP.

[0005] During a migraine attack, CGRP levels significantly increase in blood and cerebrospinal fluid, triggering neurogenic inflammation, cerebral vasodilation, and pain signal transduction. Intravenous injection of CGRP can induce migraine-like attacks. Serum CGRP levels are elevated during the acute phase of migraine and decrease during remission. The preparation and application of CGRP monoclonal antibodies and CGRP receptor antagonists have demonstrated that blocking the CGRP pathway can effectively prevent or treat migraines, thus validating its core pathological role.

[0006] Therefore, the development of CGRP as a migraine biomarker is a product driven by both in-depth analysis of pathological mechanisms and unmet clinical needs. Its core value lies in: providing the first objective diagnostic tool directly related to the core pathology of migraine; propelling migraine diagnosis from "symptom diagnosis" to "mechanism diagnosis" in the era of precision medicine; and providing scientific evidence for the personalized application of targeted therapies (such as CGRP inhibitors). With the optimization of detection technology and the advancement of large-scale clinical validation, CGRP testing is expected to become an important tool in migraine management, reshaping the diagnostic and treatment paradigm.

[0007] Currently, the main methods for detecting CGRP are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Among them, the ELISA method mainly uses the double antibody sandwich method and the competitive inhibition method. Existing commercial CGRP ELISA kits still have some shortcomings. Because CGRP is homologous to peptides such as amyloid and adrenomedullin, poor antibody design may lead to cross-reactivity, affecting the accuracy of the results.

[0008] Therefore, there is an urgent need in this field for a CGRP detection kit with higher sensitivity, higher specificity and stronger resistance to matrix interference to meet the clinical needs for precise diagnosis and treatment of migraine. Summary of the Invention

[0009] To address the shortcomings of the existing technology, this invention provides a method for preparing a CGRP detection kit and its application in the diagnosis of migraines. The purpose is to solve the technical problem that in existing CGRP detection ELISA kits, CGRP has homology with peptides such as amyloid and adrenomedullin, and poor antibody design may lead to cross-reactions, affecting the accuracy of the results.

[0010] The first technical solution provided by this invention is an anti-calcitonin gene-related peptide antibody, which includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a CDR (Cellular Dependent Ratio) L1, CDR L2, CDR The light chain CDR consists of L3, and the heavy chain variable region has CDR. H1, CDR H2, CDR The heavy chain CDR composed of H3, the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR The amino acid sequence of H3 is shown in any one of (A) to (B): (A) SEQ ID NO:3~5, SEQ ID NO:7~9; (B) SEQ ID NO: 11~13, SEQ ID NO: 15~17.

[0011] In some embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in any one of groups (C) to (D): (C) SEQ ID NO:2, SEQ ID NO:6; (D) SEQ ID NO: 10, SEQ ID NO: 14.

[0012] The second technical solution provided by the present invention is a gene encoding the anticalcitonin gene-related peptide antibody described in the first technical solution.

[0013] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0014] In some embodiments, the recombinant vector uses plasmid pcDNA3.1 as the expression vector.

[0015] The fourth technical solution provided by the present invention is a recombinant cell expressing an antibody against the anticalcitonin gene-related peptide as described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0016] In some embodiments, the recombinant cells use fungi, bacteria, animal cells, or plant cells as hosts.

[0017] In some embodiments, the fungus includes yeast or mold, and the bacteria includes Escherichia coli.

[0018] In some embodiments, the animal cells include, but are not limited to, 293 cells.

[0019] The fifth technical solution provided by the present invention is a method for preparing an anti-calcitonin gene-related peptide antibody, wherein the method involves culturing the recombinant cells described in the fourth technical solution to obtain a culture containing the anti-calcitonin gene-related peptide antibody described in the first technical solution.

[0020] The sixth technical solution provided by this invention is a biomarker or chemically marked product, wherein the product is an antibody marked by a marker, and the antibody originates from any of the following sources: (1) The anti-calcitonin gene-related peptide antibody described in the first technical solution; (2) The culture of recombinant cells described in the fourth technical solution.

[0021] In some embodiments, the labeling agents include, but are not limited to, enzymes, biotinylate, luciferase, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-antialkaline phosphatase bridges, and β-galactosidase-antiβ-galactosidase bridges; the biotinylate class includes, but is not limited to, biotin and its derivatives; the luciferase class includes, but is not limited to, AF350, AF488, AF532, AF546, AF555, and AF56. 8. AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, PerCP dye; the chemiluminescent dyes include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives, etc.; the isotope dyes include, but are not limited to, iodine labeling; the colloidal labelings include, but are not limited to, colloidal gold, colloidal carbon, colloidal selenium, etc.

[0022] The seventh technical solution provided by the present invention is a kit containing the anti-calcitonin gene-related peptide antibody described in the first technical solution or the biomarker or chemically labeled product described in the sixth technical solution.

[0023] In some embodiments, the kit includes an enzyme-linked immunosorbent assay (ELISA) kit and an immunochromatographic assay kit.

[0024] In some embodiments, the kit includes a microplate coated with a capture antibody, an enzyme-labeled detection antibody, CGRP standards, sample diluent, washing buffer, chromogenic substrate, and stop solution; the capture antibody is an anti-calcitonin gene-related peptide with a heavy chain variable region amino acid sequence as shown in SEQ ID NO:2 and a light chain variable region amino acid sequence as shown in SEQ ID NO:6, and the detection antibody is an anti-calcitonin gene-related peptide antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO:10 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:14.

[0025] Furthermore, the method for preparing the microplate coated with the capture antibody is as follows: (1) Coating: Dilute the capture antibody to the optimal concentration with carbonate coating buffer (pH 9.6), add 100-200 µL to each well of a microplate, and incubate overnight at 2-8°C; (2) Washing and blocking: Discard the coating solution and wash the microplate 3-5 times with washing solution. Then add PBS solution containing 1-5% BSA or 5% skim milk powder for blocking, 200-300 µL per well, and incubate at 37°C for 1-2 hours; (3) Drying and packaging: Discard the sealing liquid, wash and pat dry, vacuum seal the microplate in an aluminum foil bag, and add desiccant.

[0026] Furthermore, the preparation method of the detection antibody label is as follows: HRP and purified detection antibody are coupled at the optimal molar ratio using the sodium periodate method, and the resulting enzyme-labeled detection antibody working solution is obtained after purification.

[0027] The eighth technical solution provided by the present invention is the application of the anti-calcitonin gene-related peptide antibody described in the first technical solution, or the gene described in the second technical solution, or the expression recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the biomarker or chemically labeled product described in the sixth technical solution in the preparation of products for detecting calcitonin gene-related peptide.

[0028] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.

[0029] The ninth technical solution provided by this invention is the application of the anti-calcitonin gene-related peptide antibody described in the first technical solution, or the gene described in the second technical solution, or the expression recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the application of the biomarker or chemically labeled product described in the sixth technical solution in the preparation of a diagnostic migraine product.

[0030] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes efficient animal immunization with α-CGRP peptide conjugated to KLH protein, followed by screening for antibodies with good affinity using enzyme-linked immunosorbent assay (ELISA) and cross-pairing analysis. Sequencing yields the final antibody sequences, containing both heavy and light chain variable regions, with amino acid sequences shown in SEQ ID NO: 2, 6, 10, and 14. The selected paired antibodies target different and unique epitopes of CGRP and exhibit no cross-reactivity with β-CGRP or β-amyloid peptide analogs, ensuring accurate detection results. The kit performance was validated using clinical samples from multiple migraine-related patients and various cross-reactive substances, all showing good results. The paired monoclonal antibodies screened in this invention possess high sensitivity (K... DWith a detection limit (LoD) <1 nM and a detection limit (LoD) as low as pg / mL, it can effectively detect low-concentration CGRP samples. Furthermore, the optimized sample dilution formulation effectively neutralizes the matrix effect of human serum / plasma, exhibiting high reproducibility (intra- and inter-plate CV values ​​both <10%). This invention is the first to systematically link a kit prepared with this specific paired antibody to the indication function of migraine, providing a diagnostic direction for clinical practice. Attached Figure Description

[0032] Figure 1 This is a standard curve for detecting CGRP using the kit of the present invention.

[0033] Figure 2 Box plot showing the statistical comparison of CGRP levels in plasma of migraine patients during attack, remission, and healthy controls using the kit of this invention. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Raw materials used in the examples: 1. pET28a vector and pcDNA3.1 vector were purchased from Takara.

[0036] 2. Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Takara.

[0037] 3. LB liquid medium: prepared with 10 g / L peptone, 5 g / L yeast extract, and 5 g / L sodium chloride, with the pH adjusted to 7.3±0.1; LB solid medium: prepared with 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder, with the pH adjusted to 7.3±0.1; Serum-free DMEM medium was purchased from Gibco; fetal bovine serum was purchased from Sijiqing; and trypsin was purchased from Gibco.

[0038] 4. The immunization process for BALB / c mice was commissioned to Jiangsu Dongkang Biotechnology Co., Ltd.

[0039] Example 1: Preparation of anti-CGRP monoclonal antibody Peptide synthesis: The amino acid sequence of the synthesized α-CGRP is shown in SEQ ID NO: 1.

[0040] SEQ ID NO: 1: MPFLALSILVLLQAGSLHAAPFRSALESSPADPATLSEDEARLLLAALVQDYVQMKASELEQEQEREGSRIIAQKRACDTATCVTHRLAGL.

[0041] Immunogen preparation: Mix CGRP peptide solution and KLH solution at a molar ratio of CGRP:KLH = 20:1 to 50:1 in an ice bath until homogeneous. While gently stirring, add freshly prepared EDC solution dropwise to the mixture (the amount of EDC is 10 times the molar amount of CGRP). Incubate the reaction system at room temperature, protected from light, and gently stir for 2-4 hours. After the reaction is complete, transfer the reaction solution to a pre-treated dialysis bag (molecular weight cutoff 10-14 kDa). Dialyze at 4°C using a large volume of PBS (pH 7.4) for 24-48 hours, changing the dialysis buffer 3-4 times during this period to thoroughly remove unreacted EDC, byproducts, and free CGRP peptides.

[0042] Animal immunization: 6-8 week old female BALB / c mice were immunized with CGRP-KLH conjugate protein. Three to four immunizations were administered according to standard immunization methods. Mouse spleen cells were fused with SP2 / 0 myeloma cells using PEG. Positive hybridoma cell lines were screened using indirect ELISA.

[0043] Hybridoma screening: ELISA plates were coated with CGRP-KLH conjugate protein and washed three times with PBST. Blocking was performed with PBST containing 2% BSA (Sigma) for 2 hours, followed by three more washes with PBST. Fusion cell supernatant, 1:1000 diluted immunized mouse positive serum, and 1:1000 diluted mouse negative serum were added separately, and incubated at 37°C for 1 hour. After washing with PBST, HRP-labeled goat anti-mouse IgG and goat anti-mouse IgM (Sigma) were added, and incubation was performed for 1 hour. After three washes with PBST, TMB substrate was added for color development in the dark for 10 minutes, and the reaction was terminated with sulfuric acid. OD450nm values ​​were measured using an ELISA reader. With a baseline of OD ≤0.1 for negative serum, wells with an OD value ≥2 times that of negative wells were considered positive. Positive hybridoma cells were then used for subsequent cloning.

[0044] Cloning of positive cell lines: Positive hybridoma cells were counted, diluted to 100 cells / 10 mL of culture medium, and seeded into 96-well plates (100 µL / well). The cells were incubated at 37°C in a 5% CO2 incubator for 6-7 days. Single-clone wells were labeled under a microscope, and the cell supernatant was used for ELISA detection (method as before) to screen for positive single clones. Cloning was performed using a three-stage limiting dilution method to obtain hybridoma cell lines that stably secrete anti-CGRP antibodies. A total of 5 positive cell clones secreting antibodies were obtained, with clone numbers: 2F1, 3E8, 6B3, 12C2, and 10G5.

[0045] Antibody gene cloning and vector construction: mRNA was extracted from positive hybridoma cells and reverse transcribed into cDNA; the heavy chain variable region (VH) and light chain variable region (VL) genes of mouse monoclonal antibodies were amplified by PCR and sequenced to confirm the sequences. The VH and VL genes were cloned into a eukaryotic expression vector to construct heavy chain and light chain recombinant plasmids.

[0046] Construction of stable expression cell lines: Heavy chain and light chain plasmids were co-electrotransduced into CHO cells, and cultured in pressure selection medium containing 50 µM MSX for 20 days. The supernatant was collected and CHO cell lines with high antibody expression were screened by ELISA (coating antigen was CGRP).

[0047] Large-scale antibody preparation and purification: A roll-on / roll-off culture technique was used to expand the culture of positive CHO cell lines: cells were seeded at 0.2-0.3 × 10⁶ cells / mL using Vega CHO medium. 6 Cells / mL (300mL culture medium in a 1L roller bottle) were cultured at 37℃, 5% CO2, and 900 rpm for 7-9 days. When the cell viability was below 50%, the supernatant was collected by centrifugation. Antibodies were purified by Protein A affinity chromatography to obtain anti-CGRP monoclonal antibodies (clone numbers: 2F1, 3E8, 6B3, 12C2, 10G5).

[0048] Antibody titer assay: Dilute CGRP protein to 1-2 µg / mL with 0.05 mol / L pH 9.6 carbonate buffer, add 100 µL / well to a 96-well microplate, and incubate overnight at 4°C. Discard the coating solution and wash the microplate three times with PBST (PBS containing 0.05% Tween-20), soaking for 1-2 minutes each time and then pat dry on absorbent paper. Add 150 µL / well of PBST containing 2% BSA (Sigma), and incubate at 37°C for 2 hours to block non-specific sites. Wash three times with PBST and pat dry. Serially dilute the anti-CGRP monoclonal antibody to be tested with 0.02 M PBS (pH 7.4). Add 100 µL / well of the diluted antibody sample to the microplate and incubate at 37°C for 1 hour. Wash three times with PBST and pat dry. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (or IgM, selected according to antibody subtype, purchased from Sigma) was added, diluted 1:4000 with diluent, 100 µL / well, and incubated at 37°C for 1 hour. The wells were washed three times with PBST and blotted dry. TMB substrate solution was added, 100 µL / well, and incubated at room temperature in the dark for 10 minutes. The reaction was stopped by adding 50 µL of 2 mol / L sulfuric acid to each well. The OD450 nm value was immediately measured using a microplate reader. The positive cut-off value was defined as 2.1 times the OD value of the negative control (irrelevant antibody or blocking solution). The antibody titer was defined as the reciprocal of the highest antibody dilution that produced this cut-off OD value. A titer greater than 1:10000 is generally required to indicate high antibody binding activity.

[0049] Antibody purity assay (SDS-PAGE): SDS-PAGE was used for analysis. Under the condition that all components in the sample were eluted, the purified anti-CGRP monoclonal antibody sample was loaded and separated by electrophoresis to check antibody purity.

[0050] Affinity assay: The equilibrium dissociation constant (K0) of antibody-CGRP binding was determined using surface plasmon resonance. D ), to assess its binding strength.

[0051] Table 1. Results of purity, titer, and affinity tests for monoclonal antibodies.

[0052] Specificity detection: The enzyme-labeled plates were coated with KLH protein, BSA and other possible cross-linking substances (β-CGRP, amyloid, calcitonin) respectively, and the antibodies in Table 1 above were detected. The results showed that the antibodies specifically bound to the target CGRP and did not bind to other substances.

[0053] Table 2. Information on specific detection reagents and results of cross-interference.

[0054] Subtype identification: The antibody was identified as belonging to the IgG1 subtype using the mouse monoclonal antibody Ig class / subclass identification ELISA kit (IgG1\IgG2a\IgG2b\IgG3\IgM\IgA) manufactured by Bio-Rad Laboratories.

[0055] Antibody pairing: Following the ELISA reaction system described above, the double-antibody sandwich method was used for antibody cross-pairing (pairing combinations are shown in Table 3). The pair of antibodies with the best detection sensitivity was selected, namely capture antibody 2F1 and detection antibody 3E8.

[0056] Table 3 Results of antibody cross-pairing detection

[0057] Note: G1~G12 represent the color intensity of colloidal gold, with the color intensity increasing sequentially from G1 to G12. The samples were sent to General Biotech for sequencing.

[0058] The amino acid sequences of the light and heavy chain variable regions of the capture antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 6.

[0059] SEQ ID NO:2: DIVLSQSPTTMAASPGEKLTCSASSGLSSNYLHWYQQKPGFSPKLLIYRTSNLASGYVPARFGSGSGTSYSLTIGTMAFEDVATYYCQQGSSIPFTFGSGTKLEIK; CDR-L1 (SEQ ID NO:3):SASSGLSSNYLH; CDR-L2 (SEQ ID NO:4): RTSNLAS; CDR-L3 (SEQ ID NO:5): QQGSSIPFT.

[0060] SEQ ID NO: 6 DVQLQQSGADLARPGASVRMSCKASGYRTLLRYTMHWIKERPGQGLEWIGYIYPSSGYLTYNQKFKDKVTLTADKSSGGTAYMRMTSETSFDSAVYYCATITEGHYAGNSNYEGYWGQGTLTVSS; CDR-H1 (SEQ ID NO:7): RYTMH; CDR-H2 (SEQ ID NO:8): YIYPSSGYTYNQKFKD; CDR-H3 (SEQ ID NO:9): ITEGHYAGNSNYEGY.

[0061] The amino acid sequences of the light chain and heavy chain variable regions of the detection antibody are shown in SEQ ID NO:10 and SEQ ID NO:14.

[0062] SEQ ID NO:10 DAVVTQESALTTGPGETVTITCRSSTSGAVTTNNYANWVQEKPDILFTGLMGATSNHRAPSVPARGSGSLIGDKAFALTITGAQTADEAIYFCALWYSDHLVLGGSTKITVF; CDR-L1 (SEQ ID NO:11): RSSTSGAVTTNNYAN; CDR-L2 (SEQ ID NO:12): ATSNHRAP; CDR-L3 (SEQ ID NO:13): ALWYSDHLV.

[0063] SEQ ID NO:14 DVKLEQQPGAELVRPGTSVKLSCKASGYRFNYWMNSVKQRPGQGLEWIGMIRPSETRLHQKFKDKATITVDKSSSTAYMQLSSLTSEDSAVAYCARYISLGGFDYWGQGTTLTVSS; CDR-H1 (SEQ ID NO:15):GYSFNYWMN; CDR-H2 (SEQ ID NO:16): MIRPSETRLHQKFKD; CDR-H3 (SEQ ID NO:17): YISLGGFDY.

[0064] The antibody was humanized using conventional plasmid construction methods. The heavy chain variable region obtained from sequencing was ligated with the human IgG heavy chain constant region, the light chain variable region and the human IgG light chain constant region, and then constructed into the pcDNA3.1 plasmid to obtain pcDNA3.1-2F1-H, pcDNA3.1-2F1-L and pcDNA3.1-3E8-H, pcDNA3.1-3E8-L expression plasmids. The heavy chain expression plasmid was transfected into the Expi 293F mammalian cell line for expression at a molar ratio of 1:1.

[0065] The process of constructing eukaryotic expression plasmids and the reagents or parameters used in each process are not limited. Any existing technology that can achieve the above purpose is acceptable and will not be elaborated here. Antibody purification is carried out according to the standard operating procedure for protein purification, and recombinant antibodies are obtained by expression and are still named 2F1 and 3E8.

[0066] Example 2: Assembly of CGRP Double Antibody Sandwich ELISA Kit High-purity capture antibody (clone 2F1) was diluted to the optimal concentration using a specific coating buffer (carbonate buffer, pH 9.6). 100 µL of the coating buffer was precisely added to each well of a microplate, and the plate was capped or sealed with a membrane. The plate was incubated overnight at 2–8 °C to allow the capture antibody to firmly adhere to the surface of the 96-well plate. The liquid in the wells was discarded, and the plate was washed three times with washing buffer (1×PBS-T) to remove unbound coating proteins. Blocking buffer (PBS containing 1%–5% BSA) was added to each well, and the plate was incubated for 1–2 hours to block unoccupied sites, preventing the adsorption of non-specific proteins in subsequent steps and reducing background signal. The plate was washed three more times with washing buffer following the same steps, and the liquid in the wells was thoroughly blotted dry. The plate was then vacuum-dried under sterile conditions. Finally, the dried plate was placed in an aluminum foil bag, desiccant was added, and the bag was vacuum-sealed.

[0067] Preparation of standards: Take the previously purified CGRP peptide and dilute it serially with preservation solution (1×PBS, 1% glycerol, 5% trehalose) to prepare 5-7 standards of different concentrations. Each concentration is aliquoted into EP tubes.

[0068] Biotinylation of the detection antibody: The purified detection antibody (clone number 3E8) was mixed with biotin solution at a molar ratio (biotin:antibody = 10:1) in a reaction buffer (pH 7.2-8.5, 0.1M borate). The mixture was incubated overnight at 2-8°C in the dark. After the reaction, the reaction solution was placed in a dialysis bag and dialyzed in a large amount of PBS (4°C) for 24-48 hours, with the buffer changed twice during the process. Unreacted biotin molecules were separated from the labeled antibody. BSA was added to the purified biotinylated detection antibody at a final concentration of 0.1% as a stabilizer to prevent antibody adsorption and degradation.

[0069] Determine the antibody concentration (usually recommended to be 1-2 mg / mL) and measure the volume accurately.

[0070] Enzyme conjugate dilution: Dilute the HRP-labeled streptavidin (purchased from Beyotime) to the working concentration using diluent.

[0071] Other reagent preparation: Prepare concentrated washing buffer (10×PBST), TMB colorimetric solution (purchased from Tiangen Biotech), stop solution (1M H2SO4), etc.

[0072] Example 3: Reagent Kit Performance Evaluation Standard curve establishment: CGRP standards were serially diluted using sample dilution buffer. Concentrations were 0 (blank well), 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 pg / mL. 100 µL of different concentrations of standard or test sample were added to each well, with two replicates per concentration. Incubation was performed at 37°C for 2 hours. The liquid was discarded, and the sample was washed five times thoroughly with PBST. Biotinylated detection antibodies were diluted to 0.5–1 µg / mL using dilution buffer, and 100 µL was added to each well. The sample was incubated at 37°C for 1–2 hours. The liquid was discarded, and the sample was washed five times with PBST. HRP-labeled streptavidin was diluted 1:5000 using dilution buffer, and 100 µL was added to each well. The sample was incubated at 37°C in the dark for 30 minutes. Discard the liquid, wash with PBST 5-6 times, and then perform color development. Add 100 µL of TMB substrate solution to each well and develop the color at room temperature (22-25℃) in the dark for 10 min. Add 50 µL of stop solution to each well to stop the reaction. Read the OD450nm value with a microplate reader (main wavelength 450nm, reference wavelength 620nm or 650nm to subtract interference from the plate background).

[0073] Table 3 Standard Product Test Results

[0074] From Table 3 and Figure 1 The linear range of the kit of this invention is 5.94 - 200 pg / mL. The limit of detection (LoD): LoD = Mean(Blank) + 3 × SD(Blank) = 0.0525. The concentration corresponding to OD = 0.0525 on the curve is 3.54 pg / mL. The limit of quantitation (LoQ): LoQ = Mean(Blank) + 10 × SD(Blank) = 0.07. The concentration corresponding to OD = 0.07 on the curve is 5.94 pg / mL. Precision: Three concentrations of quality control samples (high (200 pg / mL), medium (25 pg / mL), and low (6.25 pg / mL)) were tested, and the intra- and inter-segment coefficients of variation (CV) were calculated; all were less than 10%.

[0075] Example 4: Application in indicating migraine To verify the clinical applicability of the reagent kit of this invention, a clinical case-control study was completed: 1. Study subjects: Thirty patients with migraine without aura who met the ICHD-3 diagnostic criteria were recruited (15 males and 15 females, mean age 35 ± 8 years), and 30 age- and sex-matched healthy volunteers.

[0076] 2. Sample Collection: For the patient group, blood samples were collected at two separate visits: V1 within 4 hours of headache onset (attack period, pain severity ≥ 6 points on VAS score of moderate to severe), and V2 at least 72 hours later when there were no headache symptoms (intermittent period). Blood samples from the healthy control group were collected at a single visit. All blood samples were anticoagulated with EDTA, centrifuged at 3000 rpm for 15 minutes to separate plasma, and stored at -80°C until analysis.

[0077] 3. Detection method: Using the kit prepared in Example 2 of this invention, the collected samples were serially diluted (1:5, 1:10, 1:50, 1:100), with two replicates for each concentration, and the CGRP concentration in all serum / plasma samples was detected.

[0078] 4. Results: such as Figure 2 As shown, there were significant differences in plasma CGRP levels among the three groups. The median CGRP level in patients during an attack (85.0 pg / mL) was significantly higher than that in patients during their remission period (35.0 pg / mL) and the level in the healthy control group (18.5 pg / mL). All comparisons between groups were statistically significant using the Kruskal-Wallis test (attack period vs. remission period: p < 0.001; attack period vs. control group: p < 0.001; remission period vs. control group: p = 0.005).

[0079] Table 4 Summary of Data

[0080] like Figure 2 As shown, the plasma CGRP level in patients during a migraine attack (median: 93 pg / mL) was significantly higher than that in patients during an interictal period (median: 55.4 pg / mL) and in healthy controls (median: 22.9 pg / mL) (p<0.001). This indicates that this kit can effectively differentiate migraine disease states and can be used as an auxiliary diagnostic tool.

[0081] Further monitoring of CGRP levels in patients receiving CGRP monoclonal antibody therapy before and after treatment revealed a significant decrease in CGRP levels after treatment, which was positively correlated with the reduction in the frequency of headaches recorded in headache diaries. This indicates that this kit can be used for efficacy monitoring.

[0082] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Anti-calcitonin gene-related peptide antibodies, characterized in that, The anti-Calcitonin Gene-Related Peptide antibody comprises a light chain variable region having light chain CDRs consisting of CDR L1, CDR L2, CDR L3, and a heavy chain variable region having heavy chain CDRs consisting of CDR H1, CDR H2, CDR H3, wherein the amino acid sequences of the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, CDR H3 are as shown in any one of (A)~(B): (A) SEQ ID NO: 3~5, SEQ ID NO: 7~9; (B) SEQ ID NO: 11~13, SEQ ID NO: 15~17.

2. The antibody of claim 1, wherein The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in any one of (C)~(D): (C) SEQ ID NO: 2, SEQ ID NO: 6; (D) SEQ ID NO: 10, SEQ ID NO:

14.

3. A gene encoding the anti-CGRP antibody of claim 1 or 2.

4. A recombinant vector carrying the gene of claim 3.

5. A recombinant cell expressing the anti-CGRP antibody of claim 1 or 2, or containing the gene of claim 3, or transformed with the recombinant vector of claim 4.

6. A method of producing an anti-Calcitonin Gene-Related Peptide antibody, characterized by, The method is to culture the recombinant cell of claim 5 to obtain a culture containing the anti-CGRP antibody of claim 1 or 2.

7. A product marked biologically or chemically, characterized in that, The product is an antibody labeled with a marker, and the antibody is derived from any one of the following: (1) the anti-CGRP antibody of claim 1 or 2; (2) the culture of the recombinant cell of claim 6.

8. A kit characterized in that, The kit contains the anti-CGRP antibody of claim 1 or 2 or the product labeled with a biological marker or a chemical marker of claim 7.

9. The kit of claim 8, wherein The kit comprises a microplate coated with a capture antibody, an enzyme-labeled detection antibody, a CGRP standard, a sample diluent, a washing solution, a color developing substrate, and a stop solution; the capture antibody is an anti-CGRP antibody with a heavy chain variable region amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region amino acid sequence shown in SEQ ID NO: 6, and the detection antibody is an anti-CGRP antibody with a light chain variable region amino acid sequence shown in SEQ ID NO: 10 and a heavy chain variable region amino acid sequence shown in SEQ ID NO:

14.

10. The kit of claim 9, wherein The preparation method of the microplate coated with the capture antibody is as follows: (1) Coating: dilute the capture antibody to an optimal concentration with a carbonate coating buffer (pH 9.6), add 100-200 μL per well to the microplate, and incubate at 2-8°C overnight; (2) Washing and blocking: discard the coating solution, wash the microplate 3-5 times with a washing solution, and then add a PBS solution containing 1-5% BSA or 5% skimmed milk powder for blocking, 200-300 μL per well, and incubate at 37°C for 1-2 hours; (3) Drying and packaging: discard the blocking solution, wash and dry the microplate, vacuum seal the microplate in an aluminum foil bag, and add a desiccant.

11. The kit of claim 9, wherein The preparation method of the detection antibody label is as follows: use the sodium periodate method to couple HRP with the purified detection antibody, and obtain the enzyme-labeled detection antibody working solution after purification.

12. Use of the anti-Calcitonin Gene Related Peptide antibody of claim 1 or 2, or the gene of claim 3, or the expression recombinant vector of claim 4, or the recombinant cell of claim 5, or the method of claim 6, or the product of biological marker or chemical marker of claim 7 in the preparation of a product for detecting Calcitonin Gene Related Peptide.

13. Use of the anti-Calcitonin Gene Related Peptide antibody of claim 1 or 2, or the gene of claim 3, or the expression recombinant vector of claim 4, or the recombinant cell of claim 5, or the method of claim 6, or the product of biological marker or chemical marker of claim 7 in the preparation of a product for diagnosing migraine.

14. Use according to claim 12 or 13, characterized in that, The product includes a reagent, a kit, a detection chip or a biosensor.

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