Application of ON detection reagent in auxiliary diagnosis of acute chest pain disease

Abstract

The invention belongs to the field of biological detection, and particularly relates to application of an ON detection reagent in auxiliary diagnosis of acute chest pain diseases. It is found that the ON protein is closely related to acute chest pain such as AAD, AMI and APE, the ON serves as a marker, a detection reagent of the ON is prepared into an auxiliary diagnosis product, and great clinical value and practical significance are achieved for early diagnosis and long-term prognosis of the three diseases (especially AAD) and improvement of life quality.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to the application of ON detection reagent in auxiliary diagnosis of acute chest pain disease. Background Art

[0002] Acute chest pain is one of the most common clinical symptoms. It is a group of heterogeneous diseases with chest pain as the main manifestation. Acute chest pain has many causes, the severity of the disease varies greatly, and the risks are different. Although non-cardiac chest pain accounts for the majority of acute chest pain, due to the high risk of cardiac chest pain, especially life-threatening and fatal chest pain such as acute aortic dissection (AAD), acute myocardial infarction (AMI), and acute pulmonary embolism (APE), it is the focus of rapid assessment of the condition in the emergency department. Among them, although the incidence of AAD and APE is low, the condition is critical, progresses rapidly, and is easily missed and misdiagnosed clinically, with a very high mortality rate. Therefore, for patients with acute chest pain, it is necessary to enter the rescue process immediately after a clear diagnosis in the emergency department. Rapid treatment helps to improve the patient's survival rate and prognosis.

[0003] With the promotion of chest pain center construction, chest pain diagnosis and treatment are becoming more standardized, but misdiagnosis and missed diagnosis still occur from time to time. Therefore, it is particularly important to quickly assess the condition, accurately differentiate and treat scientifically, and then efficiently treat and reasonably divert patients with chest pain. At present, patients with acute chest pain are mainly diagnosed and differentiated based on medical history, physical examination and auxiliary examination results. Imaging examinations such as electrocardiogram and chest X-ray, computed tomography imaging and coronary angiography can help to clarify the diagnosis. However, there are differences in emergency diagnosis and treatment capabilities in different regions, especially in grassroots hospitals in remote areas, which do not have perfect imaging examination conditions, which to a certain extent limits the differential diagnosis of acute chest pain. For patients with acute chest pain, rapid laboratory testing of cardiovascular biomarkers is one of the important tools for emergency diagnosis and treatment, which helps to quickly diagnose patients with early chest pain and improve the ability to treat acute chest pain. Commonly used myocardial injury markers include cardiac troponin (cTn), creatine kinase isoenzymes MB (CK-MB) and myoglobin (MYO), which play an important role in the early diagnosis and evaluation of acute coronary syndrome. High-sensitivity cTn (hs-cTn) is more sensitive to AMI and is often used for early screening and exclusion of AMI. The increase in D-dimer levels reflects the activation of the coagulation system and fibrinolytic system in plasma. Clinically, D-dimer is regarded as a marker of hypercoagulable state and hyperfibrinolysis in the body and can be used as the preferred screening indicator for the diagnosis of APE. D-dimer detection can also be used for screening and exclusion of AAD, but due to the low D-dimer values ​​in the plasma of young patients and patients with short tear length and pseudoembolism, misdiagnosis may occur, and clinical application has certain limitations.

[0004] Therefore, seeking new biomarkers for assisting the diagnosis of acute chest pain, especially biomarkers that can be used to assist in the diagnosis of multiple types of acute chest pain, is of great significance for clinical diagnosis. Summary of the invention

[0005] The first object of the present invention is to provide an application of an ON (Osteonectin) detection reagent in the preparation of a kit, wherein the kit is used to assist in the diagnosis of acute chest pain diseases, and the acute chest pain diseases include at least one of AAD, AMI and APE.

[0006] Furthermore, the present invention also provides a method for assisting in the diagnosis of acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE, and the method comprises: detecting the ON content in the patient's serum.

[0007] Furthermore, the present invention also provides a method for assisting in distinguishing acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE, and the method comprises: detecting the ON content in the patient's serum.

[0008] Furthermore, the present invention also proposes an auxiliary diagnosis product for realizing the application, which contains a reagent for detecting the ON content in serum.

[0009] The present invention finds that ON protein is closely related to acute chest pain such as AAD, AMI and APE. Using ON as a marker and preparing its detection reagent into an auxiliary diagnosis product has great clinical value and practical significance for the early diagnosis, long-term prognosis and improvement of the quality of life of the above three diseases (especially AAD). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 It is the ON expression level in the serum of patients in the AAD group, AMI group and APE group in the embodiment of the present invention.

[0012] Figure 2 This is the subject operating characteristic curve of ON as a biomarker for diagnosing AAD in patients with acute chest pain in an embodiment of the present invention. DETAILED DESCRIPTION

[0013] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0014] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0015] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0016] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0017] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0018] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0019] The “%” involved in the present invention, unless otherwise specified, refers to mass or volume percentage (Mass / Volume Percent, abbreviated as m / v%). Specifically, when the solute is a solid, “%” refers to the percentage of the mass (g) of the solute in the solution to the total volume (L) of the solution; when the solute is a liquid, “%” refers to the percentage of the volume (L) of the solute in the solution to the total volume (L) of the liquid.

[0020] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0021] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0022] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0023] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0024] In the present invention, the term "ON" refers to Osteonectin, also known as osteonectin or Bm-40, which is a matrix cell secretory protein composed of three domains.

[0025] In the present invention, the term "auxiliary diagnosis" refers to the process of medical diagnosis, in which various auxiliary tools and means are combined with the doctor's clinical judgment to improve the accuracy and reliability of diagnosis. In specific implementation, other auxiliary tools may include imaging examinations (such as X-rays, CT, MRI), laboratory tests (such as blood, biochemistry, immunity, etc.), pathological examinations, etc. The main purpose of auxiliary diagnosis is to support and supplement the doctor's clinical diagnosis, help determine the nature, extent and development stage of the disease, and then guide subsequent treatment plans.

[0026] In the present invention, the term "acute myocardial infarction (AMI)" refers to a disease in which myocardial ischemia and necrosis are caused by a sudden interruption of coronary blood flow. Typical symptoms include chest pain (usually squeezing pain), dyspnea, sweating, nausea, vomiting, etc.

[0027] In the present invention, the term "acute aortic dissection (AAD)" refers to a tear in the aortic intima, blood entering the middle layer of the aortic wall, and forming a dissection. Typical symptoms include severe chest pain or back pain, and the pain may move as the dissection expands.

[0028] In the present invention, the term "acute pulmonary embolism (APE)" refers to the blockage of the pulmonary artery or its branches by thrombus or other substances, resulting in pulmonary circulation disorder. Typical symptoms include dyspnea, chest pain, hemoptysis, syncope, etc.

[0029] In the present invention, the term "Streptavidin-Phycoerythrin (SA-PE)" is a binding protein that combines strong-affinity streptavidin and fluorescent phycoerythrin. It is widely used in techniques such as flow cytometry and fluorescence microscopy, and can effectively label and detect cells or molecules, especially in immunology and biomarker research. Its efficient binding and sensitive fluorescence properties make SA-PE a key tool in biomedical research.

[0030] The present invention first provides an application of an ON detection reagent in preparing a kit, wherein the kit is used for assisting in the diagnosis of acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE.

[0031] The present invention finds that ON protein is closely related to acute chest pain such as AAD, AMI and APE. Using ON as a marker and preparing its detection reagent into a detection kit can assist in the diagnosis of the above three diseases, especially AAD, which has great clinical value and practical significance for its early diagnosis, long-term prognosis and improvement of quality of life.

[0032] In some embodiments, the kit is used to assist in the diagnosis of one of AAD, AMI and APE. For example, the kit is used to assist in the diagnosis of AAD.

[0033] In some embodiments, the kit is used to assist in identifying at least one of AAD, AMI, and APE from patients with acute chest pain.

[0034] In some embodiments, the kit is used to assist in identifying patients with acute aortic dissection from patients with acute chest pain.

[0035] In a specific embodiment, the detection reagent is a reagent for detecting ON content in blood.

[0036] In some preferred embodiments, the detection reagent is a reagent for detecting ON content in serum. The present invention finds that different concentration ranges are present in the serum of patients with three critical chest pain diseases, namely AAD, AMI and APE. Detecting ON content in serum will further help to accurately distinguish the above three diseases.

[0037] In some embodiments, when the ON content in the serum sample to be tested is 6.34-20.14 ng / mL, more preferably 12.75±9.78 ng / mL, the patient to be tested is judged to have a risk of AAD; when the ON content in the serum sample to be tested is 13.25-83.69 ng / mL, more preferably 42.31±33.04 ng / mL, the patient to be tested is judged to have a risk of AMI; when the ON content in the serum sample to be tested is 35.50-99.30 ng / mL, more preferably 62.26±27.33 ng / mL, the patient to be tested is judged to have a risk of APE. Through the study of serum samples of relevant patients, the present invention found that the ON content in the serum samples of the vast majority (at least 80% or more) of patients with corresponding symptoms falls within the above corresponding range, so the range value can be used to assist in judging the above three diseases.

[0038] Furthermore, the present invention also provides a method for assisting in the diagnosis of acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE, and the method comprises: detecting the ON content in the patient's serum.

[0039] Furthermore, the present invention also provides a method for assisting in distinguishing acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE, and the method comprises: detecting the ON content in the patient's serum.

[0040] The above further limitation on the application scheme also applies to the method for assisting in diagnosing acute chest pain diseases and the method for assisting in distinguishing acute chest pain diseases.

[0041] Furthermore, the present invention also provides an auxiliary diagnosis product for realizing the application, which contains a reagent for detecting the ON content in serum.

[0042] In some specific embodiments, the auxiliary diagnostic product may be a reagent or a kit, etc. As an example, the auxiliary diagnostic product may be an enzyme-linked immunosorbent assay (ELISA) kit, an immunofluorescence detection product, a flow cytometry reagent, a protein chip, etc.

[0043] In some embodiments, the reagents include an antibody pair for specifically binding ON, wherein the antibody pair includes a capture antibody coupled to magnetic fluorescent microspheres and a biotinylated detection antibody.

[0044] In some embodiments, the capture antibody and the detection antibody are both human ON antibodies.

[0045] In some preferred embodiments, the capture antibody and the detection antibody are respectively selected from two different antibodies selected from antibody 1, antibody 2, antibody 3, antibody 4, antibody 5, antibody 6, antibody 7, and antibody 8; the light chain variable region sequence of antibody 1 is shown as SEQ ID No.2, and the heavy chain variable region sequence is shown as SEQ ID No.4; preferably, the full-length light chain sequence of antibody 1 is shown as SEQ ID No.1, and the full-length heavy chain sequence is shown as SEQ ID No.3; the light chain variable region sequence of antibody 2 is shown as SEQ ID No.6, and the heavy chain variable region sequence is shown as SEQ ID No.8; preferably, the full-length light chain sequence of antibody 2 is shown as SEQ ID No.5, and the full-length heavy chain sequence is shown as SEQ ID No.7; the light chain variable region sequence of antibody 3 is shown as SEQ ID No.10, and the heavy chain variable region sequence is shown as SEQ ID No.12; preferably, the full-length light chain sequence of antibody 3 is shown as SEQ ID No.9, and the full-length heavy chain sequence is shown as SEQ ID No.11; the light chain variable region sequence of antibody 4 is shown as SEQ ID No. ID No.14, and the heavy chain variable region sequence is shown in SEQ ID No.16; preferably, the full-length light chain sequence of antibody 4 is shown in SEQ ID No.13, and the full-length heavy chain sequence is shown in SEQ ID No.15; the light chain variable region sequence of antibody 5 is shown in SEQ ID No.18, and the heavy chain variable region sequence is shown in SEQ ID No.20; preferably, the full-length light chain sequence of antibody 5 is shown in SEQ ID No.17, and the full-length heavy chain sequence is shown in SEQ ID No.19; the light chain variable region sequence of antibody 6 is shown in SEQ ID No.22, and the heavy chain variable region sequence is shown in SEQ ID No.24; preferably, the full-length light chain sequence of antibody 6 is shown in SEQ ID No.21, and the full-length heavy chain sequence is shown in SEQ ID No.23; the light chain variable region sequence of antibody 7 is shown in SEQ ID No.26, and the heavy chain variable region sequence is shown in SEQ ID No.28; preferably, the full-length light chain sequence of antibody 7 is shown in SEQ ID No.25, and the full-length heavy chain sequence is shown in SEQ ID No.27; the light chain variable region sequence of antibody 8 is shown in SEQ ID No.30, and the heavy chain variable region sequence is shown in SEQ ID No.32; preferably, the full-length light chain sequence of antibody 8 is shown in SEQ ID No.29, and the full-length heavy chain sequence is shown in SEQ ID No.31.

[0046] The present invention further found that the above antibody pairs can be used to better detect ON.

[0047] In some further preferred embodiments, the antibody pair is selected from any one of the following antibody pairs 1 to 9:

[0048]

[0049] In some embodiments, the detection antibody is used at a concentration of 200 to 1000 ng / mL, more preferably 250 ± 50 ng / mL. As an example, the detection antibody may be used at a concentration of 200 ng / mL, 225 ng / mL, 250 ng / mL, 275 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL, or any value between 200 and 1000 ng / mL. In specific implementation, those skilled in the art may directly configure the detection antibody solution in the kit, or may configure the solid detection antibody and the detection antibody diluent, and provide the recommended concentration of the detection antibody in the accompanying instructions.

[0050] In some embodiments, the reagents further include ON standards. In specific embodiments, those skilled in the art can select commercially available ON proteins as standards based on common sense.

[0051] In some preferred embodiments, the amino acid sequence of the ON standard is shown as SEQ ID No.33.

[0052] In some preferred embodiments, the ON standard is selected from at least one of protein 1 and protein 2 (the amino acid sequences of which are both shown in SEQ ID No. 33); wherein the protein 1 is RP00217 purchased from Wuhan ABclonal Biotechnology Co., Ltd.; and the protein 2 is RP01973 purchased from Wuhan ABclonal Biotechnology Co., Ltd. In specific implementation, it is more preferred that the ON standard is protein 1.

[0053] In some embodiments, the reagent includes the protein 1 and the antibody pair 5; or includes the protein 1 and the antibody pair 6. The above combination has a better signal-to-noise ratio in detection.

[0054] In some embodiments, the reagent further includes Streptavidin-Fluorescein (SA-FITC).

[0055] In a specific embodiment, those skilled in the art can select commercially available streptavidin-phycoerythrin (SAPE) in combination with common sense, which can realize the detection of ON in the present invention. In some specific embodiments, the streptavidin-fluorescein is streptavidin-phycoerythrin (SAPE).

[0056] In some preferred embodiments, the streptavidin-phycoerythrin is selected from at least one of the following SAPE 1 to SAPE 6, more preferably SAPE 2; wherein SAPE 1 is S866 purchased from Thermo Fisher Scientific; SAPE 2 is 12-4317-87 purchased from Thermo Fisher Scientific; SAPE 3 is S21388 purchased from Thermo Fisher Scientific; SAPE 4 is L600-001 purchased from Shanghai Laya Biotech Co., Ltd.; SAPE 5 is LF600-001 purchased from Shanghai Laya Biotech Co., Ltd.; SAPE 6 is S406169 purchased from Aladdin Biochemical Technology Co., Ltd.

[0057] In some preferred embodiments, the concentration of streptavidin-phycoerythrin is 25 to 2000 ng / mL, more preferably 100 ± 50 ng / mL. As an example, the concentration of streptavidin-phycoerythrin can be 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 125 ng / mL, 150 ng / mL, 200 ng / mL, 400 ng / mL, 800 ng / mL, 1000 ng / mL, 1500 ng / mL, 200 ng / mL, or any value of 25 to 2000 ng / mL. In specific implementation, those skilled in the art can directly configure the SAPE solution in the kit, or can configure the solid SAPE and SAPE diluent, and provide the recommended concentration of SAPE in the accompanying instructions.

[0058] In some embodiments, the reagent further comprises a sample diluent. In a specific embodiment, a person skilled in the art can identify the sample diluent in combination with common sense, which can realize the detection of ON in the present invention.

[0059] In some preferred embodiments, the sample diluent contains: 30±5 mM PBS, 0.25±0.05% BSA, 0.1±0.05% sucrose, 0.25±0.05% PEG6000, and 0.05±0.02% Proclin300, and the pH value is 7.4±0.2.

[0060] In a specific implementation, those skilled in the art can combine the above-mentioned solutions with common sense to obtain a preferred embodiment of the auxiliary diagnostic product of the present invention.

[0061] Furthermore, the present invention also provides an auxiliary diagnosis method, which comprises: using the auxiliary diagnosis product.

[0062] When specifying the implementation method, those skilled in the art can determine the specific method of using the auxiliary diagnostic product based on common sense.

[0063] In some specific embodiments, the auxiliary diagnosis method includes: mixing the serum sample to be tested and the magnetic fluorescent microsphere suspension bound to the capture antibody, and the ON in the serum sample to be tested specifically binds to the capture antibody on the magnetic fluorescent microsphere; then adding the biotinylated detection antibody, and specifically binding the detection antibody to the reaction product of the first step; then adding the streptavidin-phycoerythrin solution, and binding the streptavidin to the biotin on the detection antibody, and finally forming a "capture antibody on the surface of the magnetic fluorescent microsphere-antigen (ON)-biotinylated detection antibody-streptavidin-phycoerythrin" complex. When using a flow cytometer to detect it, read the fluorescence signal intensity value of the magnetic fluorescent microsphere and the corresponding coated antibody, and different microsphere complexes can be identified as specific analyte types and analyze the corresponding concentrations to achieve quantitative detection of ON in the sample. The method of using the kit is a high-throughput, low-consumption, low-cost detection method with comprehensive detection, high specificity, and good sensitivity, which can improve detection efficiency.

[0064] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0065] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0066] Example 1: Using magnetic fluorescent microspheres to couple anti-human ON antibodies

[0067] Activation of magnetic fluorescent microspheres: Take 100 μL of uniformly mixed magnetic fluorescent microspheres (10 610 μL of 50mg / mL NHS (N-hydroxysuccinimide ester) solution (prepared with buffer 1) and vortexed, 10 μL of 50mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) solution (prepared with buffer 1) and vortexed, mixed on a rotator at room temperature for 20 minutes, magnetic separation and discarding the supernatant, adding buffer 2 and mixing evenly, and magnetic separation and discarding the supernatant. Add 500 μL of buffer 2 and mix evenly, add 5 μg of human ON antibody and vortexed, mix on a rotator at room temperature for 2h, magnetic separation and discarding the supernatant, wash twice with 500 μL of 0.01M pH7.4 PBS-T buffer, and magnetic separation and discarding the supernatant.

[0068] Microsphere blocking: Add 1 mL of blocking solution and mix well, then mix well on a rotator at room temperature for 2 hours. Discard the supernatant after magnetic separation, and wash twice with 500 μL of 0.01M pH7.4 PBST buffer. Discard the supernatant after magnetic separation, add 100 μL of preservation solution, mix well, and store at 2-8°C in the dark for later use.

[0069] Buffer 1: The buffer reagent is acetate buffer with a concentration of 50 mM and a pH of 5.0.

[0070] Buffer 2: The buffer reagent is borate buffer with a concentration of 100 mM and a pH of 6.8.

[0071] Blocking solution: PBS solution containing 0.5% BSA 0.01M pH 8.0.

[0072] Storage solution: PBST solution containing 0.5% BSA 0.01M pH 8.0.

[0073] Example 2: Biotinylated anti-human ON antibody

[0074] Take 50μg of ON antibody, add it to 200μL labeling buffer and mix well, then prepare Sulfo-NHS-LC-Biotin to 10mM with labeling buffer, take 10μL and add it to the ON antibody solution, then mix well on a rotator at room temperature for 120 minutes. Use salt exchange buffer to ultrafilter three times, add biotinylated antibody storage solution to the antibody concentration of 0.25mg / mL, and store at -20℃ for later use.

[0075] Labeling buffer: Use borate buffer with a concentration of 50 mM and a pH of 7.8.

[0076] Salt exchange buffer: Use TBS with a concentration of 10mM and a pH of 8.0.

[0077] Biotinylated antibody storage solution: PBS solution with a concentration of 10 mM and a pH value of 7.4 containing 40% glycerol.

[0078] Example 3: Effect of Antibodies on

[0079] Antibody description: In Table 1, the full-length sequence of the light chain of the antibody 1 is shown in SEQ ID No.1, the sequence of the light chain variable region is shown in SEQ ID No.2, the full-length sequence of the heavy chain is shown in SEQ ID No.3, and the sequence of the heavy chain variable region is shown in SEQ ID No.4; the full-length sequence of the light chain of the antibody 2 is shown in SEQ ID No.5, the sequence of the light chain variable region is shown in SEQ ID No.6, the full-length sequence of the heavy chain is shown in SEQ ID No.7, and the sequence of the heavy chain variable region is shown in SEQ ID No.8; the full-length sequence of the light chain of the antibody 3 is shown in SEQ ID No.9, the sequence of the light chain variable region is shown in SEQ ID No.10, the full-length sequence of the heavy chain is shown in SEQ ID No.11, and the sequence of the heavy chain variable region is shown in SEQ ID No.12; the full-length sequence of the light chain of the antibody 4 is shown in SEQ ID No.13, the sequence of the light chain variable region is shown in SEQ ID No.14, the full-length sequence of the heavy chain is shown in SEQ ID No.15, and the sequence of the heavy chain variable region is shown in SEQ ID The full-length sequence of the light chain of the antibody 5 is shown in SEQ ID No.17, the sequence of the light chain variable region is shown in SEQ ID No.18, the full-length sequence of the heavy chain is shown in SEQ ID No.19, and the sequence of the heavy chain variable region is shown in SEQ ID No.20; the full-length sequence of the light chain of the antibody 6 is shown in SEQ ID No.21, the sequence of the light chain variable region is shown in SEQ ID No.22, the full-length sequence of the heavy chain is shown in SEQ ID No.23, and the sequence of the heavy chain variable region is shown in SEQ ID No.24; the full-length sequence of the light chain of the antibody 7 is shown in SEQ ID No.25, the sequence of the light chain variable region is shown in SEQ ID No.26, the full-length sequence of the heavy chain is shown in SEQ ID No.27, and the sequence of the heavy chain variable region is shown in SEQ ID No.28; the full-length sequence of the light chain of the antibody 8 is shown in SEQ ID No.29, the sequence of the light chain variable region is shown in SEQ ID No.30, the full-length sequence of the heavy chain is shown in SEQ ID No.31, and the sequence of the heavy chain variable region is shown in SEQ ID No.32.

[0080] Preparation of coupled microsphere working solution (capture antibody): The magnetic fluorescent microspheres coupled with ON antibody were diluted with the preservation solution in Example 1 at a dilution factor of 200 times.

[0081] Preparation of detection antibody working solution: dilute with detection antibody diluent, the dilution multiple is 250 times.

[0082] Preparation of Streptavidin-R-Phycoerythrin (SAPE) working solution: dilute 500 times with SAPE diluent.

[0083] Detection antibody diluent: HEPES was used with a concentration of 10 mM and a pH of 7.4.

[0084] SAPE diluent: a PBS solution containing 10 mM glucose and a pH value of 6.5, wherein the glucose content is 0.02%.

[0085] Experimental process: 20 μL of capture antibody was added to each well of a 96-well polystyrene plate, and then 20 μL of clinical sample was added. Incubated in a thermostatic oscillator at 37°C and 1200 rpm for 45 min. Placed on a magnetic plate for 2 min and discarded the supernatant. Added 100 μL PBS-T and washed in a thermostatic oscillator at 37°C and 1200 rpm for 1 min. Placed on a magnetic plate for 2 min and discarded the supernatant. Then, 50 μL of detection antibody working solution was added to the well and incubated in a thermostatic oscillator at 37°C and 1200 rpm for 30 min. Placed on a magnetic plate for 2 min and discarded the supernatant. Added 100 μL PBS-T and washed in a thermostatic oscillator at 37°C and 1200 rpm for 1 min. Placed on a magnetic plate for 2 min and discarded the supernatant. Add 50 μL SAPE working solution and incubate in a constant temperature oscillator at 37°C and 1200 rpm for 15 min. Place on a magnetic plate and let it stand for 2 min, then discard the supernatant. Add 100 μL PBS-T and wash in a constant temperature oscillator at 37°C and 1200 rpm for 1 min. Place on a magnetic plate and let it stand for 2 min, then discard the supernatant, add 70 μL PBS-T and mix in a constant temperature oscillator at 37°C and 1200 rpm for 1 min. Detect the signal value on a flow cytometer or a flow cytometer. A flow cytometer is preferred for this experiment.

[0086] Test method: For example, antibody 1 coupled with ON is used as capture antibody 1, and antibody 2 labeled with biotin is used as detection antibody 2. Prepare the corresponding working solution according to the above process of preparing the working solution for coupled microspheres and the working solution for detection antibodies (other paired antibodies are prepared according to the above operation). Using the diluent as a blank control and the clinical serum sample as a reference, a cross-pairing experiment of the capture antibody and the detection antibody is performed, and the results are shown in Table 1; finally, with the requirements of the blank being less than 2000 and the clinical sample being not less than 14000, 9 pairs of antibodies were found to be available, and their pairing results are shown in Table 2.

[0087] Table 1 Antibody pair test data

[0088]

[0089]

[0090] Table 2 Available antibody pair combinations

[0091]

[0092] Example 4: Effect of ON protein standards

[0093] The 9 pairs of antibodies screened in Example 3 were used as antibody pairs, and ON protein 1 (Aibotek Biotechnology Co., Ltd., RP00217) and protein 2 (Aibotek Biotechnology Co., Ltd., RP01973) were used as standard products, and their amino acid sequences were all shown in SEQ ID No. 33. The obtained detection results are shown in Tables 3 and 4. From the results, it can be seen that when protein 1, protein 2 and the aforementioned antibody pairs are used for detection, good detection effects are obtained. In specific implementation, based on the consideration of better signal-to-noise ratio, the combination of protein 1 and antibody pair 5 or protein 1 and antibody pair 6 is preferred.

[0094] Table 3 Detection results of protein 1 combined antibody pairs

[0095]

[0096]

[0097] Table 4 Detection results of protein 2 combined antibody pairs

[0098]

[0099] Example 5: Effect of SAPE

[0100] In this embodiment, six different SAPEs were compared in performance: protein 1 was diluted to 1000ng / mL, 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, and 15.625ng / mL; using sample diluent as a blank control, antibody pair 6 as a test antibody pair, comparing the detection results of six different SAPEs, comparing the strength of the signal value and the background signal, and selecting a SAPE with a lower background and a relatively higher overall signal. It can be seen from the results that the application of various SAPEs has a good detection effect. Among them, SAPE 2, SAPE 4, and SAPE 6 are preferred, and SAPE 2 with better cost performance is more preferred, and the results are shown in Table 5.

[0101] SAPE 1 was S866 purchased from Thermo Fisher Scientific;

[0102] SAPE 2 was 12-4317-87 purchased from Thermo Fisher Scientific;

[0103] SAPE 3 was S21388 purchased from Thermo Fisher Scientific;

[0104] SAPE 4 was L600-001 purchased from Shanghai Langya Biotechnology Co., Ltd.;

[0105] SAPE 5 was LF600-001 purchased from Shanghai Langya Biotechnology Co., Ltd.;

[0106] SAPE 6 was S406169 purchased from Aladdin Biotechnology Co., Ltd.;

[0107] Table 5 Comparison of six different SAPE test signal values

[0108]

[0109] This example further studies the use concentration of SAPE 2: protein 1 is diluted to 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, and 15.625 ng / mL; the sample diluent is used as a blank control, and antibody pair 6 is used as a test antibody pair to compare seven concentrations (25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 800 ng / mL, and 2000 ng / mL) of SAPE 2, and the strength of the signal value and the background signal are compared. The concentration with a lower background and a relatively higher overall signal is selected, and the preferred concentration is 100 ng / mL. The results are shown in Table 6.

[0110] Table 6 SAPE 2 concentration study

[0111]

[0112] Example 6: Study on detection antibody concentration

[0113] Protein 1 was diluted to 1000ng / mL, 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, and 15.625ng / mL; the sample diluent was used as a blank control, antibody pair 6 was used as a test antibody pair, SAPE 2 was selected, and its working concentration was selected as 100ng / mL, and the concentration of detection antibody 3 was studied. Conclusion: All three concentrations meet the experimental requirements, and the signal values ​​of the three concentration tests are similar, but the background is better when the detection antibody concentration is 250ng / mL, and the amount of detection antibody consumed is less. The preferred detection antibody concentration is 250ng / mL. See Table 7.

[0114] Table 7 Detection antibody concentration study

[0115]

[0116] Example 7: Sample dilution study

[0117] Protein 1 was diluted to 1000ng / mL, 500ng / mL, and 31.25ng / mL with the following solutions 1-6; the corresponding solutions were used as blank controls, antibody pair 6 was used as the test antibody pair, detection antibody 3 was diluted to 250ng / mL, and SAPE 2 was selected to be diluted to 100ng / mL, and the signal values ​​of the six sample dilutions (solutions 1-6) were studied. Solutions 1-6 met the experimental requirements in terms of background value and overall signal intensity, and solution 6, which had relatively better signal performance and background value, was selected as the sample dilution, and the results are shown in Table 8.

[0118] Solution 1: 50 mM borate buffer, 0.1% BSA, 0.1% PEG8000, 0.02% Tween 20, 0.05% Proclin 300, the remainder is purified water, pH 8.5.

[0119] Solution 2: 50 mM MES, 0.1% BSA, 0.1% sucrose, 0.02% Tween 20, 0.05% Proclin 300, the remainder is purified water, pH 6.5.

[0120] Solution 3: 50 mM HEPES, 0.1% BSA, 0.02% Tween 20, 0.05% Proclin 300, the balance being purified water, pH 7.4.

[0121] Solution 4: 25 mM TRIS, 0.2% BSA, 0.1% PEG8000, 0.05% Tween 20, 0.05% Proclin 300, the remainder is purified water, pH 7.8.

[0122] Solution 5: 50 mM PBS, 0.1% BSA, 0.05% Proclin 300, the balance being purified water, pH 7.4.

[0123] Solution 6: 30 mM PBS, 0.25% BSA, 0.1% sucrose, 0.25% PEG6000, 0.05% Proclin300, the remainder is purified water, pH 7.4.

[0124] Table 8 Reaction solution research

[0125]

[0126] Example 8: Analytical Performance Evaluation

[0127] Protein 1 was diluted to 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, and 15.625 ng / mL with solution 6 in Example 7; solution 6 was used as a blank control, antibody pair 6 was used as a test antibody pair, detection antibody 3 was diluted to 250 ng / mL, and SAPE 2 was selected and diluted to 100 ng / mL. The linearity, precision, minimum detection limit, and hook effect of the interference test machine of the reagent were evaluated:

[0128] (1) Linear analysis: Prepare samples of 8 concentrations according to the above method, use the theoretical concentration as the X-axis and the detected concentration as the Y-axis, use the least squares method to fit, and calculate the linear correlation coefficient R 2 , the data between each gradient are clearly distinguished, and R 2 >0.99, the results are shown in Table 9.

[0129] Table 9 ON kit linearity data

[0130]

[0131] (2) Precision: Use samples with the same concentration level as precision sample 1 and precision sample 2 for 10 repeated tests. Calculate the mean (M) and standard deviation (SD) of the 10 test results. Calculate the coefficient of variation (CV) according to the following formula.

[0132] CV=SD / M×100%

[0133] Where: CV is the coefficient of variation; SD is the standard deviation of 10 measurement results; M is the average of 10 measurement results, and the CV value is required to be less than 10%. The analysis results show that CV < 10%, which meets the requirements. The results are shown in Table 10 (only one set of data is required).

[0134] Table 10 ON kit precision analysis

[0135]

[0136] (3) Minimum detection limit: The zero-concentration sample was tested and repeated 20 times to obtain 20 measurement result signal values. The mean value (M) and standard deviation (SD) were calculated to obtain the signal value corresponding to M+2SD. A linear equation was obtained by regression fitting based on the concentration-signal value results between the zero-concentration sample and the adjacent calibrator. The signal value corresponding to M+2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit. The detection limit of ON was calculated to be 1.64 ng / mL. The results are shown in Table 11.

[0137] Table 11 Minimum detection limit test of ON kit

[0138]

[0139]

[0140] (4) Interference experiment: The presence of endogenous and exogenous interfering substances in serum samples, such as hemoglobin, triglycerides, bilirubin, HAMA (Human Anti-Mouse Antibody), etc., may affect the test results of serum samples. Therefore, interfering substances were added to the samples to evaluate the interference effect. 2240g / L hemoglobin, 60000mg / dL triglycerides, 1000mg / dL bilirubin, and 200ng / dL HAMA were prepared, and 5 working concentration gradients were set to add to the samples with concentrations of 500ng / mL and 125ng / mL for repeated testing. The average value of the test results was compared with the normal sample test results for deviation. The results showed that if the deviation of the interference test results of the two concentrations of 500ng / mL and 125ng / mL was within ±20%, it means that these substances in the serum sample test did not interfere with the test results, see Table 12, Table 13, and Table 14. Conclusion: The test results are not affected by hemolysis (hemoglobin <56mg / dL), lipemia (triglyceride concentration <1500mg / dL), jaundice (bilirubin <25mg / dL), and HAMA <5ng / dL.

[0141] Table 12 Preparation of working concentrations of interfering substances

[0142]

[0143] Table 13 ON kit high value interference test results

[0144]

[0145]

[0146] Table 14 ON kit median interference test results

[0147]

[0148]

[0149] (5) Hook effect: Set the sample with a concentration 40 times higher than 1000 ng / mL as the high-value test sample for the hook effect, and gradually perform gradient dilution until the detection signal shows a linear increase or approaches a stable state. After testing, the sample concentration reached 20000 ng / mL, and there was no hook effect on the detection. The results are shown in Table 15.

[0150] Table 15ON kit hook effect test

[0151]

[0152]

[0153] Example 9: Serum ON detection in patients with acute chest pain

[0154] 124 AAD patients, 80 AMI patients and 31 APE patients were selected, and serum ON expression was detected using a liquid phase chip kit to determine whether it could distinguish the above diseases. The experimental steps are as follows:

[0155] (1) Sample processing: Serum samples are used for testing. The specimens are collected in yellow blood collection tubes containing separation gel and centrifuged at 4°C, 3000 rpm for 10 minutes within 30 minutes after collection. The supernatant can be taken for testing, or the supernatant can be stored at -80°C, but repeated freezing and thawing should be avoided.

[0156] (2) Inclusion and exclusion criteria: 124 patients diagnosed with AAD, 80 patients with AMI, and 31 patients with APE were included. Peripheral blood serum was collected for liquid chip detection to verify whether ON can distinguish the above chest pain diseases. Inclusion criteria for patients in the AAD group: patients diagnosed with AAD by aortic CT angiography, aged ≥18 years; inclusion criteria for patients in the APE group: confirmed by positive spiral computed tomography or pulmonary angiography, with a high possibility of ventilation perfusion scintigraphy, or compression ultrasound or angiography of proximal deep vein thrombosis recorded in pulmonary angiography; inclusion criteria for patients in the AMI group: symptoms of ischemic chest pain, characteristic ST-T dynamic changes or abnormal Q waves on the electrocardiogram, and elevated and decreased serum myocardial enzyme spectrum. Patients who met two of the following criteria could be diagnosed. Exclusion criteria for the third group: patients with a history of rheumatism, autoimmune diseases, aortitis, pregnancy or hereditary syndrome, severe lung infection, abnormal liver and kidney function, and tumors were excluded. This study was approved by the Ethics Committee of the People's Hospital of Xinjiang Uygur Autonomous Region, and all included subjects signed informed consent for biological sample collection.

[0157] (3) Preparation before testing:

[0158] 1. Take out the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature.

[0159] 2. Prepare 7 1.5mL EP tubes and label them S7-S0, and arrange them in order. Take 120ul from the calibrator and transfer it to S7. Add 60ul of sample diluent to each of tubes S6-S0, take 60ul from tube S7 and transfer it to tube S6, vortex mix for 5-10s, take 60ul from tube S6 and transfer it to tube S5, then transfer it to tube S4, continue serial dilution, and mix thoroughly. Add 60ul of sample diluent to tube S6 as the zero value calibrator.

[0160] 3. Mix the sample and the microsphere suspension bound to the capture antibody, and the analyte in the sample binds to the specific capture antibody on the microsphere;

[0161] 4. Add detection antibodies, which specifically bind to the reaction products of the first step;

[0162] 5. Add fluorescein solution, and the streptavidin on the fluorescein will bind to the biotin on the detection antibody, eventually forming a "capture antibody-antigen-biotinylated antibody-streptavidin-fluorescein on the surface of the microsphere" complex.

[0163] 6. When using flow cytometry to detect it, different microsphere complexes can be identified as specific analyte types and the corresponding concentrations can be analyzed to achieve quantitative detection of ON in the sample.

[0164] a. The composition of the liquid phase chip kit is shown in Table 16. The reagents used in the ON kit are the optimal choices confirmed in the above examples:

[0165] Table 16 ON kit composition

[0166]

[0167] b. Experimental equipment required by the experiment: flow cytometer (Nova HT); high-precision pipette and gun tips: 0.5-10μL, 5-50μL, 20-200μL, 200-1000μL; incubator, etc.

[0168] c. Operation and testing steps:

[0169] Step 1 (Power on): Add sheath liquid to the scale line and discard the waste liquid. Turn on the instrument switch, sample tray switch and software. After the software progress bar ends, click Initialize. Wait for the instrument to be used 30 minutes after it is turned on.

[0170] Step 2 (Instrument Quality Control): Use a quality control ball to control the instrument. Only after confirming that the quality control data is normal can sample testing be performed.

[0171] Step 3 (Circle Ball): Test a blank control ball, determine the signal reading range, and then name and save it (if the report range of subsequent tests is consistent, this step can be skipped).

[0172] Step 4 (Establish standard curve): Create a new test project file with the circle ball file, enter the corresponding calibration concentration and perform standard test. After the test is completed, make a fitting curve and save it.

[0173] Step 5 (Test samples and analysis): Use the project file from step 4 to test the samples, and export the raw data for analysis.

[0174] Step 6 (Shutdown): After the test, perform cleaning and maintenance, and finally turn off all switches.

[0175] Through the above method, ON was used as a biomarker to detect and analyze the AAD group, AMI group, and APE group. The obtained data were analyzed using GraphPad Prism 9.0 statistical software and the receiver operating characteristic (ROC) curve was drawn to clarify whether ON can differentiate and diagnose the above three groups of diseases, and to explore the diagnostic value of ON for acute chest pain.

[0176] (4) Results analysis

[0177] like Figure 1As shown in Table 17, the figure shows the expression level of ON as a biomarker in the serum of patients in the AAD group, AMI group, and APE group. The results show that ON has a good degree of differentiation for the above three groups, especially for distinguishing AAD patients.

[0178] like Figure 2 As shown in Table 18, this figure is the receiver operating characteristic curve of ON as a biomarker for diagnosing AAD in patients with acute chest pain (AAD, AMI and APE). The results show that the area under the receiver operating characteristic curve is 0.907, and ON has a good diagnostic value for AAD.

[0179] Table 17 Serum ON expression in AAD, AMI and APE groups

[0180]

[0181]

[0182] Table 18 ROC analysis of ON kit for identification of AAD in acute chest pain

[0183]

[0184] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Application of ON detection reagent in the preparation of a kit, wherein: The kit is used for assisting diagnosis of acute chest pain diseases, wherein the acute chest pain diseases include at least one of AAD, AMI and APE.

2. The use according to claim 1, wherein: The kit is used to assist in identifying at least one of AAD, AMI and APE from patients with acute chest pain.

3. The use according to claim 1 or 2, wherein: The detection reagent is a reagent for detecting ON content in serum.

4. The use according to claim 3, wherein: When the ON content in the serum sample to be tested is 6.34-20.14 ng / mL, more preferably 12.75±9.78 ng / mL, it is determined that the patient to be tested has a risk of AAD; When the ON content in the serum sample to be tested is 13.25-83.69 ng / mL, more preferably 42.31±33.04 ng / mL, it is determined that the patient to be tested has a risk of AMI; When the ON content in the serum sample to be tested is 35.50-99.30 ng / mL, more preferably 62.26±27.33 ng / mL, it is determined that the patient to be tested has a risk of APE.

5. An auxiliary diagnostic product for realizing the use according to any one of claims 1 to 4, comprising a reagent for detecting ON content in serum.

6. The auxiliary diagnostic product according to claim 5, wherein: The reagent includes an antibody pair for specifically binding to ON, wherein the antibody pair includes a capture antibody coupled to a magnetic fluorescent microsphere and a biotinylated detection antibody; Wherein, the capture antibody and the detection antibody are respectively selected from two different antibodies among antibody 1, antibody 2, antibody 3, antibody 4, antibody 5, antibody 6, antibody 7, and antibody 8; The light chain variable region sequence of the antibody 1 is shown in SEQ ID No.2, and the heavy chain variable region sequence is shown in SEQ ID No.4; preferably, the full-length light chain sequence of the antibody 1 is shown in SEQ ID No.1, and the full-length heavy chain sequence is shown in SEQ ID No.3; The light chain variable region sequence of the antibody 2 is shown in SEQ ID No.6, and the heavy chain variable region sequence is shown in SEQ ID No.8; preferably, the full-length light chain sequence of the antibody 2 is shown in SEQ ID No.5, and the full-length heavy chain sequence is shown in SEQ ID No.7; The light chain variable region sequence of the antibody 3 is shown in SEQ ID No.10, and the heavy chain variable region sequence is shown in SEQ ID No.12; preferably, the full-length light chain sequence of the antibody 3 is shown in SEQ ID No.9, and the full-length heavy chain sequence is shown in SEQ ID No.11; The light chain variable region sequence of antibody 4 is shown in SEQ ID No.14, and the heavy chain variable region sequence is shown in SEQ ID No.16; preferably, the full-length light chain sequence of antibody 4 is shown in SEQ ID No.13, and the full-length heavy chain sequence is shown in SEQ ID No.15; The light chain variable region sequence of the antibody 5 is shown in SEQ ID No.18, and the heavy chain variable region sequence is shown in SEQ ID No.20; preferably, the full-length light chain sequence of the antibody 5 is shown in SEQ ID No.17, and the full-length heavy chain sequence is shown in SEQ ID No.19; The light chain variable region sequence of the antibody 6 is shown in SEQ ID No.22, and the heavy chain variable region sequence is shown in SEQ ID No.24; preferably, the full-length light chain sequence of the antibody 6 is shown in SEQ ID No.21, and the full-length heavy chain sequence is shown in SEQ ID No.23; The light chain variable region sequence of the antibody 7 is shown in SEQ ID No.26, and the heavy chain variable region sequence is shown in SEQ ID No.28; preferably, the full-length light chain sequence of the antibody 7 is shown in SEQ ID No.25, and the full-length heavy chain sequence is shown in SEQ ID No.27; The light chain variable region sequence of the antibody 8 is shown as SEQ ID No.30, and the heavy chain variable region sequence is shown as SEQ ID No.32; preferably, the full-length light chain sequence of the antibody 8 is shown as SEQ ID No.29, and the full-length heavy chain sequence is shown as SEQ ID No.

31.

7. The auxiliary diagnostic product according to claim 6, wherein: The antibody pair is selected from any one of the following antibody pairs 1 to 9: 。 8. The auxiliary diagnostic product according to claim 6 or 7, wherein: The detection antibody is used at a concentration of 200 to 1000 ng / mL, more preferably 250±50 ng / mL.

9. The auxiliary diagnostic product according to any one of claims 6 to 8, wherein: The reagent also includes an ON standard, the amino acid sequence of the ON standard is shown in SEQ ID No. 33; preferably at least one selected from protein 1 and protein 2; Wherein, the protein 1 is RP00217 purchased from Wuhan Abotek Biotechnology Co., Ltd.; The protein 2 is RP01973 purchased from Wuhan Abotek Biotechnology Co., Ltd.

10. The auxiliary diagnostic product according to claim 9, wherein: The reagent includes the protein 1 and the antibody pair 5; or includes the protein 1 and the antibody pair 6.

11. The auxiliary diagnostic product according to any one of claims 6 to 10, wherein: The reagent further comprises streptavidin-phycoerythrin, wherein the streptavidin-phycoerythrin is selected from at least one of the following SAPE 1 to SAPE 6, more preferably SAPE 2; Among them, SAPE 1 was S866 purchased from Thermo Fisher Scientific; SAPE 2 was 12-4317-87 purchased from Thermo Fisher Scientific; SAPE 3 was S21388 purchased from Thermo Fisher Scientific; SAPE 4 was L600-001 purchased from Shanghai Langya Biotechnology Co., Ltd.; SAPE 5 was LF600-001 purchased from Shanghai Langya Biotechnology Co., Ltd.; SAPE 6 was S406169 purchased from Aladdin Biotechnology Co., Ltd.; Optionally, the streptavidin-phycoerythrin is used at a concentration of 25 to 2000 ng / mL, more preferably 100±50 ng / mL.

12. The auxiliary diagnostic product according to any one of claims 6 to 11, wherein: The reagent further includes a sample diluent, which contains: 30±5 mM PBS, 0.25±0.05% BSA, 0.1±0.05% sucrose, 0.25±0.05% PEG6000, and 0.05±0.02% Proclin300, and has a pH value of 7.4±0.2.

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