Biomarker on for auxiliary diagnosis of acute chest pain diseases, and use thereof

Abstract

A biomarker osteonectin (ON) for auxiliary diagnosis of acute chest pain diseases, and a use thereof. ON is closely associated with acute chest pain diseases such as AAD, AMI, and APE, ON is used as a biomarker, and a detection reagent thereof is prepared into an auxiliary diagnostic product, facilitating early diagnosis, long-term prognosis, and quality-of-life improvement for patients with the three diseases, especially AAD.

Description

ON, a biomarker for the auxiliary diagnosis of acute chest pain and its application Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to osteonectin (ON), a biomarker for the auxiliary diagnosis of acute chest pain, and its application. Background Technology

[0002] Acute chest pain is one of the most common clinical symptoms, representing a heterogeneous group of diseases with chest pain as the primary manifestation. The causes of acute chest pain are numerous, the severity varies greatly, and the risks differ significantly. Although non-cardiac chest pain accounts for the majority of acute chest pain cases, cardiac chest pain, especially life-threatening conditions such as acute aortic dissection (AAD), acute myocardial infarction (AMI), and acute pulmonary embolism (APE), is a key focus for rapid assessment in the emergency department. While AAD and APE have a lower incidence, they are critical, progress rapidly, and are easily missed or misdiagnosed clinically, resulting in extremely high mortality rates. Therefore, for patients with acute chest pain, immediate resuscitation procedures are necessary after a definitive diagnosis in the emergency department. Rapid treatment helps improve patient survival rates and prognosis.

[0003] With the promotion of chest pain center construction, chest pain diagnosis and treatment are becoming increasingly standardized; however, misdiagnosis and missed diagnosis still occur from time to time. Therefore, rapid assessment of the condition, accurate differential diagnosis, and scientific management are crucial for efficient treatment and rational triage of chest pain patients. Currently, the diagnosis and differential diagnosis of acute chest pain patients mainly rely on medical history, physical examination, and auxiliary examination results. Imaging examinations such as electrocardiogram, chest X-ray, computed tomography (CT) imaging, and coronary angiography are helpful in clarifying the diagnosis. However, emergency medical capabilities vary across different regions, especially in remote primary hospitals, which lack comprehensive imaging examination facilities, thus limiting the ability to differentiate acute chest pain to some extent. For patients with acute chest pain, rapid laboratory testing of cardiovascular biomarkers is one of the important tools for emergency diagnosis and treatment, helping to quickly diagnose early-stage chest pain and improve the ability to treat acute chest pain. Commonly used biomarkers for myocardial injury include cardiac troponin (cTn), creatine kinase isoenzymes MB (CK-MB), and myoglobin (MYO), which play an important role in the early diagnosis and assessment of acute coronary syndromes. High-sensitivity cardiac troponin (hs-cTn) has higher sensitivity for acute myocardial infarction (AMI) and is often used for early screening and exclusion of AMI. Elevated D-dimer levels reflect the activation of the coagulation and fibrinolytic systems in plasma. Clinically, D-dimer is considered a marker of hypercoagulable state and hyperfibrinolysis and can be used as the first-line screening indicator for the diagnosis of acute myocardial embolism (APE). D-dimer testing can also be used for screening and exclusion of acute arterial disease (AAD), but because the plasma D-dimer levels are low in young patients and patients with short tear lengths and pseudoembolism, it may lead to misdiagnosis, thus limiting its clinical application.

[0004] Therefore, the search for new biomarkers to aid in the diagnosis of acute chest pain, especially those that can be used to aid in the diagnosis of multiple types of acute chest pain, is of great significance for clinical diagnosis. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of an ON (Osteonectin) detection reagent in the preparation of a kit for the auxiliary diagnosis of acute chest pain, particularly for the auxiliary identification of AAD in patients with acute chest pain.

[0006] Furthermore, the present invention also provides an antibody pair for the specific detection of ON in serum.

[0007] Furthermore, the present invention also proposes a kit for assisting in the diagnosis of acute chest pain, particularly for assisting in the identification of AAD from patients with acute chest pain, which contains a reagent for detecting the ON content in serum.

[0008] This invention discovers that ON protein is closely related to acute chest pain such as AAD, AMI, and APE. Using ON as a biomarker and preparing its detection reagent into an auxiliary diagnostic product has significant clinical value and practical significance for the early diagnosis, long-term prognosis, and improvement of quality of life of the above three diseases (especially AAD). Attached Figure Description

[0009] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0010] Figure 1 shows the ON expression levels in the serum of patients in the AAD, AMI, and APE groups in Example 9.

[0011] Figure 2 shows the operating characteristic curve of subjects in Example 9 who used ON as a biomarker to diagnose AAD in patients with acute chest pain.

[0012] Figure 3 shows the ON expression levels in the serum of patients in the AAD, AMI, and APE groups in Example 10.

[0013] Figure 4 shows the operating characteristic curves of subjects in Example 10 who used ON as a biomarker to diagnose AAD in patients with acute chest pain.

[0014] Figure 5 shows the ON expression levels in the serum of patients in the AAD, AMI, and APE groups in Example 11.

[0015] Figure 6 shows the operating characteristic curves of subjects in Example 11 who used ON as a biomarker to diagnose AAD in patients with acute chest pain. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0017] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0018] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These 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 connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0019] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0020] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0021] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0022] In this invention, the term "%" refers to mass or volume percentage (m / v%) unless otherwise specified. Specifically, when the solute is a solid, "%" refers to the percentage of the mass (grams) of the solute in the solution to the total volume (liters) of the solution; when the solute is a liquid, "%" refers to the percentage of the volume (liters) of the solute in the solution to the total volume (liters) of the liquid.

[0023] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0024] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0025] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0026] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0027] In this invention, the term "ON" refers to Osteonectin, also known as osteonectin or Bm-40, which is a stromal cell secreted protein composed of three domains.

[0028] In this invention, the term "auxiliary diagnosis" refers to the process of improving the accuracy and reliability of diagnosis by combining various auxiliary tools and methods with the doctor's clinical judgment during medical diagnosis. In practice, other auxiliary tools may include imaging examinations (such as X-rays, CT scans, and MRI), laboratory tests (such as blood tests, biochemical tests, and immunological tests), and pathological examinations. The main purpose of auxiliary diagnosis is to support and supplement the doctor's clinical diagnosis, helping to determine the nature, extent, and stage of the disease, thereby guiding subsequent treatment plans.

[0029] In this invention, the term "acute myocardial infarction (AMI)" refers to a disease caused by a sudden interruption of coronary blood flow, leading to myocardial ischemia and necrosis. Typical symptoms include chest pain (usually a squeezing pain), shortness of breath, sweating, nausea, and vomiting.

[0030] In this invention, the term "acute aortic dissection (AAD)" refers to a tear in the aortic intima, allowing blood to enter the aortic wall media and form a dissection. Typical symptoms include severe chest or back pain, which may shift as the dissection progresses.

[0031] In this invention, the term "acute pulmonary embolism (APE)" refers to the obstruction of the pulmonary artery or its branches by a thrombus or other substance, leading to pulmonary circulatory disorders. Typical symptoms include dyspnea, chest pain, hemoptysis, and syncope.

[0032] In this invention, the term "streptavidin-Phycoerythrin (SA-PE)" refers to a binding protein that combines the highly affinity streptavidin with the fluorescent phycoerythrin. It is widely used in techniques such as flow cytometry and fluorescence microscopy, enabling the effective labeling and detection of cells or molecules, and is particularly important in immunological and biomarker research. Its efficient binding and sensitive fluorescence properties make SA-PE a key tool in biomedical research.

[0033] This invention first provides the application of an ON detection reagent in the preparation of a kit for the auxiliary diagnosis of acute chest pain.

[0034] This invention discovers that ON protein is closely related to acute chest pain such as AAD, AMI, and APE. Using ON as a biomarker and preparing a detection kit based on its detection reagent can assist in the diagnosis of the above three diseases. In particular, it can help identify AAD in patients with acute chest pain, which has great clinical value and practical significance for early diagnosis, long-term prognosis and improvement of quality of life.

[0035] In some specific embodiments, the kit is used to assist in the diagnosis of at least one of AAD, AMI, and APE.

[0036] In some embodiments, the kit is used to assist in the identification of patients with acute aortic dissection (AAD) from patients with acute chest pain.

[0037] In a specific embodiment, the detection reagent is a reagent for detecting the ON content in blood.

[0038] In some preferred embodiments, the detection reagent is a reagent for detecting the ON content in serum. This invention has found that different concentration ranges exist in the serum of patients with AAD, AMI, and APE, three critical chest pain diseases. Detecting the ON content in serum will further help to accurately differentiate between these three diseases.

[0039] In some embodiments, when the ON content in the serum sample is 6.34–20.14 ng / mL, more preferably 12.75 ± 9.78 ng / mL, the patient is assessed as having a risk of AAD; when the ON content is 13.25–83.69 ng / mL, more preferably 42.31 ± 33.04 ng / mL, the patient is assessed as having a risk of AMI; and when the ON content is 35.50–99.30 ng / mL, more preferably 62.26 ± 27.33 ng / mL, the patient is assessed as having a risk of APE. Through studies of serum samples from relevant patients, this invention has found that the ON content in the serum samples of the vast majority (at least 80%) of patients with the corresponding diseases falls within the above-mentioned ranges, therefore, this range can be used to assist in the assessment of the above three diseases.

[0040] Furthermore, the present invention also provides a kit for assisting in the diagnosis of acute chest pain, which contains a reagent for detecting the ON content in the blood.

[0041] In some specific embodiments, the kit may also include reagents for conventional detection methods in the art, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and flow cytometry.

[0042] In some embodiments, the reagent includes an antibody pair for specifically binding ON, the antibody pair including a capture antibody coupled to a magnetic fluorescent microsphere and a biotinylated detection antibody.

[0043] In some preferred embodiments, the capture antibody and the detection antibody are each selected from two different antibodies chosen 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 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 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 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 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 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 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. 8. As shown in No. 14, the heavy chain variable region sequence is as shown in SEQ ID No. 16; preferably, the full-length light chain sequence of antibody 4 is as shown in SEQ ID No. 13, and the full-length heavy chain sequence is as shown in SEQ ID No. 15; the light chain variable region sequence of antibody 5 is as shown in SEQ ID No. 18, and the heavy chain variable region sequence is as shown in SEQ ID No. 20; preferably, the full-length light chain sequence of antibody 5 is as shown in SEQ ID No. 17, and the full-length heavy chain sequence is as shown in SEQ ID No. 19; the light chain variable region sequence of antibody 6 is as shown in SEQ ID No. 22, and the heavy chain variable region sequence is as shown in SEQ ID No. 24; preferably, the full-length light chain sequence of antibody 6 is as shown in SEQ ID No. 21, and the full-length heavy chain sequence is as shown in SEQ ID No. 23; the light chain variable region sequence of antibody 7 is as shown in SEQ ID No. 26, and the heavy chain variable region sequence is as shown in SEQ ID No. 28; preferably, the full-length light chain sequence of antibody 7 is as shown in SEQ ID No. 25, and the full-length heavy chain sequence is as shown in SEQ ID No. 19. As shown in No. 27; the light chain variable region sequence of the 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 the antibody 8 is shown in SEQ ID No. 29, and the full-length heavy chain sequence is shown in SEQ ID No. 31.

[0044] The present invention further discovers that by applying the above-mentioned antibody pairs, the detection of ON can be achieved well.

[0045] In some further preferred embodiments, the antibody pair is selected from any one of antibody pairs 1 to antibody pairs 9:

[0046] In some embodiments, the concentration of the detection antibody used is 200–1000 ng / mL, more preferably 250 ± 50 ng / mL. As an example, the concentration of the detection antibody can be any value between 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 200–1000 ng / mL. In practice, those skilled in the art can directly prepare the detection antibody solution in the kit, or prepare a solid detection antibody and a detection antibody diluent, and provide recommended concentrations of the detection antibody in the accompanying instructions.

[0047] In some embodiments, the reagent also includes an ON standard. In specific embodiments, those skilled in the art can select commercially available ON proteins as standards based on common knowledge.

[0048] In some preferred embodiments, the amino acid sequence of the ON standard is shown in SEQ ID No. 33.

[0049] In some preferred embodiments, the ON standard is selected from at least one of protein 1 and protein 2 (both with amino acid sequences as shown in SEQ ID No. 33); wherein protein 1 is RP00217 purchased from Wuhan ABclonal Biotechnology Co., Ltd.; and protein 2 is RP01973 purchased from Wuhan ABclonal Biotechnology Co., Ltd. In specific implementations, protein 1 is more preferably selected as the ON standard.

[0050] In some embodiments, the reagent comprises protein 1 and antibody pair 5; or comprises protein 1 and antibody pair 6. The above combinations offer a superior signal-to-noise ratio in detection.

[0051] In some embodiments, the reagent further includes streptavidin-fluorescein (SA-FITC).

[0052] In specific embodiments, those skilled in the art can use commercially available streptavidin-phycoerythrin (SAPE) based on common knowledge, as both can be used to detect ON in this invention. In some specific embodiments, the streptavidin-luciferin is streptavidin-phycoerythrin (SAPE).

[0053] In some preferred embodiments, the streptavidin-phycoerythrin is selected from at least one of 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 Laya Biotech Co., Ltd.; SAPE 5 is LF600-001 purchased from Laya Biotech Co., Ltd.; and SAPE 6 is S406169 purchased from Aladdin Biochemical Technology Co., Ltd.

[0054] In some preferred embodiments, the concentration of streptavidin-phycoerythrin used is 25–2000 ng / mL, more preferably 100 ± 50 ng / mL. As an example, the concentration of streptavidin-phycoerythrin used can be any value from 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 25–2000 ng / mL. In practice, those skilled in the art can directly prepare the SAPE solution in the kit, or prepare solid SAPE and SAPE diluent, and provide recommended SAPE concentrations in the accompanying instructions.

[0055] In some embodiments, the reagent further includes a sample diluent. In specific embodiments, those skilled in the art can confirm, based on common knowledge, that all sample diluents used in this invention can be used to detect ON.

[0056] In some preferred embodiments, the sample diluent contains: 30±5mM 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 of 7.4±0.2.

[0057] In specific embodiments, those skilled in the art can combine the above-mentioned solutions with common sense to obtain preferred embodiments of the auxiliary diagnostic product of the present invention.

[0058] In specific implementations, those skilled in the art can confirm the specific usage methods of the auxiliary diagnostic product based on common sense.

[0059] In some specific embodiments, the auxiliary diagnostic method includes: mixing the serum sample to be tested with a suspension of magnetic fluorescent microspheres bound to a capture antibody, wherein the ON in the serum sample specifically binds to the capture antibody on the magnetic fluorescent microspheres; then adding a biotinylated detection antibody, which specifically binds to the reaction product from the first step; subsequently adding a streptavidin-phycoerythrin solution, which binds streptavidin to the biotin on the detection antibody, ultimately forming a "capture antibody on the surface of magnetic fluorescent microspheres - antigen (ON) - biotinylated detection antibody - streptavidin - phycoerythrin" complex. When detected using a flow cytometer, the fluorescence signal intensity values ​​of the magnetic fluorescent microspheres and the corresponding coated antibody are read. Different microsphere complexes can be identified to determine the specific analyte type and analyze the corresponding concentration, achieving quantitative detection of ON in the sample. The method using this kit is a high-throughput, low-consumption, and low-cost detection method with comprehensive detection, high specificity, and good sensitivity, which can improve detection efficiency.

[0060] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0061] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0062] Example 1: Conjugation of anti-human ON antibody to magnetic fluorescent microspheres

[0063] Activation of magnetic fluorescent microspheres: Take 100 μL of uniformly mixed magnetic fluorescent microspheres (10 6100 μL of magnetic fluorescent microspheres were washed twice with 200 μL of buffer 1. 80 μL of buffer 1 was added and mixed thoroughly. Then, 10 μL of 50 mg / mL NHS (N-hydroxysuccinimide ester) solution (prepared with buffer 1) was added and vortexed. 10 μL of 50 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) solution (prepared with buffer 1) was added and vortexed. The mixture was then incubated at room temperature for 20 minutes on a rotary vibrator. The supernatant was discarded after magnetic separation. Buffer 2 was added and mixed thoroughly, followed by magnetic separation and discarding of the supernatant. 500 μL of buffer 2 was added and mixed thoroughly. 5 μg of human ON antibody was added and vortexed. The mixture was then incubated at room temperature for 2 hours on a rotary vibrator. The supernatant was discarded after magnetic separation. The microspheres were washed twice with 500 μL of 0.01 M pH 7.4 PBS-T buffer, followed by magnetic separation and discarding of the supernatant.

[0064] Microsphere blocking: Add 1 mL of blocking solution, mix well, and then mix on a rotary apparatus at room temperature for 2 hours. Magnetic separation: Discard the supernatant and wash twice with 500 μL of 0.01 M pH 7.4 PBST buffer. Magnetic separation: Discard the supernatant and add 100 μL of storage solution, mix well, and store at 2-8℃ protected from light for later use.

[0065] Buffer 1: The buffer reagent used is acetate buffer, with a concentration of 50 mM and a pH of 5.0.

[0066] Buffer 2: The buffer reagent used is borate buffer with a concentration of 100mM and a pH of 6.8.

[0067] Blocking solution: PBS solution containing 0.5% BSA at pH 8.0.

[0068] Preservative solution: PBST solution containing 0.5% BSA at pH 8.0, 0.01M.

[0069] Example 2: Biotinylated anti-human ON antibody

[0070] Take 50 μg of ON antibody and add it to 200 μL of labeling buffer. Mix well. Then, prepare Sulfo-NHS-LC-Biotin to 10 mM using labeling buffer. Add 10 μL of this solution to the ON antibody solution and mix thoroughly at room temperature using a rotary oscillator for 120 minutes. Ultrafilter three times using salt replacement buffer. Add biotinylated antibody preservation solution to a final antibody concentration of 0.25 mg / mL and store at -20°C for later use.

[0071] Labeling buffer: Use borate buffer with a concentration of 50 mM and a pH of 7.8.

[0072] Salt change buffer: Use TBS at a concentration of 10 mM and a pH of 8.0.

[0073] Biotinylated antibody preservation solution: PBS solution containing 40% glycerol at a concentration of 10 mM and a pH of 7.4.

[0074] Example 3: Effects of Antibody Pairs

[0075] Antibody Description: In Table 1, the full-length light chain sequence of antibody 1 is shown in SEQ ID No. 1, the variable region sequence of the light chain is shown in SEQ ID No. 2, the full-length heavy chain sequence is shown in SEQ ID No. 3, and the variable region sequence of the heavy chain is shown in SEQ ID No. 4; the full-length light chain sequence of antibody 2 is shown in SEQ ID No. 5, the variable region sequence of the light chain is shown in SEQ ID No. 6, the full-length heavy chain sequence is shown in SEQ ID No. 7, and the variable region sequence of the heavy chain is shown in SEQ ID No. 8; the full-length light chain sequence of antibody 3 is shown in SEQ ID No. 9, the variable region sequence of the light chain is shown in SEQ ID No. 10, the full-length heavy chain sequence is shown in SEQ ID No. 11, and the variable region sequence of the heavy chain is shown in SEQ ID No. 12; the full-length light chain sequence of antibody 4 is shown in SEQ ID No. 13, the variable region sequence of the light chain is shown in SEQ ID No. 14, the full-length heavy chain sequence is shown in SEQ ID No. 15, and the variable region sequence of the heavy chain is shown in SEQ ID No. 16. The sequence of antibody 5 is shown in SEQ ID No. 16; the full-length light chain sequence of antibody 5 is shown in SEQ ID No. 17, the variable region sequence of the light chain is shown in SEQ ID No. 18, the full-length heavy chain sequence is shown in SEQ ID No. 19, and the variable region sequence of the heavy chain is shown in SEQ ID No. 20; the full-length light chain sequence of antibody 6 is shown in SEQ ID No. 21, the variable region sequence of the light chain is shown in SEQ ID No. 22, the full-length heavy chain sequence is shown in SEQ ID No. 23, and the variable region sequence of the heavy chain is shown in SEQ ID No. 24; the full-length light chain sequence of antibody 7 is shown in SEQ ID No. 25, the variable region sequence of the light chain is shown in SEQ ID No. 26, the full-length heavy chain sequence is shown in SEQ ID No. 27, and the variable region sequence of the heavy chain is shown in SEQ ID No. 28; the full-length light chain sequence of antibody 8 is shown in SEQ ID No. 29, the variable region sequence of the light chain is shown in SEQ ID No. 30, the full-length heavy chain sequence is shown in SEQ ID No. 31, and the variable region sequence of the heavy chain is shown in SEQ ID No. 32.

[0076] Preparation of working solution for conjugated microspheres (capture antibody): Dilute the magnetic fluorescent microspheres conjugated with ON antibody by 200-fold using the preservation solution from Example 1.

[0077] Preparation of working solution for detection antibody: Dilute with detection antibody diluent, with a dilution factor of 250 times.

[0078] Preparation of working solution for streptavidin-R-Phycoerythrin (SAPE): Dilute with SAPE diluent 500 times.

[0079] Antibody dilution solution: HEPES with a concentration of 10 mM and a pH of 7.4 was selected.

[0080] SAPE dilution: A PBS solution containing 10 mM glucose at pH 6.5, with a glucose content of 0.02%.

[0081] Experimental Procedure: 20 μL of capture antibody was added to each well of a 96-well polystyrene plate, followed by 20 μL of clinical sample. The plates were incubated at 37°C and 1200 rpm for 45 min using a temperature-controlled shaker. After incubation on a magnetic plate for 2 min, the supernatant was discarded. 100 μL of PBS-T was added, and the plates were washed for 1 min at 37°C and 1200 rpm using a temperature-controlled shaker. After incubation on a magnetic plate for 2 min, the supernatant was discarded. Next, 50 μL of the detection antibody working solution was added to each well, and the plates were incubated at 37°C and 1200 rpm for 30 min using a temperature-controlled shaker. After incubation on a magnetic plate for 2 min, the supernatant was discarded. 100 μL of PBS-T was added, and the plates were washed for 1 min at 37°C and 1200 rpm using a temperature-controlled shaker. After incubation on a magnetic plate for 2 min, the supernatant was discarded. Add 50 μL of SAPE working solution and incubate for 15 min at 37°C and 1200 rpm in a constant-temperature shaker. Place on a magnetic plate and allow to stand for 2 min, then discard the supernatant. Add 100 μL of PBS-T and wash for 1 min at 37°C and 1200 rpm in a constant-temperature shaker. Place on a magnetic plate and allow to stand for 2 min, then discard the supernatant. Add 70 μL of PBS-T and mix for 1 min at 37°C and 1200 rpm in a constant-temperature shaker. Detect the signal value using a flow cytometer or flow fractionator; a flow fractionator is preferred in this experiment.

[0082] Test method: For example, antibody 1 conjugated with ON is used as capture antibody 1, and antibody 2 labeled with biotin is used as detection antibody 2. The corresponding working solutions were prepared according to the above procedures for preparing the conjugated microsphere working solution and the detection antibody working solution (other paired antibodies were prepared according to the above procedures). Using the diluent as a blank control and clinical serum samples as references, a cross-pairing experiment was performed between the capture antibody and the detection antibody. The results are shown in Table 1. Finally, based on the requirement of a blank control of less than 2000 and a clinical sample control of not less than 14000, 9 antibody pairs were found to be usable, and their pairing results are shown in Table 2.

[0083] Table 1 Antibody test data

[0084] Table 2. Available Antibody Pair Combinations

[0085] Example 4: Effect of ON protein standards

[0086] Nine antibody pairs screened in Example 3 were used as antibody pairs. ON protein 1 (Abbott Biotechnology Co., Ltd., RP00217) and protein 2 (Abbott Biotechnology Co., Ltd., RP01973) were used as standards. Their amino acid sequences are shown in SEQ ID No. 33. The detection results are shown in Tables 3 and 4. These results show that using protein 1, protein 2, and the aforementioned antibody pairs all provide good detection results. In practical implementation, for better signal-to-noise ratio considerations, the combination of protein 1 with antibody pair 5 or protein 1 with antibody pair 6 is preferred.

[0087] Table 3. Detection results of protein 1 combined antibody pairs

[0088] Table 4. Detection results of protein 2 combined antibody pairs

[0089] Example 5: The Effect of SAPE

[0090] In this embodiment, the performance of six different SAPEs was compared: 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; the sample dilution was used as a blank control, and antibody pair 6 was used as the test antibody pair. The detection results of the six different SAPEs were compared, and the signal strength and background signal were compared. The SAPE with lower background and relatively higher overall signal was selected. The results show that all SAPEs have good detection effects. Among them, SAPE 2, SAPE 4, and SAPE 6 are preferred, and SAPE 2, which has a better cost performance, is even more preferred. The results are shown in Table 5.

[0091] SAPE 1 is an S866 purchased from Thermo Fisher Scientific.

[0092] SAPE 2 is 12-4317-87 purchased from Thermo Fisher Scientific.

[0093] SAPE 3 is an S21388 purchased from Thermo Fisher Scientific.

[0094] SAPE 4 was purchased from Shanghai Langya Biotechnology Co., Ltd. as L600-001.

[0095] SAPE 5 was purchased from Shanghai Langya Biotechnology Co., Ltd. as LF600-001.

[0096] SAPE 6 is S406169 purchased from Aladdin Biotechnology Co., Ltd.

[0097] Table 5 Comparison of Six Different SAPE Test Signal Values

[0098] This embodiment further investigated the concentration of SAPE 2 used: 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; using the sample dilution as a blank control and antibody pair 6 as a test antibody pair, the 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 were compared. The signal strength and background signal were compared, and the concentration with lower background and relatively higher overall signal was selected. The preferred concentration was 100 ng / mL. The results are shown in Table 6.

[0099] Table 6. SAPE 2 Concentration Study

[0100] Example 6: Study on the detection of antibody concentration

[0101] 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. The sample dilutions served as blank controls. Antibody pair 6 was used as the test antibody pair. SAPE 2 was selected with a working concentration of 100 ng / mL. The optimal concentration for detecting antibody 3 was investigated. Conclusion: All three concentrations met the experimental requirements, and the signal values ​​were similar across the three concentrations. However, the background was better at a detection antibody concentration of 250 ng / mL, and less antibody was consumed. Therefore, the preferred detection antibody concentration is 250 ng / mL. See Table 7.

[0102] Table 7. Study on antibody concentration detection

[0103] Example 7: Study of Sample Diluent

[0104] Protein 1 was diluted to 1000 ng / mL, 500 ng / mL, and 31.25 ng / mL using solutions 1-6. The corresponding solutions served as blank controls. Antibody pair 6 was used as the test antibody pair. Detection antibody 3 was diluted to 250 ng / mL, and SAPE2 was diluted to 100 ng / mL. The signal values ​​of the six sample dilutions (solutions 1-6) were investigated. Solutions 1-6 met the experimental requirements in terms of background values ​​and overall signal intensity. Solution 6, which showed relatively better signal performance and background values, was selected as the preferred sample dilution. The results are shown in Table 8.

[0105] Solution 1: 50mM borate buffer, 0.1% BSA, 0.1% PEG8000, 0.02% Tween 20, 0.05% Proclin 300, balance purified water, pH 8.5.

[0106] Solution 2: 50 mM MES, 0.1% BSA, 0.1% sucrose, 0.02% Tween 20, 0.05% Proclin 300, balance is purified water, pH 6.5.

[0107] Solution 3: 50 mM HEPES, 0.1% BSA, 0.02% Tween 20, 0.05% Proclin 300, balance: purified water, pH 7.4.

[0108] Solution 4: 25mM TRIS, 0.2% BSA, 0.1% PEG8000, 0.05% Tween 20, 0.05% Proclin 300, balance is purified water, pH 7.8.

[0109] Solution 5: 50 mM PBS, 0.1% BSA, 0.05% Proclin 300, balance purified water, pH 7.4.

[0110] Solution 6: 30mM PBS, 0.25% BSA, 0.1% sucrose, 0.25% PEG6000, 0.05% Proclin300, balance purified water, pH 7.4.

[0111] Table 8. Study of reaction solutions

[0112] Example 8: Performance Evaluation Analysis

[0113] Protein 1 was diluted with solution 6 from Example 7 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; solution 6 served as a blank control, antibody pair 6 served as the test antibody pair, detection antibody 3 was diluted to 250 ng / mL, and SAPE 2 was diluted to 100 ng / mL. The linearity, precision, limit of detection, and interference with the hook effect of the testing apparatus were evaluated.

[0114] (1) Linear analysis: Eight samples of different concentrations were prepared using the above method. The theoretical concentration was used as the X-axis and the detected concentration as the Y-axis. The least squares method was used for fitting, and the linear correlation coefficient R was calculated. 2 The data between each gradient is clearly distinguishable, and R 2 >0.99, the results are shown in Table 9.

[0115] Table 9. Linear data for ON reagent kit

[0116] (2) Precision: Samples at the concentration levels of precision sample 1 and precision sample 2 were tested repeatedly 10 times each. The mean (M) and standard deviation (SD) of the 10 test results were calculated, and the coefficient of variation (CV) was calculated using the following formula: CV = SD / M × 100%

[0117] In the formula: CV is the coefficient of variation; SD is the standard deviation of 10 measurements; M is the average of 10 measurements, 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 needed).

[0118] Table 10 Precision analysis of ON reagent kit

[0119] (3) Limit of detection: The zero-concentration sample was tested and repeated 20 times to obtain the signal values ​​of the 20 measurement results. The mean (M) and standard deviation (SD) were calculated to obtain the signal value corresponding to M+2SD. The 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 limit of detection. The calculated limit of detection was 1.64 ng / mL. The results are shown in Table 11.

[0120] Table 11. Limit of Detection Test of ON Reagent Kit

[0121] (4) Interference experiment: There are endogenous and exogenous interfering substances in serum samples, such as hemoglobin, triglycerides, bilirubin, and HAMA (Human Anti-Mouse Antibody), which may affect the test results of serum samples. Therefore, interfering substances were added to the samples to evaluate the interference effect. 2240 g / L hemoglobin, 60000 mg / dL triglycerides, 1000 mg / dL bilirubin, and 200 ng / dL HAMA were prepared and added to samples with concentrations of 500 ng / mL and 125 ng / mL for repeated testing. The average test results were compared with the test results of normal samples. The results showed that if the deviation of the two concentrations of 500 ng / mL and 125 ng / mL in the interference experiment test results was within ±20%, it indicated that these substances in the serum sample test did not interfere with the test results. See Tables 12, 13, and 14. Conclusion: The test results are not affected by hemolysis (hemoglobin <56mg / dL), lipemia (triglyceride concentration <1500mg / dL), jaundice (bilirubin <25mg / dL), or HAMA <5ng / dL.

[0122] Table 12 Working Concentration of Interference Substances

[0123] Table 13 Results of high-value interference experiments using the ON reagent kit

[0124] Table 14. Results of median interference test using the ON reagent kit

[0125] (5) Hook effect: Samples with a concentration 40 times higher than 1000 ng / mL were designated as high-value samples for the hook effect. Gradual dilution was performed until the detection signal showed a linear increase or approached a stable state. Testing showed that a sample concentration of 20000 ng / mL did not produce a hook effect for detection. The results are shown in Table 15.

[0126] Table 15 Hook effect test of ON reagent kit

[0127] Example 9: Differential expression of serum ON in different patients with acute chest pain

[0128] Eighty-five patients with acute arterial disease (AAD), 68 patients with acute myocardial infarction (AMI), and 18 patients with acute pelvic inflammatory disease (APE) from the People's Hospital of Xinjiang Uygur Autonomous Region were selected to form a test set. Serum ON expression was detected using a liquid chromatography-array kit to determine whether it could differentiate between these diseases. The experimental steps are as follows:

[0129] (1) Sample processing: Serum samples are used for all tests. The specimens are collected using yellow blood collection tubes containing separation gel. The specimens are centrifuged at 4°C and 3000 rpm for 10 minutes within 30 minutes after collection. The supernatant can be used for testing, or the supernatant can be stored at -80°C, but repeated freeze-thaw cycles should be avoided.

[0130] (2) Inclusion and Exclusion Criteria: 85 patients with confirmed AAD, 68 patients with AMI, and 18 patients with APE were included. Peripheral blood serum was collected from the subjects for liquid phase microarray analysis to verify whether ON could differentiate the above-mentioned chest pain diseases. Inclusion criteria for AAD group: Patients diagnosed with AAD by aortic CT angiography and aged ≥18 years; Inclusion criteria for APE group: Confirmed by positive spiral computed tomography or pulmonary angiography, with a high probability of ventilation-perfusion scintillation, or recording proximal deep vein thrombosis compression ultrasound or angiography in pulmonary angiography; Inclusion criteria for AMI group: Symptoms of ischemic chest pain, characteristic ST-T dynamic evolution on electrocardiogram or with abnormal Q waves, and elevated and decreased serum myocardial enzyme levels. Meeting two of these criteria was sufficient for diagnosis. Exclusion criteria for the three groups: Patients with a history of rheumatic diseases, autoimmune diseases, aortitis, pregnancy, or hereditary syndromes, as well as patients with severe pulmonary infections, abnormal liver and kidney function, or tumors were excluded. This study was approved by the Ethics Committee of the People's Hospital of Xinjiang Uygur Autonomous Region, and all participants signed informed consent forms for biological sample collection.

[0131] (3) Preparatory work before testing:

[0132] 1. Remove the kit from the refrigerator 10 minutes in advance and allow it to equilibrate to room temperature.

[0133] 2. Prepare seven 1.5mL EP tubes and label them S7-S0, arranging them in sequence. Transfer 120µl from the calibrator to tube S7. Add 60µl of sample diluent to each of tubes S6-S0. Transfer 60µl from tube S7 to tube S6, vortex to mix for 5-10 seconds. Transfer 60µl from tube S6 to tube S5, then to tube S4, continuing the serial dilution process and mixing thoroughly. Add 60µl of sample diluent to tube S6 as the zero-value calibrator.

[0134] 3. Mix the sample with the microsphere suspension containing the capture antibody. The analytes in the sample bind to the specific capture antibodies on the microspheres.

[0135] 4. Add detection antibody; the detection antibody specifically binds to the reaction product of the first step.

[0136] 5. Add fluorescein solution. The streptavidin on the fluorescein binds to the biotin on the detection antibody, ultimately forming a "capture antibody-antigen-biotinylated antibody-streptavidin-fluorescein complex on the surface of the microspheres".

[0137] 6. When using a flow cytometer to detect it, different microsphere complexes can be identified as specific analyte types and their corresponding concentrations can be analyzed, thus achieving quantitative detection of ON in the sample.

[0138] a. The composition of the liquid phase chip kit is shown in Table 16. All reagents used in this ON kit are the optimal choices confirmed in the above examples.

[0139] Table 16 Composition of the ON Reagent Kit

[0140] b. Experimental equipment required (self-provided): flow cytometer (Nova HT); high-precision pipettes and tips: 0.5-10μL, 5-50μL, 20-200μL, 200-1000μL; incubator, etc.

[0141] c. Operation and testing procedures:

[0142] Step 1 (Power-on): Add sheath fluid to the mark and discard the waste fluid. Turn on the instrument switch, sample tray switch, and software. After the software progress bar finishes, click Initialize. The instrument is ready to use after 30 minutes of startup.

[0143] Step 2 (Instrument Quality Control): Use a quality control ball to control the quality data. Only after confirming that the quality control data is normal can sample testing be carried out.

[0144] Step 3 (Circle): 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).

[0145] Step 4 (Establishing a Standard Curve): Create a new test project file using the sphere file, input the corresponding calibration concentration, and perform a standard test. After the test is completed, generate a fitted curve and save it.

[0146] Step 5 (Test Samples and Analysis): Perform sample testing using the project files from Step 4, and then export the raw data for analysis.

[0147] Step 6 (Power off): After the test is finished, perform cleaning and maintenance, and finally turn off all switches.

[0148] Using the above methods, ON was used as a biomarker to detect and analyze the AAD, AMI, and APE groups. The obtained data were analyzed using GraphPad Prism 9.0 statistical software to plot receiver operating characteristic (ROC) curves, 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.

[0149] (4) Results Analysis

[0150] As shown in Figure 1 and Table 17, the figure shows the expression level of ON as a biomarker in the serum of patients in the AAD, AMI, and APE groups. The results show that ON has good differentiation between the three groups, especially in distinguishing AAD patients.

[0151] As shown in Figure 2 and Table 18, this figure is the receiver operating characteristic (ROC) curve of ON as a biomarker in diagnosing AAD in patients with acute chest pain (AAD, AMI, and APE). The results show that the area under the ROC curve (AUC) is 0.8774, indicating that ON has good diagnostic value for AAD.

[0152] Table 17 Serum ON expression in the AAD, AMI, and APE groups

[0153] Table 18 ROC analysis of the ON kit for differentiating AAD in acute chest pain.

[0154] Example 10: Validation of ON-assisted diagnosis of acute chest pain

[0155] Using the inclusion and exclusion criteria described in Example 9, an additional 39 patients with AAD, 30 patients with AMI, and 13 patients with APE from the People's Hospital of Xinjiang Uygur Autonomous Region were selected to form a validation set. The ability of ON to assist in the diagnosis of acute chest pain was further validated using the same detection methods and data analysis methods as in Example 9.

[0156] The results are shown in Figures 3-4 and Tables 19-20. In the validation data, ON also showed significant expression differences among patients with acute chest pain (AAD), AMI, and APE, effectively distinguishing AAD patients. The ROC curves showed an AUC of 0.8137, a sensitivity of 69.77%, and a specificity of 94.87%, demonstrating that ON has good diagnostic value for AAD and great potential in the auxiliary diagnosis of acute chest pain.

[0157] Table 19 Serum ON expression in the AAD, AMI, and APE groups

[0158] Table 20 ROC analysis of the ON kit for differentiating AAD in acute chest pain.

[0159] Example 11: Chemiluminescence detection of ON protein in clinical samples

[0160] Using the inclusion and exclusion criteria described in Example 9, another 60 patients with AAD, 35 patients with AMI, and 27 patients with APE from the People's Hospital of Xinjiang Uygur Autonomous Region were selected, and the ability of ON to assist in the diagnosis of acute chest pain was further verified by chemiluminescence immunoassay.

[0161] (1) Detection procedure of chemiluminescence detection method:

[0162] Step 1: Immobilization and Blocking: Add the coated antibody to the wells and incubate overnight at 4°C or for 2 hours at 37°C. After incubation, wash the wells 3-5 times. Then add blocking buffer (such as PBS containing BSA) and incubate at 37°C for 1 hour. After incubation, wash again 3-5 times.

[0163] Step 2: Target Capture and Impurity Removal: Add standards or samples to the wells, along with enzyme-labeled antibodies. Incubate at 37°C for 30-60 minutes to allow the coating antibody, target material, and enzyme-labeled antibody to form a "sandwich" complex. After incubation, wash 3-5 times to remove unbound enzyme-labeled antibodies to avoid background interference.

[0164] Step 3: Add freshly prepared luminescent substrate to each well, mix gently (avoiding air bubbles), and incubate for 5-10 minutes. Then place the microplate in the analyzer and read the relative luminescence intensity (RLU) of each well.

[0165] Step 4: Data Calculation: Plot a standard curve with the concentration of the standard on the x-axis and the corresponding RLU value on the y-axis. Substitute the RLU value of the sample into the standard curve to calculate the concentration of the target substance in the sample (if the sample has been diluted, multiply by the corresponding dilution factor); simultaneously, use quality control samples for verification to ensure the validity of the test results.

[0166] (2) Results Analysis

[0167] The detection results were analyzed using the data analysis method described in Example 9. As shown in Figures 5-6 and Tables 21-22, the detection results of chemiluminescence and liquid-phase chip methods were consistent. ON also showed significant expression differences in patients with acute chest pain (AAD), AMI, and APE. The ROC curve showed an AUC of 0.9922, a sensitivity of 93.55%, and a specificity of 90.22%. The chemiluminescence detection results further validated that ON protein can assist in the diagnosis of acute chest pain, especially in effectively differentiating AAD.

[0168] Table 21 Serum ON expression in the AAD, AMI, and APE groups

[0169] Table 22 ROC analysis of the ON kit for differentiating AAD in acute chest pain

[0170] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

[0171] Sequence list (for informational purposes only)

[0172] Antibody 1 (A27152) clone number: 1F9

[0173] >A27152-Light chain full-length (FL) sequence (SEQ ID No. 1):

[0174] >A27152-Light chain variable region (VL) sequence (SEQ ID No. 2):

[0175] >A27152- Heavy chain full-length (FH) sequence (SEQ ID No. 3):

[0176] >A27152-Heavy chain variable region (VH) sequence (SEQ ID No. 4):

[0177] Antibody 2 (A27153) clone number: 1G8

[0178] >A27153-Light chain full-length (FL) sequence (SEQ ID No. 5):

[0179] >A27153-Light chain variable region (VL) sequence (SEQ ID No. 6):

[0180] >A27153- Heavy chain full-length (FH) sequence (SEQ ID No. 7):

[0181] >A27153-Heavy chain variable region (VH) sequence (SEQ ID No. 8):

[0182] Antibody 3 (A26850) clone number: 1E8

[0183] >A26850-Light Chain Full-Length (FL) Sequence (SEQ ID No. 9):

[0184] >A26850 - Light chain variable region (VL) sequence (SEQ ID No. 10):

[0185] >A26850- Heavy chain full-length (FH) sequence (SEQ ID No. 11):

[0186] >A26850-heavy chain variable region (VH) sequence (SEQ ID No. 12):

[0187] Antibody 4 (A27154) clone number: 1D10

[0188] >A27154-Light chain full-length (FL) sequence (SEQ ID No. 13):

[0189] >A27154-Light chain variable region (VL) sequence (SEQ ID No. 14):

[0190] >A27154- Heavy chain full-length (FH) sequence (SEQ ID No. 15):

[0191] >A27154-Heavy chain variable region (VH) sequence (SEQ ID No. 16):

[0192] Antibody 5 (A27155) clone number: 1B12

[0193] >A27155 - Light chain full-length (FL) sequence (SEQ ID No. 17):

[0194] >A27155-Light chain variable region (VL) sequence (SEQ ID No. 18):

[0195] >A27155- Heavy chain full-length (FH) sequence (SEQ ID No. 19):

[0196] >A27155-Heavy chain variable region (VH) sequence (SEQ ID No. 20):

[0197] Antibody 6 (A27156) clone number: 1B5

[0198] >A27156-Light chain full-length (FL) sequence (SEQ ID No. 21):

[0199] >A27156-Light chain variable region (VL) sequence (SEQ ID No. 22):

[0200] >A27156- Heavy chain full-length (FH) sequence (SEQ ID No. 23):

[0201] >A27156-Heavy chain variable region (VH) sequence (SEQ ID No. 24):

[0202] Antibody 7 (A27157) clone number: 1B10

[0203] >A27157-Light chain full-length (FL) sequence (SEQ ID No. 25):

[0204] >A27157-Light chain variable region (VL) sequence (SEQ ID No. 26):

[0205] >A27157- Heavy chain full-length (FH) sequence (SEQ ID No. 27):

[0206] >A27157-Heavy chain variable region (VH) sequence (SEQ ID No. 28):

[0207] Antibody 8 (A26849) clone number: 1H8

[0208] >A26849-Light chain full-length (FL) sequence (SEQ ID No. 29):

[0209] >A26849-Light chain variable region (VL) sequence (SEQ ID No. 30):

[0210] >A26849- Heavy chain full-length (FH) sequence (SEQ ID No. 31):

[0211] >A26849-Heavy chain variable region (VH) sequence (SEQ ID No. 32):

[0212] The amino acid sequence of the ON standard is (SEQ ID No. 33):

Claims

1. The use of an ON detection reagent in the preparation of a kit for the auxiliary identification of AAD in patients with acute chest pain.

2. The application according to claim 1, characterized in that, The detection reagent includes an antibody pair for specifically binding ON, the antibody pair comprising a capture antibody coupled to magnetic fluorescent microspheres and a biotinylated detection antibody.

3. The application according to claim 2, characterized in that, The capture antibody and detection antibody are each selected from two different antibodies chosen 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 in SEQ ID No. 2, and the heavy chain variable region sequence is shown in SEQ ID No. 4; the light chain variable region sequence of antibody 2 is shown in SEQ ID No. 6, and the heavy chain variable region sequence is shown in SEQ ID No. 8; the light chain variable region sequence of antibody 3 is shown in SEQ ID No. 10, and the heavy chain variable region sequence is shown in SEQ ID No. 12; 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; 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; 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; the light chain variable region sequence of antibody 6 is shown in SEQ ID No.

8. As shown in No. 22, the heavy chain variable region sequence is shown in SEQ ID No. 24; 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; 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.

4. The application according to claim 3, characterized in that, The antibody pair is selected from any one of the following antibody pairs 1 to 9:

5. The application according to any one of claims 1-4, characterized in that, The kit also includes a sample diluent comprising 30±5mM PBS, 0.25±0.05% BSA, 0.1±0.05% sucrose, 0.25±0.05% PEG6000, and 0.05±0.02% Proclin300, with a pH of 7.4±0.

2.

6. A kit for assisting in the identification of acute analgesia (AAD) in patients with acute chest pain, characterized in that, Including ON detection reagents.

7. The reagent kit according to claim 6, characterized in that, The detection reagent includes an antibody pair for specifically binding ON, the antibody pair comprising a capture antibody coupled to magnetic fluorescent microspheres and a biotinylated detection antibody.

8. The reagent kit according to claim 7, characterized in that, The capture antibody and detection antibody are each selected from two different antibodies chosen 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 in SEQ ID No. 2, and the heavy chain variable region sequence is shown in SEQ ID No. 4; the light chain variable region sequence of antibody 2 is shown in SEQ ID No. 6, and the heavy chain variable region sequence is shown in SEQ ID No. 8; the light chain variable region sequence of antibody 3 is shown in SEQ ID No. 10, and the heavy chain variable region sequence is shown in SEQ ID No. 12; 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; 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; 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; the light chain variable region sequence of antibody 6 is shown in SEQ ID No.

8. As shown in No. 22, the heavy chain variable region sequence is shown in SEQ ID No. 24; 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; 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.

9. The reagent kit according to claim 8, characterized in that, The antibody pair is selected from any one of the following antibody pairs 1 to 9:

10. The kit according to any one of claims 6-9, characterized in that, The kit also includes a sample diluent comprising 30±5mM PBS, 0.25±0.05% BSA, 0.1±0.05% sucrose, 0.25±0.05% PEG6000, and 0.05±0.02% Proclin300, with a pH of 7.4±0.2.

Images