Calcitonin gene related peptide specific antigen epitope peptide, antigen, antibody and kit

Abstract

The invention belongs to the field of biology, and discloses a calcitonin gene related peptide specific epitope peptide, an antigen, an antibody and a kit. The antigen epitope peptide is at least one of an antigen epitope peptide I with an amino acid sequence as shown in SEQ ID NO.1 and an antigen epitope peptide II with an amino acid sequence as shown in SEQ ID NO.2. An antibody obtained by immunizing an animal with an antigen obtained by using the antigen epitope peptide is used for qualitatively or quantitatively detecting the calcitonin gene-related peptide. The kit containing the antibody has good diagnostic sensitivity, specificity and repeatability, and can be used for preparing products for diagnosing migraine.

Description

Technical Field

[0001] This invention belongs to the field of biology, and specifically relates to a calcitonin gene-related peptide specific antigenic epitope peptide, antigen, antibody and kit. Background Technology

[0002] Migraine is a common neurovascular disorder characterized by unilateral throbbing pain in the head. Severe cases may experience nausea, vomiting, and even anxiety and fear due to recurrent headaches. During a migraine attack, activation of the trigeminal nerve leads to the release of vasoactive neuropeptides, particularly calcitonin gene-related peptide (CGRP), from its presynaptic nerve endings. CGRP, as a neurotransmitter, plays a crucial regulatory role in pain transmission, vasodilation, and inflammatory responses.

[0003] In recent years, the role of CGRP in migraine has received considerable attention. It induces vasodilation and neurogenic inflammation of the pia mater and extracranial vessels, thereby causing the typical pulsatile migraine. Electrophysiological and imaging studies of evoked and reflex responses during migraine have revealed potential changes in extensive neural networks within the central nervous system (CNS), including the cerebral cortex, brainstem, hypothalamus, and thalamus. The peripheral and central portions of the trigeminal nerve system, which transmits headache signals to the brain, are also affected.

[0004] Migraine is defined as a headache lasting 4–72 hours and characterized by at least two of the following features: unilateral localization, throbbing, moderate to severe pain intensity, and motor aggravation. In addition, the headache must be accompanied by at least one of the following two symptoms: nausea and / or vomiting, or photophobia and phonophobia. However, many migraine sufferers experience chronic daily headaches, occurring at least 15 days a month, often accompanied by migraine-like features. A prodromal phase almost always precedes a migraine, which can last for several hours before the onset of the headache. The most common reported symptoms are fatigue, gastrointestinal problems, and mood changes, which can persist throughout the migraine attack. Approximately 20% of migraine sufferers also experience a prodromal phase, including visual, sensory, or motor disturbances. The headache is often accompanied by a recovery or syndrome phase, characterized by fatigue and persistent sensory disturbances. In summary, migraine sufferers typically experience a prolonged period of incapacitation before, during, and after the headache.

[0005] With advancements in migraine research both domestically and internationally, particularly the discovery of novel therapeutic targets such as calcitonin gene-related peptide (CGRP), migraine treatment methods are constantly being updated. Therefore, establishing standardized clinical diagnostic and treatment procedures for migraines and updating treatment concepts are of great significance for the accurate diagnosis, assessment, and effective treatment of the disease.

[0006] Although there are preliminary studies on the use of CGRP in the auxiliary diagnosis and treatment of migraine, the lack of an objective gold standard for migraine diagnosis in clinical practice makes clinical trials difficult, and there is currently no kit for measuring calcitonin gene-related peptide for migraine diagnosis. Therefore, there is a need in this field to develop a kit that can measure the concentration level of CGRP in human plasma to overcome the shortcomings of subjective diagnosis of migraine in clinical practice, and to provide a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring, and efficacy evaluation of anti-migraine drugs. Summary of the Invention

[0007] To address the problems existing in the prior art, the primary objective of this invention is to provide a calcitonin gene-related peptide-specific antigenic epitope peptide.

[0008] Another object of the present invention is to provide an antigen containing the above-mentioned calcitonin gene-related peptide-specific antigenic epitope peptide.

[0009] Another object of the present invention is to provide an antibody prepared using the above-described antigen.

[0010] Another object of the present invention is to provide a kit containing the above-mentioned antibody, which can be used to detect calcitonin gene-related peptide.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A calcitonin gene-related peptide-specific antigenic epitope peptide, comprising at least one of antigenic epitope peptide I with an amino acid sequence as shown in SEQ ID NO.1 and antigenic epitope peptide II with an amino acid sequence as shown in SEQ ID NO.2.

[0013] The amino acid sequence of the antigenic epitope peptide I is shown below (SEQ ID NO.1): KRCVTHRLAGLLSRSG.

[0014] The amino acid sequence of the antigenic epitope peptide II is shown below (SEQ ID NO.2): CKNNFVPTNV.

[0015] An antigen containing the above-mentioned calcitonin gene-related peptide-specific antigenic epitope peptide can be used to immunize animals to obtain antibodies that specifically bind to the calcitonin gene-related peptide.

[0016] The antigen is preferably obtained by conjugating the above-mentioned calcitonin gene-related peptide-specific antigenic epitope peptide with a carrier protein.

[0017] The carrier protein is preferably keyhole hemocyanin.

[0018] An antibody that specifically binds to calcitonin gene-related peptide is a monoclonal or polyclonal antibody, obtained by immunizing animals with the above-mentioned antigen according to the preparation method of polyclonal or monoclonal antibodies. It has the activity of binding to calcitonin gene-related peptide, and can therefore be used for qualitative or quantitative detection of calcitonin gene-related peptide.

[0019] A calcitonin gene-related peptide (CGRP) assay kit containing the aforementioned antibody.

[0020] The calcitonin gene-related peptide assay kit further comprises at least one of a solid-phase carrier, a marker, a calcitonin gene-related peptide quality control, and a calcitonin gene-related peptide calibrator.

[0021] The solid support is preferably a microporous reaction plate or magnetic microspheres.

[0022] The preferred marker is horseradish peroxidase (HRP) or alkaline phosphatase (AP).

[0023] The horseradish peroxidase HRP described herein is used for enzyme-linked immunosorbent assay (ELISA).

[0024] The alkaline phosphatase AP is used for chemiluminescent immunoassay (CLIA).

[0025] The calcitonin gene-related peptide quality control product and the calcitonin gene-related peptide calibrator are diluted recombinant or extracted calcitonin gene-related peptides.

[0026] The above-mentioned calcitonin gene-related peptide assay kit is a chemiluminescent immunoassay kit, which is developed using the principle of chemiluminescent immunoassay (CLIA) combined with the double antibody sandwich method. It contains magnetic microspheres directly or indirectly linked to the antibody to form a magnetic separation reagent, or contains magnetic microspheres directly or indirectly linked to the antibody to form a magnetic separation reagent and a luminescent substrate.

[0027] The magnetic separation reagent is preferably an immunomagnetic bead coated with the above-mentioned antibody or obtained by linking the above-mentioned antibody to magnetic bead microspheres via streptavidin.

[0028] The luminescent substrate is preferably luminol, isoluminol and its derivatives or (goldenon)-1,2-dioxane and its derivatives.

[0029] The kit contains magnetic separation reagent, AP-labeled CGRP polyclonal antibody, CGRP quality control, CGRP calibrator, washing solution, and chemiluminescent substrate.

[0030] The above-mentioned antibody or calcitonin gene-related peptide assay kit that can specifically bind to calcitonin gene-related peptide is used in the preparation of products for diagnosing migraines.

[0031] When the kit of the present invention is used to prepare a product for diagnosing migraine, the concentration of CGRP in the subject sample measured by the kit is compared with the control reference range (i.e., the concentration level of healthy subjects). If the concentration of CGRP in the subject sample is higher than that in the control reference range, the subject is at risk of developing migraine, and the severity of the condition is positively correlated with the concentration level of CGRP in the sample.

[0032] The concentrations of CGRP in patient samples before and after treatment were compared using the kit. If the concentration of CGRP in the patient samples remained unchanged or increased with the progress of treatment, it indicated that the drug treatment was ineffective. If the concentration of CGRP in the patient samples decreased with the progress of treatment, it indicated that the drug treatment was effective.

[0033] In chemiluminescence immunoassay, this method offers high detection sensitivity (limit of detection is 10 pg / mL), a wide linear range of 3-6 orders of magnitude, and the ability to directly measure samples with concentrations up to 1000 pg / mL without dilution. It also boasts high accuracy (96.54% recovery rate in recovery tests), high precision (CV < 10%), and a short reaction time (results available in 15 minutes), significantly improving detection efficiency. Furthermore, it is highly automated, requiring no manual operation and reducing human error.

[0034] Compared with the prior art, the present invention has the following advantages and positive effects:

[0035] 1. This invention applies CGRP as a biomarker to the preparation of migraine auxiliary diagnostic or efficacy evaluation kits, overcoming the shortcomings of the indicators currently used in the clinical diagnosis of migraine being too subjective and lacking objectivity, and providing a more accurate and reliable objective indicator for the auxiliary diagnosis, disease monitoring and drug efficacy evaluation of migraine.

[0036] 2. The two CGRP-specific antigenic epitope peptides screened in this invention are characterized by hydrophilicity, strong antigenicity, and ease of synthesis. The antigens (immunogens) prepared from them can produce highly specific polyclonal antibodies when used to immunize animals.

[0037] 3. The CGRP-specific polyclonal antibody prepared in this invention can bind to CGRP in the sample with high specificity.

[0038] 4. The reagent kit prepared by this invention should have good diagnostic sensitivity, specificity and reproducibility, and meet the needs of clinical application. Attached Figure Description

[0039] Figure 1 The results of detecting plasma CGRP levels in healthy controls and untreated migraine patients using the kit prepared for Example 3 are shown in the figure.

[0040] Figure 2 The ROC curve of the kit prepared in Example 3 for detecting the effect of plasma CGRP on migraine diagnosis in subjects (without medication).

[0041] Figure 3 The results of detecting plasma CGRP levels in healthy controls and patients with migraines treated with medication using the kit prepared for Example 3 are shown in the figure.

[0042] Figure 4 The ROC curve of the kit prepared in Example 3 for detecting the effect of plasma CGRP on migraine diagnosis in subjects (medication recipients).

[0043] Figure 5 A graph showing plasma CGRP levels in healthy controls, patients with migraines treated with medication, and patients without medication.

[0044] Figure 6 ROC curves for CGRP in subjects diagnosed with migraine with and without medication. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for the purposes of this invention only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0046] Example 1: Synthesis of CGRP-specific antigenic epitope peptides I and II

[0047] 1. Raw materials used:

[0048] HMP resin (P-hydroxymethylphenoxymethyl polyethylene resin, Sigma-Aldrich); Fmoc-AA (9-fluorenylmethoxycarbonyl-protected amino acid, Merck); NMP (N-methylpyrrolidone, Sigma-Aldrich); DCM (dichloromethane, Zhongyuan Chemical Co., Ltd.); MeOH (methanol, Zhongyuan Chemical Co., Ltd.); Piperidine (piperidine, Sigma-Aldrich); DMAP (dimethylaminopyridine, Sigma-Aldrich); HOBT (hydroxybenzotriazole, Sigma-Aldrich); DCC (dicyclohexylcarbodiimide, Sigma-Aldrich); TFA (trifluoroacetic acid, Sigma-Aldrich); EDT (1,2-ethylenedithiol, Sigma-Aldrich); thioanisole, Guangzhou Weber Chemical Co., Ltd.; crystalline phenol, Sinopharm Chemical Reagent Co., Ltd.; acetonitrile, Sinopharm Chemical Reagent Co., Ltd.

[0049] 2. Instruments used:

[0050] Automated peptide synthesizer, model 431A, ABI Corporation; rotary evaporator, model R-201, Shanghai Shenshun Company; high performance liquid chromatograph, Waters 600, Waters Corporation, USA; freeze dryer, model VFD-2000, Beijing Boyikang Company.

[0051] 3. Synthesis method and process:

[0052] Weigh 100 mg of HMP resin (equivalent to 1.0 mEq), or 0.1 mmol, and place it in the reaction chamber of an ABI 431A automated peptide synthesizer. The synthesizer automatically links specific amino acids in different sequences, achieving a coupling rate of 99%. The reaction is as follows:

[0053] (1) Activation of amino acids (HOBt / DCC method);

[0054] (2) Connecting amino acids to the resin;

[0055] (3) Remove the Fmoc protecting group from amino acids;

[0056] (4) Activation of another amino acid (HOBt / DCC method);

[0057] (5) Coupled to amino acids already bound to the resin;

[0058] (6) Repeat steps (3) to (5) until the synthesis is complete, and obtain peptide resins with the following amino acids respectively;

[0059] Lys-Arg-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly;

[0060] Cys-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val.

[0061] (7) Peptide cleavage resin:

[0062] The peptide chain was cleaved with TFA (trifluoroacetic acid), and EDT (2.5 vol%) and thioanisole (2.5 vol%) were used as scavenging agents. The reaction was carried out at room temperature for 3.0 hours. After removing the cleavage reagent, the peptides were extracted with diethyl ether to obtain crude CGRP antigen epitope peptides I and II, respectively.

[0063] 4. Purification of crude CGRP antigenic epitope peptides I and II:

[0064] High-performance liquid chromatography (HPLC) was used for separation and purification: the chromatographic column was a C8 10×100 mm, which could be purchased from Waters Corporation, USA; the chromatograph was a Waters 600, also from Waters Corporation, USA; the mobile phase was: phase A was 0.1% v / v TFA (trifluoroacetic acid) aqueous solution, and phase B was 0.1% TFA (trifluoroacetic acid) 60% acetonitrile solution; the detection wavelength was 214 nm, the flow rate was 4 mL / min, the elution gradient was 20-60% B, and the reaction time was 30 min.

[0065] HPLC (High Performance Liquid Chromatography) analysis: The chromatographic column was a C18 4.6×150mm, available from Waters Corporation, USA; the mobile phase was: Phase A was 0.1% TFA (trifluoroacetic acid) aqueous solution, and Phase B was 0.1% TFA (trifluoroacetic acid) acetonitrile solution; the detection wavelength was 214nm, the flow rate was 1 mL / min, the elution gradient was 0-60% B, and the reaction time was 30 min. The eluent was freeze-dried to obtain CGRP-specific antigenic epitope peptide lyophilized powder.

[0066] Peptide analysis results show that the purity of CGRP antigenic epitope peptides I and II of the present invention is both above 95%.

[0067] Example 2: Preparation of CGRP-specific antibodies

[0068] 1. Preparation of antigens:

[0069] CGRP specific antigenic epitope peptides I and II are conjugated to the carrier protein KLH (keyhole hemocyanin) using the BDB (Bis-diazotized benzidine dichloride) method to prepare CGRP antigen I or II. The specific steps are as follows:

[0070] Dissolve 20.0 mg of CGRP-specific epitope peptide I or II in 0.2 M borate buffer (pH 9.0). Take 7.36 mL of a 6.25 mg / mL KLH solution, cool to 0°C, add 1 mL of bisphenylamine (BDB) to the solution, and mix in an ice-water mixture protected from light. React on a shaker for 1–1.5 h. After the reaction is complete, adjust the pH to 9.0 with 0.2 mol / L NaOH solution. Dialyze overnight using an MD44 dialysis bag, then aliquot and store at -20°C. The borate buffer is prepared by mixing 80 mL of a 0.05 mol / L borax solution and 20 mL of a 0.2 mol / L boric acid solution.

[0071] 2. Preparation of polyclonal antibodies by immunizing animals:

[0072] 3.1. Three-month-old New Zealand white rabbits weighing approximately 2 kg (purchased from Guangdong Provincial Medical Laboratory Animal Center) were selected as immunization animals. For the primary immunization, the prepared CGRP antigen I or II (immunogen) was mixed with an equal volume of Freund's complete adjuvant, thoroughly emulsified, and then injected subcutaneously at multiple sites on the rabbit's back, with each rabbit receiving 1 mL (antigen content 1-2 mg). The resulting adjuvant, obtained by thoroughly emulsifying the antigen with incomplete Freund's adjuvant, was used for booster immunization. Booster immunizations were administered every 2-3 weeks, with 1 mL / rabbit injected subcutaneously at multiple sites on the back, for a total of three booster immunizations. Ten days after the last booster immunization, blood was excised from the carotid artery, plasma was separated, and serum was collected.

[0073] 3.2. Antibody titer determination: The titer of CGRP polyclonal antibody I or II in serum was determined by indirect ELISA. The results showed that the titers of CGRP polyclonal antibody I or II were both above 1:32000.

[0074] 3.3 Blood collection and serum separation: Blood was collected by carotid artery catheterization and serum was separated.

[0075] 4. Isolation and purification of antibodies:

[0076] Serum containing CGRP polyclonal antibody I or II, to be loaded, is first dialyzed with a equilibration buffer (pH 8.0, 0.02M PB) until the pH is consistent. The column is then loaded into an ion exchange chromatography column and connected to the protein chromatography system, also washed with the equilibration buffer until pH 8.0. For sample loading, the serum containing CGRP polyclonal antibody I or II to be purified is added to the column. After all the sample has been injected, it is washed with an elution buffer (pH 8.0, 0.05M PB) until the protein analyzer shows the beginning of elution. Protein solution collection begins when the peak declines gradually. Collection is stopped when the peak becomes gradual. The concentration of CGRP polyclonal antibody I or II is measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision Technology). The purified CGRP polyclonal antibody I or II is then aliquoted and stored at -20℃.

[0077] Example 3: Preparation of a chemiluminescent reagent kit for calcitonin gene-related peptides

[0078] 1. Preparation of magnetic separation reagent (working solution for CGRP-coated polyclonal antibody I immunomagnetic beads):

[0079] (1) Washing of magnetic beads: Take 1 mL of 0.1 M MES (pH 6.0) buffer into the coated tube, add 2 mg of JSR magnetic bead stock solution (Shenzhen Ruisi Biotechnology Co., Ltd.), and vortex mix for 1 min. Place the coated tube on a magnetic separator for 1 min and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex mix for 1 min. Repeat the above process twice.

[0080] (2) Activation of magnetic beads: Add 800 μL of 0.1M MES (pH 6.0) buffer to the coated tube, then add 100 μL of NHS (10 mg / L) solution and vortex mix for 1 min. Add 100 μL of EDC (10 mg / L) solution to the coated tube. Place the rotary mixer in a constant temperature environment of 25±1℃, place the coated tube on the rotary mixer, set the rotary mixer speed to 50±1 rpm, and react for 30 minutes.

[0081] (3) Coating CGRP polyclonal antibody I with activated magnetic beads: Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 12–16 μL of CGRP polyclonal antibody I (10 mg / mL) to the coating tube and vortex for 1 min. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coating tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 2 hours.

[0082] (4) Magnetic bead blocking: After placing the coated tube on the magnetic separator for 1 min, discard the supernatant and add 1 mL of 0.05 M TRIS (pH 7.4) buffer solution to the coated tube and vortex mix. Repeat the operation twice. Place the rotary mixer in a constant temperature environment of 25±1℃, place the coated tube on the rotary mixer, set the rotary mixer speed to 50±1 rpm, and react for 30 minutes.

[0083] (5) Cleaning: After sealing, place the coated tube on a magnetic separator for 1 min and discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min.

[0084] (6) Preparation of magnetic separation reagent: The magnetic beads coated with CGRP polyclonal antibody I were diluted with enzyme-labeled dilution buffer and 0.05 MTTRIS (pH 7.4) to obtain a final concentration of 0.2 mg / mL.

[0085] 2. Preparation of enzyme-labeled antibody (CGRP polyclonal antibody II labeled with alkaline phosphatase):

[0086] (1) Take 250 μL of 0.1M MES (pH4.5) buffer and soak the ultrafiltration centrifuge tube (30KD) for 2 min.

[0087] (2) Add 250 μL of 0.1M MES (pH 4.5) buffer to the centrifuge column to bring the volume to 500 μL.

[0088] (3) Add 12.5 μL of AP enzyme to an ultrafiltration centrifuge tube, mix well, centrifuge at 13000 rpm for 15 min and then discard the waste liquid.

[0089] (4) Add 200 μL of 0.1M MES (pH 4.5) to the ultrafiltration centrifuge tube, then centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0090] (5) Add 50 μL EDC (10 mg / mL) and 6 μL NHS (10 mg / mL) to the ultrafiltration centrifuge tube, then add 100 μL 0.1M MES (pH 4.5) to make up to 250 μL and mix well. Place on a shaker to activate for 1.5 hours.

[0091] (6) Take 10 μL of CGRP polyclonal antibody II 10 mg / L, mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0092] (7) Add 250 μL of 0.1 M PB (pH 9.0), mix well, centrifuge at 13000 rpm for 20 min and discard the waste liquid.

[0093] (8) Repeat the experimental steps (6) once.

[0094] (9) Add 0.1M PB (pH 9.0) to a final volume of 250μL and place on a shaker at 120rpm for 2h.

[0095] (10) Add 250 μL of 0.05 M TRIS (pH 8.0) and mix well. Place on a shaker at 120 rpm for 30 min.

[0096] Take out about 500 μL of the coupled solution from the centrifuge column, then add an equal volume of glycerol, mix well, and store at -20°C.

[0097] The concentration of alkaline phosphatase-labeled antibody was measured and calculated using an L5S UV-Vis spectrophotometer (Boda Precision Technology). The antibody was then diluted 1:1000 by volume with 0.05M TRIS (pH 7.4) enzyme-labeled dilution buffer to obtain the enzyme-labeled antibody.

[0098] 3. Preparation of washing solution: It consists of 10mM PBS (pH 7.2), 0.08% v / v Tween-20 and 0.03% v / v Proclin-300.

[0099] 4. Preparation of calibrators / quality control samples for calcitonin gene-related peptide: Calcitonin gene-related peptide was serially diluted with calibrator diluent (containing 10mM, pH 7.2 phosphate-buffered saline PBS, 1% w / v BSA, and 0.03% v / v Proclin-300 biological preservative) to prepare a series of calibrators (0-1000 pg / mL) and two quality control samples of high and low concentrations.

[0100] 5. Preparation of luminescent substrate: It consists of 0.01% w / v (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacrylamide) disodium phosphate (APS-5) and 0.3M Tris buffer containing 0.0003% w / v gloss extract, 0.001% w / v sodium sulfite, 0.1% w / v sodium dodecyl sulfate (SDS), and 0.03% w / v Tween 20.

[0101] 6. Kit Composition: The kit mainly consists of reagent compartments (Reagent 1, Reagent 2), calcitonin gene-related peptide calibrator (1.0mL×1 vial), quality control sample (1.0mL×1 vial), substrate solution (12.0mL / 25.0mL), concentrated wash buffer (25×) (60.0mL×1 vial / 60.0mL×2 vials), and calibration / quality control information cards. Reagent 1 in the reagent compartment contains magnetic beads coated with CGRP polyclonal antibody I, and Reagent 2 contains CGRP polyclonal antibody II labeled with alkaline phosphatase AP. The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0102] Example 4: Preparation of a chemiluminescent reagent kit for calcitonin gene-related peptides

[0103] 1. Preparation of immunomagnetic beads coated with monoclonal antibodies

[0104] (1) Washing of magnetic beads: Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube, add 2 mg of JSR magnetic bead stock solution (Shenzhen Ruisi Biotechnology Co., Ltd.), and vortex for 1 min. Place the coated tube on a magnetic separator for 1 min and discard the supernatant. Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coated tube and vortex for 1 min. Repeat the above process twice.

[0105] (2) Activation of magnetic beads: Add 800 μL of 0.1M MES (pH 6.0) buffer to the coated tube, then add 100 μL of NHS (10 mg / L) solution and vortex mix for 1 min. Add 100 μL of EDC (10 mg / L) solution to the coated tube. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coated tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 30 min.

[0106] (3) Coating CGRP monoclonal antibody with activated magnetic beads: Add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Add 50-100 μL of CGRP monoclonal antibody (C9487, purchased from Shanghai Xibao Company) to the coating tube. Vortex for 1 min. At room temperature, add 1 mL of 0.1 M MES (pH 6.0) buffer to the coating tube and vortex for 1 min. Place the rotary mixer in a constant temperature environment of 25 ± 1 °C, place the coating tube on the rotary mixer, set the rotary mixer speed to 50 ± 1 rpm, and react for 2 hours.

[0107] (4) Magnetic bead blocking: After placing the coated tube on the magnetic separator for 1 min, discard the supernatant and add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex mix. Repeat the operation twice. Place the rotary mixer in a constant temperature environment of 25±1℃, place the coated tube on the rotary mixer, set the rotary mixer speed to 50±1 rpm, and react for 30 minutes.

[0108] (5) Cleaning: After sealing, place the coated tube on a magnetic separator for 1 min and discard the supernatant. Add 1 mL of 0.05 M TRIS (pH 7.4) buffer to the coated tube and vortex for 1 min. Repeat this operation twice. Add 1 mL of 0.05 M TRIS (pH 7.4) to the coated tube and vortex for 1 min.

[0109] (6) Preparation of magnetic separation reagent: The magnetic beads coated with CGRP monoclonal antibody were diluted with enzyme-labeled dilution buffer and 0.05 MTTRIS (pH 7.4) to obtain a final concentration of 0.2 mg / mL.

[0110] 2. Preparation of enzyme-labeled antibody (CGRP polyclonal antibody II labeled with alkaline phosphatase): Same as in Example 3.

[0111] 3. Preparation of washing solution: Same as in Example 3.

[0112] 4. Preparation of calibrators / quality control products for calcitonin gene-related peptides: Same as in Example 3.

[0113] 5. Preparation of luminescent substrate: Same as in Example 3.

[0114] 6. Kit Composition: The kit mainly consists of reagent compartments (Reagent 1, Reagent 2), calcitonin gene-related peptide calibrator (1.0mL×1 vial), quality control sample (1.0mL×1 vial), substrate solution (12.0mL / 25.0mL), concentrated wash buffer (25×) (60.0mL×1 vial / 60.0mL×2 vials), and calibration / quality control information cards. Reagent 1 in the reagent compartment contains magnetic beads coated with CGRP monoclonal antibody, and Reagent 2 contains CGRP polyclonal antibody II labeled with alkaline phosphatase AP. The kit is available in two packaging specifications: 50 doses / box and 100 doses / box.

[0115] Example 5: Preparation of a calcitonin gene-related peptide ELISA assay kit

[0116] 1. Preparation of various buffer solutions and reagents:

[0117] 1.1 Coating buffer: 0.05M, pH 9.6, CB (carbonate buffer): Na2CO3 16.0 g, NaHCO3 29.0 g, deionized water to a final volume of 1000 mL.

[0118] 1.2, pH 7.2, 10×PBS / Tween-20: 58 g Na2HPO4·12H2O, 4 g KH2PO4, 100 g NaCl, 4 g KCl, 20 mL Tween-20, and deionized water to a final volume of 1000 mL.

[0119] 1.3 Blocking buffer / antibody dilution buffer: 10×PBS / Tween-20 100mL, BSA (bovine plasma albumin) 10g, biological preservative (Proclin-300, purchased from Shanghai Xibao Company) 1mL, deionized water to a final volume of 1000mL.

[0120] 1.4 Enzyme label dilution buffer: 10×PBS / Tween-20 10 mL, FCS (fetal calf plasma) 20 mL, enzyme stabilizer (purchased from Shanghai Xibao Company, model ACE0070A) 1 g, biological preservative (Proclin-300, purchased from Shanghai Xibao Company) 1 mL, and deionized water to a final volume of 1000 mL.

[0121] 1.5. Color reagent A: 35.5 g citric acid, 10 g urea peroxide, 10 mL Tween-20, and deionized water to a final volume of 1000 mL.

[0122] 1.6 Color reagent B: 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, and deionized water to a final volume of 1000 mL.

[0123] 1.7 Concentrated washing solution (pH 7.2, 25×PBS / Tween-20): 145 g Na2HPO4·12H2O, 10 g KH2PO4, 250 g NaCl, 10 g KCl, 50 mL Tween-20, and dilute to 1000 mL with deionized water.

[0124] 1.8 Termination solution (2M H2SO4): 22.2 mL concentrated sulfuric acid (95-98%) and 177.3 mL deionized water. When preparing, slowly add the concentrated sulfuric acid dropwise to the deionized water and dilute to the middle while shaking.

[0125] 2. Preparation of pre-coated plates: Dissolve CGRP polyclonal antibody I in coating buffer to prepare a pre-coating solution. Add 100 μL of 0.1 μg / well to each well of an enzyme-labeled coated plate (purchased from Shenzhen Jincanhua Co., Ltd.). Incubate at 4℃ for 18-24 hours. Remove the plate, discard the coating solution, wash the plate, add 100 μL of blocking buffer to each well, and block at 4℃ for 16 hours. Discard the blocking buffer, air dry the plate, pack it into an aluminum foil bag, vacuum seal it, and store it at 4℃.

[0126] 3. Preparation of conjugated antibody and enzyme label: The conjugated antibody (CGRP polyclonal antibody II) and the enzyme label (horseradish peroxidase-labeled goat anti-rabbit IgG antibody, purchased from Beijing Zhongshan Jinqiao Company) were diluted with antibody diluent to the working concentration, which was determined by a checkerboard titration experiment.

[0127] 4. Preparation of CGRP calibrators and quality control samples: Dilute recombinant CGRP with sample diluent to prepare CGRP calibrators (0-100 pg / mL) and two quality control samples with high and low concentrations.

[0128] 5. ELISA kit composition: The kit mainly consists of pre-coated plates (48 or 96 test samples), 1 set of CGRP calibrators, CGRP quality control samples (high and low concentrations), binding antibody (10 mL), enzyme label (10 mL), chromogenic solution A (5 mL), chromogenic solution B (5 mL), concentrated washing buffer (20 mL), and stop solution (5 mL).

[0129] Application Example 1: Determination of CGRP using the chemiluminescence reagent kit prepared in Example 3 and validation of kit performance.

[0130] 1. Plasma sample: Fresh plasma samples are preferred. They can be stored for 7 days at 2℃-8℃ and for one year at -20℃. When using the sample, remove it from the refrigerator, let it stand at room temperature, and then mix it well.

[0131] 2. The chemiluminescent immunoassay (CLIA) kit is used to determine CGRP concentration. The specific steps are divided into detection and calibration procedures, as follows:

[0132] Testing procedure:

[0133] (1) Concentrated washing solution (25×): Dilute the concentrated washing solution 25 times to the cleaning concentration to be used. For a 50-person batch, bring the volume to 1.5L, and for a 100-person batch, bring the volume to 3.0L.

[0134] (2) When loading the kit onto the chemiluminescence analyzer for the first time, the magnetic beads in reagent 1 in the reagent compartment need to be mixed thoroughly to resuspend any magnetic particles that may have settled during storage and transportation.

[0135] (3) After the calibrator is taken out from 2℃~8℃, it should be equilibrated to room temperature. Before use, gently turn it over and mix it. After use, tighten the cap and put the calibrator back into storage at 2℃~8℃.

[0136] (4) Perform the calibration procedure: Place the calibrator in the sample rack, manually scan the reagent information in the reagent compartment, the main curve information on the calibration / quality control information card, and the QR code information of the calibrator, click calibration, select the location to place the calibrator, and the instrument will automatically perform the calibration procedure.

[0137] (5) Check the sample volume in the sample cup to ensure that the sample volume in the sample cup is above 300 μL before each test. If a blood collection tube is used, the liquid volume of the sample should be sufficient and not less than 300 μL.

[0138] (6) Sample injection: For information on sample injection, please refer to the instruction manual for the chemiluminescence analyzer.

[0139] (7) Click the instrument run button. The luminescence analyzer will perform the pre-set operation and detection (total detection time is about 25 minutes).

[0140] Calibration procedure:

[0141] (1) When using a new batch of reagents, the standard curve data must be entered and then calibration is performed; when the chemiluminescence analyzer is calibrated, each calibrator will be tested twice. After the test is successful, the sample can be tested directly without repeated calibration.

[0142] (2) Calibrator A and calibrator B need to be replicated for calibration experiments;

[0143] (3) Conduct the experiment according to the operation manual of the luminescence analyzer.

[0144] 3. Reagent kit performance verification

[0145] 3.1 Linearity Range Validation: Samples close to the upper limit of the kit's linear range were serially diluted with calibrator diluent. Each diluted concentration was measured twice, and the average concentration (yi) was calculated. A linear regression was performed with the dilution ratio (xi) as the independent variable and the corresponding average concentration (yi) as the dependent variable. The linear correlation coefficient (r) was calculated. The test results are shown in the table below:

[0146] Table 1. Linearity range test results and calculation units: pg / mL

[0147]

[0148] The test results show that the linear correlation coefficient of the kit in Example 3 can reach 0.9975 in the range of 0 to 1000 pg / mL, indicating that the kit has a wide linear range.

[0149] 3.2. Limit of detection: The zero-concentration reference sample was tested 20 times using the kit of Example 3. The average (M) and standard deviation (SD) of the 20 tests were calculated. The limit of detection was then calculated as M + 2SD.

[0150] The test results showed that the limit of detection was 15.625 pg / mL, indicating that the kit has high sensitivity.

[0151] 3.3 Precision: Precision was validated on samples at two concentration levels, high and low. Each concentration was tested 10 times using the kit from Example 3. The two concentrations were 40 pg / mL and 900 pg / mL, respectively.

[0152] Calculate the average of 10 measurements for each concentration sample. And standard deviation SD, according to CV=SD / The coefficient of variation (CV) was calculated by multiplying the result by 100%, and the precision of the kit was evaluated. The test results are shown in the table below:

[0153] Table 2 Precision Test Results

[0154]

[0155] Test results show that the CV% of the kit in Example 3 was less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0156] 3.4 Accuracy: The accuracy of the reagent kit was evaluated using a recovery test.

[0157] A high-concentration sample A is added to a low-concentration sample B, with a volume ratio of 1:9 between the added sample A and sample B. The concentrations of sample B and the mixed sample are then measured, and the recovery rate is calculated using the following formula.

[0158] ;

[0159] In the formula:

[0160] R - Recovery rate; V - Volume of sample A added; V0 - Volume of sample B; C - Detection concentration of sample B after adding sample A; C0 - Detection concentration of sample B; CS - The concentration of sample A.

[0161] The recovery rate was calculated to be 97.32%, indicating that the kit has high accuracy.

[0162] Verification conclusion:

[0163] The verification results of Application Example 1 show that the kit of Example 3 has a wide linear range, can directly measure samples with concentrations up to 1000 pg / mL without dilution, has high sensitivity (limit of detection is 15.625 pg / mL), high accuracy (recovery rate is 97.32%), good precision (CV% for high and low concentrations are 4.12% and 0.85%, respectively), and the entire reaction time is short, with results available in 30 minutes, which greatly improves the detection efficiency.

[0164] Application Example 2: Determination of CGRP using the chemiluminescence reagent kit prepared in Example 4 and validation of kit performance.

[0165] 1. Plasma sample: Same as application example 1.

[0166] 2. The chemiluminescent immunoassay (CLIA) kit is used to determine the CGRP concentration. The specific steps are the same as in application example 1.

[0167] 3. Reagent kit performance verification

[0168] 3.1 Linear Range Verification: Same as Application Example 1. The test results are shown in the table below:

[0169] Table 3. Linearity range test results and calculation units: pg / mL

[0170]

[0171] The test results show that the linear correlation coefficient of the kit in Example 4 can reach 0.9958 in the range of 0 to 1000 pg / mL, indicating that the kit has a wide linear range.

[0172] 3.2. Minimum detection limit: The verification method is the same as in Application Example 1.

[0173] The test results showed that the limit of detection was 30.125 pg / mL, indicating that the kit has high sensitivity.

[0174] 3.3 Precision: The verification method is the same as in Application Example 1. The two concentrations used for precision verification were 50 pg / mL and 800 pg / mL. The test results are shown in the table below:

[0175] Table 4 Precision Test Results

[0176]

[0177] Test results show that the CV% of the kit in Example 4 is less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0178] 3.4 Accuracy: The verification method is the same as in Application Example 1. The calculated recovery rate was 96.11%, indicating that the kit has high accuracy.

[0179] Verification conclusion:

[0180] The verification results of Application Example 2 show that the kit of Example 4 has a wide linear range, can directly measure samples with concentrations up to 1000 pg / mL without dilution, has high sensitivity (limit of detection is 30.125 pg / mL), high accuracy (recovery rate of 96.11%), good precision (CV% of high and low concentrations are 9.69% and 2.82%, respectively), and the entire reaction time is short, with results available in 30 minutes, which greatly improves the detection efficiency.

[0181] Application Example 3: Determination of CGRP and Performance Validation of the Enzyme-Linked Immunosorbent Assay (ELISA) Kit Prepared in Example 5

[0182] 1. Sample collection: Same as in Application Example 1.

[0183] 2. The enzyme-linked immunosorbent assay (ELISA) kit was used to determine the CGRP concentration. The specific steps are as follows:

[0184] (1) Preparation of washing solution: 25× concentrated washing solution is diluted with deionized water at a ratio of 1:25;

[0185] (2) Take out the kit prepared in Example 4 and bring it to room temperature. Prepare the samples, calibrators, and quality control materials. Dilute the clinical samples with sample diluent at a ratio of 1:20.

[0186] (3) Add 50 μL of diluted test sample / calibrator / quality control to each well of the microplate, then add 50 μL of binding antibody to the corresponding well, gently pat to mix, seal the microplate with sealing film, and incubate at 37°C for 30 minutes.

[0187] (4) Remove the reaction plate, discard the liquid in the plate, add 200-300 μL of washing solution to each well and wash the plate 5 times, then pat dry;

[0188] (5) Add two drops or 100 μL of enzyme-labeled material to each well, seal the plate with sealing film, and incubate at 37°C for 30 minutes;

[0189] (6) Remove the reaction plate, discard the liquid in the plate, add 200-300 μL of washing solution to each well and wash the plate 5 times, then pat dry;

[0190] (7) Add 50 μL of colorimetric reagent A and B solution to each well, mix thoroughly, and incubate at 37°C for 15 minutes;

[0191] (8) Add 1 drop (50 μL) of stop solution to each well as soon as possible and gently tap to mix;

[0192] (9) Measure the OD value of each well using an ELISA reader (with dual wavelengths of 450 / 630 nm);

[0193] (10) Plot the calibration curve and calculate the CGRP concentration in the sample based on the calibration curve.

[0194] 3. Reagent kit performance verification

[0195] 3.1 Linearity Range Verification: The verification method is the same as in Application Example 1. The test results are shown in the table below:

[0196] Table 5. Linearity range test results and calculation units: pg / mL

[0197]

[0198] Test results show that the kit in Example 5 has a linear correlation coefficient of 0.9923 in the range of 1.56 to 100 pg / mL.

[0199] 3.2 Limit of Detection: The verification method is the same as in Application Example 1. Test results show that the limit of detection is 2 pg / mL, indicating that the kit has high sensitivity.

[0200] 3.2 Precision: The verification method is the same as in Application Example 1. The two concentrations used for precision verification were 10 pg / mL and 70 pg / mL. The test results are shown in the table below:

[0201] Table 6 Precision Test Results

[0202]

[0203] Test results show that the CV% of the kit in Example 5 was less than 10% for both high and low concentration samples, indicating that the kit has high precision.

[0204] 3.4 Accuracy: The verification method is the same as in Application Example 1. The calculated recovery rate was 95.12%, indicating that the kit has high accuracy.

[0205] Verification conclusion:

[0206] The validation results of Example 3 show that the kit of Example 5 exhibits good linearity (up to 0.9923) in the range of 0–100 pg / mL, high sensitivity (limit of detection is 2 pg / mL), recovery rate of 95.12%, high accuracy, and good precision (CV% of high and low concentrations are 4.99% and 5.68%, respectively). It does not require complex equipment, can be used for batch detection, and can simultaneously produce more than 80 test results in 1.5 hours, demonstrating high detection efficiency.

[0207] Test Comparison Example 1:

[0208] GY-L0020 monoclonal antibody and GY-L5120 CGRP antibody (purchased from Shanghai Guyan Industrial Co., Ltd.) were used to prepare a chemiluminescence reagent kit (comparative kit) according to the method in Example 3. The analytical performance of the kit was compared with that of the kit in Example 3. The results of the performance comparison are shown in the table below:

[0209] Table 7 Detection results of different reagent kits

[0210]

[0211] In the comparison of various analytical performance indicators, the performance of the kit in Example 3 was better than that of the control kit in most aspects.

[0212] Application Example 4: CGRP assay kit for auxiliary diagnosis and disease monitoring of migraine.

[0213] Study participants were selected based on the International Classification of Headache Disorders, 3rd Revision (ICHD-3), aged 18-60 years, regardless of gender; the duration of previous attacks was used to determine the onset of the disease, with a timeframe of 5 minutes to 72 hours after the onset of the attack; head CT, head MRI, and head MRA were used to exclude intracranial organic lesions; all participants voluntarily participated in this study, signed informed consent, and completed a questionnaire.

[0214] The untreated migraine group included 46 patients with episodic cluster headache (eCH), 23 of whom were male and 23 of whom were female, with an average age of (34.3±15.2) years; and 52 patients with chronic cluster headache (cCH), 25 of whom were male and 27 of whom were female, with an average age of (35.4±14.9) years.

[0215] The medication-treated migraine group consisted of 40 patients with episodic cluster headache (eCH-1), including 21 males and 19 females, with an average age of (33.7±16.2) years; and 46 patients with chronic cluster headache (cCH-1), including 23 males and 23 females, with an average age of (34.4±12.7) years.

[0216] Healthy control group (Con): 63 healthy individuals underwent physical examination, including 30 males and 33 females, with an average age of (34.3±15.2) years.

[0217] 2. Experimental Methods

[0218] 2.1 The CGRP kit prepared in Example 3 of this invention was used to test plasma samples from the enrolled healthy control group, the untreated migraine group, and the treated migraine group.

[0219] The dosage of the medication was administered according to the drug instructions. After treatment, plasma samples were collected and tested using a CGRP kit. Plasma CGRP levels before and after treatment were compared, and ROC curves were generated to analyze the value of plasma CGRP in evaluating the efficacy of migraine treatment.

[0220] 3. Results

[0221] (1) Plasma CGRP levels in migraine patients and control group

[0222] like Figure 1 As shown, plasma CGRP levels during an attack were different between the migraine group (eCH, no medication) and the chronic migraine group (cCH, no medication), but the difference was not statistically significant (69.52±7.75 pg / mL during an attack, 61.06±4.34 pg / mL during chronic migraine). Compared with the control group (Con), plasma CGRP levels during attacks and in both the migraine and chronic migraine groups were significantly higher than those in the control group (69.52±7.75 pg / mL during an attack, 61.06±4.34 pg / mL during chronic migraine, and 10.05±1.06 pg / mL in the control group).

[0223] Plot the operating curve for subjects (without medication) diagnosed with migraine, such as... Figure 2 The area under the ROC curve (AUC) for diagnosis was 0.97 (95% CI = 0.94 ± 1.0), P < 0.001. This indicates that plasma CGRP has good value in the auxiliary diagnosis of migraine (69.52 ± 7.75 pg / mL during an attack, and 61.06 ± 4.34 pg / mL for chronic migraine).

[0224] (2) Plasma CGRP levels in migraine patients and the control group after medication

[0225] like Figure 3 The plasma CGRP levels in the episodic migraine group (eCH-1) and the chronic migraine patients (cCH-1) during the 48-hour medication period were not significantly different. However, the plasma CGRP levels in both the episodic migraine group and the chronic migraine patients were significantly higher than those in the control group (52.78±6.07 pg / mL during episodic migraine, 50.37±3.65 pg / mL for chronic migraine, and 10.05±1.06 pg / mL for the control group).

[0226] Plotting the subject (medication) operating curve for migraine diagnosis as follows: Figure 4 The area under the ROC curve (AUC) for diagnosis was 0.96 (95% CI = 0.92–1.0), P < 0.001. This indicates that plasma CGRP has good value in the auxiliary diagnosis of migraine (52.78 ± 6.07 pg / mL, chronic migraine 50.37 ± 3.65 pg / mL).

[0227] 3. Plasma CGRP levels in the untreated migraine group and the treated migraine group

[0228] like Figure 5 As shown, the plasma CGRP levels in the paroxysmal migraine group and the chronic migraine patients during the 48-hour medication period were significantly lower than those in the unmedicated paroxysmal migraine group and the chronic migraine patients, indicating that plasma CGRP has good value in the auxiliary diagnosis of migraine (unmedicated paroxysmal migraine 69.52±7.75pg / mL, unmedicated chronic migraine 61.06±4.34pg / mL, medication-treated paroxysmal migraine 52.78±6.07pg / mL, medication-treated chronic migraine 50.37±3.65pg / mL, control group 10.05±1.06pg / mL).

[0229] Plotting curves for all subjects diagnosed with migraines, as shown in the figure. Figure 6 The area under the ROC curve (AUC) for diagnosis was 0.96 (95% CI = 0.93–1.0), P < 0.001. This indicates that plasma CGRP has good value in the auxiliary diagnosis of migraine (69.52 ± 7.75 pg / mL in the untreated acute phase, 61.06 ± 4.34 pg / mL in the untreated chronic phase, 52.78 ± 6.07 pg / mL in the treated acute phase, 50.37 ± 3.65 pg / mL in the treated chronic phase, and 10.05 ± 1.06 pg / mL in the control group).

[0230] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A calcitonin gene-related peptide-specific antigenic epitope peptide, characterized in that: The calcitonin gene-related peptide-specific antigenic epitope peptide is at least one of antigenic epitope peptide I with an amino acid sequence as shown in SEQ ID NO.1 and antigenic epitope peptide II with an amino acid sequence as shown in SEQ ID NO.

2.

2. An antigen, characterized in that: The calcitonin gene-related peptide-specific antigenic epitope peptide as described in claim 1 can be used to immunize animals.

3. The antigen according to claim 2, characterized in that: The antigen is obtained by coupling the calcitonin gene-related peptide specific antigenic epitope peptide of claim 1 with a carrier protein; the carrier protein is further keyhole hemocyanin.

4. An antibody that specifically binds to calcitonin gene-related peptide, characterized in that: The antibody that specifically binds to the calcitonin gene-related peptide is a monoclonal antibody or a polyclonal antibody, obtained by immunizing animals with the antigen described in claim 2 or 3 according to the preparation method of polyclonal antibody or monoclonal antibody.

5. A kit for the assay of calcitonin gene-related peptide, characterized in that: The antibody comprising the one described in claim 4 that specifically binds to the calcitonin gene-related peptide.

6. The calcitonin gene-related peptide assay kit according to claim 5, characterized in that: It also includes at least one of a solid-phase carrier, a marker, a calcitonin gene-related peptide calibrator, and a calcitonin gene-related peptide quality control.

7. The calcitonin gene-related peptide assay kit according to claim 6, characterized in that: The solid support is a microporous reaction plate or magnetic microspheres; The markers are horseradish peroxidase (HRP) or alkaline phosphatase (AP). The calcitonin gene-related peptide calibrator is a diluted recombinant or extracted calcitonin gene-related peptide; The calcitonin gene-related peptide quality control product is a diluted recombinant or extracted calcitonin gene-related peptide.

8. The calcitonin gene-related peptide assay kit according to any one of claims 5-7, characterized in that: The kit is a chemiluminescent immunoassay kit, comprising magnetic microspheres directly or indirectly linked to the antibody of claim 4 to form a magnetic separation reagent, or comprising magnetic microspheres directly or indirectly linked to the antibody of claim 4 to form a magnetic separation reagent and a luminescent substrate.

9. The calcitonin gene-related peptide assay kit according to claim 8, characterized in that: The magnetic separation reagent is either an immunomagnetic bead coated with the antibody of claim 4 or obtained by linking the antibody of claim 4 to magnetic microspheres via streptavidin; The luminescent substrate is luminol, isoluminol and its derivatives or (goldenon)-1,2-dioxane and its derivatives.

10. The use of the antibody that specifically binds to calcitonin gene-related peptide as described in claim 4 or the calcitonin gene-related peptide assay kit as described in any one of claims 5 to 9 in the preparation of a product for diagnosing migraine.

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