Hybridoma cell lines 10B9E3-1 and 2D5C12-1 and applications of their secreted antibodies

Abstract

This invention provides a monoclonal antibody against smooth muscle myosin heavy chain (SMMHC), which specifically binds to SMMHC. The antibody is produced by hybridoma cell lines 10B9E3-1 (accession number CCTCC NO: C2021117) and 2D5C12-1 (accession number CCTCC NO: C2021119), respectively. Furthermore, the monoclonal antibodies secreted by hybridoma cell lines 10B9E3-1 and 2D5C12-1 exhibit good specificity. Based on this, an SMMHC serum detection kit developed using a chemiluminescence detection platform can be used to screen for SMMHC in patient serum, greatly improving the accuracy of early diagnosis of acute aortic dissection and providing a preliminary assessment of patient prognosis, reducing the mortality rate of aortic dissection. This kit offers advantages such as high sensitivity, good specificity, and accurate quantification.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to the application of hybridoma cell lines 10B9E3-1 and 2D5C12-1 and their secreted antibodies. Background Technology

[0002] With the modernization of people's lifestyles and the high incidence of diseases such as hypertension, diabetes, and atherosclerosis, the incidence of aortic disease in my country is rapidly increasing. Aortic disease is one of the cardiovascular diseases that seriously threaten human health. Even in the United States, with its advanced medical conditions and technology, 15,000 people die each year from aortic aneurysms and aortic dissections. Aortic dissection (AD) is an extremely dangerous acute cardiovascular disease that can cause serious complications in multiple systems (e.g., aortic rupture can cause cardiac tamponade, hemothorax, and retroperitoneal hematoma; aortic valve insufficiency can lead to left ventricular enlargement and heart failure; and impaired blood supply to vital organs can cause myocardial infarction, stroke, paraplegia, and ischemia of abdominal organs, etc.). It has an extremely high mortality rate and a high rate of misdiagnosis. AD is one of the three major critical illnesses with chest pain as the main symptom, after myocardial infarction and pulmonary embolism, especially acute aortic dissection (AAD), which has an extremely high mortality rate. In untreated aortic aortic dissection (AAD), approximately 1% of patients die every hour within the first 24 hours of onset, more than half die within one week, 70% within two weeks, and 90% within one year. With timely and appropriate drug and surgical treatment, the survival rate can be greatly improved, reducing the mortality rate to 18%–27%. Aortic dissection has multiple causes and various possible symptoms, leading to a high rate of misdiagnosis. Delayed diagnosis or inappropriate treatment due to misdiagnosis directly results in increased mortality; therefore, early diagnosis of aortic dissection is crucial to reducing mortality.

[0003] Currently, the diagnosis of aortic dissection mainly relies on a single mode of imaging examination results. CT angiography and magnetic resonance imaging can assess the entire aortic dissection with high sensitivity and specificity, but they also have significant limitations. The examination is expensive, requires patients to be transferred to a radiology department with specialized equipment, is time-consuming, and may worsen the disease, easily delaying treatment. Moreover, for patients with renal insufficiency or contrast agent allergy, it may lead to life-threatening serious conditions such as contrast agent nephropathy and anaphylactic shock, making it unsuitable for the early rapid diagnosis and exclusion of aortic dissection.

[0004] Therefore, there is an urgent need to find a breakthrough in the existing single diagnostic model of aortic dissection imaging, and to provide a simple, fast, and highly specific diagnostic model of peripheral blood for aortic dissection, which can be used for the diagnosis, exclusion, and prognostic assessment of AAD, and provide new tools for the diagnosis of AAD and early warning of death, so as to reduce the mortality rate of patients. Summary of the Invention

[0005] The purpose of this invention is to provide a simpler, faster, and more specific technical means for diagnosing, excluding, and assessing the prognosis of AAD.

[0006] In a first aspect of the invention, a monoclonal antibody against smooth muscle myosin heavy chain (SMMHC) is provided, the antibody specifically binding to SMMHC protein, and the antibody is produced by hybridoma cell line 10B9E3-1, the hybridoma cell line having accession number CCTCC NO:C2021117; or the antibody is produced by hybridoma cell line 2D5C12-1, the hybridoma cell line having accession number CCTCC NO:C2021119.

[0007] In another preferred embodiment, the antibody is used to detect SMMHC protein.

[0008] In another preferred embodiment, the SMMHC protein is human or recombinant.

[0009] In another preferred embodiment, the amino acid sequence of the SMMHC protein is shown in SEQ ID NO:1.

[0010] In another preferred embodiment, the EC50 of the antibody secreted by the hybridoma cell line 10B9E3-1 (accession number CCTCC NO: C2021117) with SMMHC protein is 0.50-2.00 ng / mL, more preferably 0.60-1.85 ng / mL.

[0011] In another preferred embodiment, the EC50 of the antibody secreted by the hybridoma cell line 2D5C12-1 (accession number CCTCC NO: C2021119) with SMMHC protein is 0.55-2.10 ng / mL, more preferably 0.70-1.80 ng / mL.

[0012] In another preferred embodiment, the antibody is of type IgG1.

[0013] In another preferred embodiment, the antibody titer is ≥1:512000.

[0014] In another preferred embodiment, the antibody specifically binds to the SMMHC protein.

[0015] In another preferred embodiment, the antibody does not bind to proteins in cardiac and smooth muscle other than SMMHC proteins.

[0016] In another preferred embodiment, the antibody does not bind to MYO protein, CTNI protein, and / or SMMHC protein.

[0017] In another preferred embodiment, the antibody is conjugated with or carries a detectable marker.

[0018] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0019] In another preferred embodiment, the antibody is used to detect SMMHC protein.

[0020] In a second aspect of the present invention, a hybridoma cell line is provided with accession number CCTCC NO:C2021117; or accession number CCTCC NO:C2021119; wherein the hybridoma cell line is capable of producing the anti-SMMHC monoclonal antibody as described in the first aspect of the present invention.

[0021] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0022] (i) a monoclonal antibody as described in the first aspect of the present invention;

[0023] (ii) Optional tag sequences to assist in expression and / or purification.

[0024] In another preferred embodiment, the tag for assisting expression and / or purification is selected from the group consisting of His tag, GST tag, HA tag, c-Myc tag, Flag tag, MBP tag, Avi tag, SUMO tag, or a combination thereof.

[0025] In a fourth aspect of the present invention, a detection system for detecting SMMHC protein is provided, the detection system comprising:

[0026] (i) Solid support Z0;

[0027] (ii) a first binding protein A, wherein the first binding protein A is a monoclonal antibody as described in the first aspect of the present invention or a recombinant protein as described in the third aspect of the present invention, and the first binding protein A is coated on the solid support Z0; and

[0028] (iii) Second binding protein B, which specifically binds to SMMHC protein and is coupled to or carries a detectable tag.

[0029] The binding between the second binding protein B and the SMMHC protein is not competitive with the binding between the first binding protein A and the SMMHC protein.

[0030] In another preferred embodiment, when the detection system contains the SMMHC protein to be detected, a complex as shown in Formula I can be formed in the detection system:

[0031] Z0-(ACB)n (I)

[0032] in,

[0033] Z0 is a solid support;

[0034] A is the first binding protein, wherein A is a monoclonal antibody as described in the first aspect of the present invention or a recombinant protein as described in the third aspect of the present invention, and is coated on the surface of a solid support.

[0035] B is a second binding protein that specifically binds to the SMMHC protein to be detected and is coupled with or carries a detectable tag; wherein the binding between B and the SMMHC protein is not competitive with the binding between A and the SMMHC protein.

[0036] C represents the SMMHC protein to be detected;

[0037] n is a positive integer greater than or equal to 1; and

[0038] "-" indicates a bond or linking group.

[0039] In another preferred embodiment, the solid support material is selected from the group consisting of metals, glass, colloids, plastics, or combinations thereof.

[0040] In another preferred embodiment, the solid support material includes: homopolymer, copolymer, or a combination thereof.

[0041] In another preferred embodiment, the solid support material is selected from the group consisting of polystyrene, polyethylene, polypropylene, or combinations thereof.

[0042] In another preferred embodiment, the solid support material is selected from the group consisting of microspheres, microporous plates, strips, test tubes, or combinations thereof.

[0043] In another preferred embodiment, the solid support is magnetic microparticles.

[0044] In another preferred embodiment, the concentration of the magnetic particles ranges from 0.1 to 10 mg / mL, more preferably from 0.3 to 3 mg / mL, and even more preferably from 0.5 to 2 mg / mL.

[0045] In another preferred embodiment, the first binding protein A is produced by the hybridoma cell line CCTCC NO:C2021117.

[0046] In another preferred embodiment, the concentration of the first binding protein A is in the range of 0.5-50 μg / mL, more preferably 1-20 μg / mL, and even more preferably 5-10 μg / mL.

[0047] In another preferred embodiment, the second binding protein B is selected from the group consisting of: rabbit anti-human SMMHC monoclonal antibody, rabbit anti-human SMMHC polyclonal antibody, mouse anti-human SMMHC monoclonal antibody, mouse anti-human SMMHC polyclonal antibody, and goat anti-human SMMHC polyclonal antibody.

[0048] In another preferred embodiment, the second binding protein B is a mouse anti-human SMMHC monoclonal antibody IgG.

[0049] In another preferred embodiment, the second binding protein B is produced by the hybridoma cell line CCTCC NO:C2021119.

[0050] In another preferred embodiment, the detectable marker is selected from the group consisting of fluorescent substances, radioactive elements, enzymes, chemiluminescent agents, colloidal gold, or combinations thereof.

[0051] In another preferred embodiment, the concentration of the second binding protein B is 0.05-5 μg / mL, more preferably 0.1-2 μg / mL, and even more preferably 0.5-1 μg / mL.

[0052] In a fifth aspect of the invention, a kit is provided, the kit comprising: a container and raw material reagents located within the container for forming a detection system as described in the fourth aspect of the invention.

[0053] In another preferred embodiment, the kit comprises:

[0054] (a) The first container and the solid support Z0 in the detection system located in the first container;

[0055] (b) The second container and the first binding protein A in the detection system located within the second container;

[0056] (c) The third container and the second binding protein B in the detection system located in the third container;

[0057] (d) Optionally, a fourth container and a buffer solution for the reaction system located in the fourth container;

[0058] (e) Optionally, a fifth container and the sample diluent contained in the fifth container; and

[0059] (f) Optionally, a sixth container and the washing liquid located in the sixth container.

[0060] In another preferred embodiment, the first container, the second container, and the third container may be the same or different containers.

[0061] In another preferred embodiment, the second binding protein B in the detection system located in the third container may be unlabeled during long-term storage, and may be labeled within a certain period of time before use as required.

[0062] In another preferred embodiment, the kit further includes a label or instruction manual indicating that the kit is used for (a) detecting SMMHC protein and / or (b) detecting or diagnosing aortic dissection.

[0063] In another preferred embodiment, the detection of aortic dissection is a plasma or serum test, preferably a serum test, and more preferably a human serum test.

[0064] In another preferred embodiment, the label or instruction manual includes the following information:

[0065] (i) If the SMMHC concentration in a sample from a test subject is higher than 1.20 ng / ml, the subject has a greater chance of developing aortic dissection than the normal population.

[0066] In another preferred embodiment, the object of detection is a person.

[0067] In another preferred embodiment, the sample is a serum or plasma sample.

[0068] In a sixth aspect of the invention, the use of monoclonal antibodies as described in the first aspect of the invention, hybridoma cell lines as described in the second aspect of the invention, or recombinant proteins as described in the third aspect of the invention, for the preparation of reagents or kits for the detection of SMMHC proteins is provided.

[0069] In another preferred embodiment, the kit is the kit as described in the fifth aspect of the invention.

[0070] In another preferred embodiment, the reagent or kit is a diagnostic reagent or kit for detecting aortic dissection.

[0071] In another preferred embodiment, the aortic dissection includes: acute aortic dissection and / or chronic aortic dissection.

[0072] In another preferred embodiment, the aortic dissection is an acute aortic dissection.

[0073] In a seventh aspect of the present invention, a method for detecting whether a sample contains SMMHC protein is provided, the method comprising:

[0074] A detection system as described in the fourth aspect of the present invention is provided, wherein the sample is added to the detection system to allow for a full reaction, and the resulting reaction solution is detected.

[0075] In another preferred embodiment, the sample is an ex vivo sample or an in vitro sample.

[0076] In another preferred embodiment, the sample is selected from the group consisting of serum samples, plasma samples, urine samples, tissue samples, or combinations thereof.

[0077] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0078] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0079] In another preferred embodiment, in the method, if a signal of a detectable marker coupled to or carried by the second binding protein B is detected, it indicates that the sample contains SMMHC protein; if no signal of a detectable marker coupled to or carried by the second binding protein B is detected, it indicates that the sample does not contain SMMHC protein.

[0080] In another preferred embodiment, the method is diagnostic.

[0081] In another preferred embodiment, the method is a method for the diagnosis and prognostic assessment of aortic dissection.

[0082] In another preferred embodiment, the method is also a chemiluminescence detection method.

[0083] In an eighth aspect of the present invention, a method for detecting or determining aortic dissection is provided, comprising the following steps:

[0084] (a) Provide an ex vivo test sample from the object being tested;

[0085] (b) Detect the expression level of SMMHC protein in the test samples; and

[0086] (c) Compare the SMMHC expression levels determined in (b) with a control reference value; wherein, compared with the control,

[0087] If the expression level of SMMHC in the test sample of the tested object is higher than the reference value A1,

[0088] This suggests that the individual being tested is more likely to have aortic dissection than the general healthy population.

[0089] In another preferred embodiment, the sample is from a test object.

[0090] In another preferred embodiment, the detection object is a human or a non-human mammal.

[0091] In another preferred embodiment, the sample is a serum sample, a plasma sample, a urine sample, a tissue sample, or a combination thereof.

[0092] In another preferred embodiment, the aortic dissection is an acute aortic dissection.

[0093] In another preferred embodiment, the detection step (b) includes detecting the amount of SMMHC mRNA; and / or detecting the amount of SMMHC protein.

[0094] In another preferred embodiment, the expression level of SMMHC protein in the sample is detected by quantitative real-time PCR or immunohistochemistry.

[0095] In another preferred embodiment, the expression level of SMMHC protein in a sample is detected using a kit as described in the fifth aspect of the invention.

[0096] In another preferred embodiment, the reference value A1 is a cut-off value.

[0097] In another preferred embodiment, the cut-off value is 1.20 ng / ml.

[0098] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0099] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0100] Figure 1 The antibody titer in mouse serum after the fourth immunization is shown. The final titer of the anti-SMMHC monoclonal antibody (10B9E3-1) after serial dilution was 1:512000.

[0101] Figure 2 The antibody titer in mouse serum after the fourth immunization is shown. The final titer of the anti-SMMHC monoclonal antibody (2D5C12-1) after serial dilution was 1:512000.

[0102] Figure 3 The SDS-PAGE protein electrophoresis report of the purified SMMHC recombinant antigen is shown. The report indicates that the molecular weight of the SMMHC recombinant antigen is approximately 90 kDa.

[0103] Figure 4The SDS-PAGE protein electrophoresis report of the purified SMMHC monoclonal antibody (10B9E3-1) shows that the heavy chain and light chain of this anti-SMMHC monoclonal antibody have molecular weights of approximately 50 kDa and 26 kDa, respectively.

[0104] Figure 5 The SDS-PAGE protein electrophoresis report of the purified SMMHC monoclonal antibody (2D5C12-1) shows that the heavy chain and light chain of this anti-SMMHC monoclonal antibody have molecular weights of approximately 50 kDa and 26 kDa, respectively.

[0105] Figure 6 The reactivity of the SMMHC monoclonal antibody (10B9E3-1) to SMMHC is shown. As can be seen from the figure, this SMMHC antibody has a good reactivity to the SMMHC antigen, and the antibody titer is 1:512000.

[0106] Figure 7 The reactivity of the SMMHC monoclonal antibody (2D5C12-1) to SMMHC is shown. As can be seen from the figure, this SMMHC antibody has a good reactivity to the SMMHC antigen, and the antibody titer is 1:512000.

[0107] Figure 8 The reactivity of the SMMHC monoclonal antibody (10B9E3-1) to MYO was shown. As can be seen from the figure, this SMMHC antibody showed no reactivity to the MYO antigen, indicating that the SMMHC monoclonal antibody has good specificity for MYO.

[0108] Figure 9 The reactivity of the SMMHC monoclonal antibody (2D5C12-1) to MYO was shown. As can be seen from the figure, this SMMHC antibody showed no reactivity to the MYO antigen, indicating that the SMMHC monoclonal antibody has good specificity for MYO.

[0109] Figure 10 The reactivity of the SMMHC monoclonal antibody (10B9E3-1) to CTNI was shown. As can be seen from the figure, this SMMHC antibody showed no reactivity to the CTNI antigen, indicating that the SMMHC monoclonal antibody has good specificity for CTNI.

[0110] Figure 11 The reactivity of the SMMHC monoclonal antibody (2D5C12-1) to CTNI was shown. As can be seen from the figure, this SMMHC antibody showed no reactivity to the CTNI antigen, indicating that the SMMHC monoclonal antibody has good specificity for CTNI.

[0111] Figure 12The reactivity of the SMMHC monoclonal antibody (10B9E3-1) to CNN1 is shown. As can be seen from the figure, this SMMHC antibody is not reactive to the CNN1 antigen, indicating that this SMMHC monoclonal antibody has good specificity for CNN1.

[0112] Figure 13 The reactivity of the SMMHC monoclonal antibody (2D5C12-1) to CNN1 is shown. As can be seen from the figure, this SMMHC antibody is not reactive to the CNN1 antigen, indicating that the SMMHC monoclonal antibody has good specificity for CNN1.

[0113] Figure 14 The ROC curve determined by the SMMHC reference value is shown. The area of ​​the ROC curve is 0.964 (95% CI, 0.940-0.989), and the p-value is 0.000 < 0.05, indicating that the model simulation is statistically significant.

[0114] Figure 15 The results show a comparison of serum SMMHC levels between healthy individuals and patients with aortic dissection. The comparison of serum SMMHC levels between healthy individuals and patients with aortic dissection was statistically significant (p<0.05). Detailed Implementation

[0115] Through extensive and in-depth research and screening, the inventors have developed for the first time a highly efficient monoclonal antibody pair and kit for detecting SMMHC protein. Specifically, this invention provides hybridoma cell lines 10B9E3-1 and 2D5C12-1 that secrete the anti-SMMHC monoclonal antibody of this invention. Results show that using the monoclonal antibody secreted by hybridoma cell line 10B9E3-1 as the primary antibody and the monoclonal antibody secreted by hybridoma cell line 2D5C12-1 as the secondary antibody, the presence of SMMHC protein in a sample can be efficiently detected using the antigen sandwich assay principle. This method has significant clinical application value. This invention was completed based on this foundation.

[0116] This invention relates to hybridoma cell lines 10B9E3-1 and 2D5C12-1, their preparation methods, and the anti-SMMHC monoclonal antibody secreted by them and its application in the serological detection of aortic dissection. Specifically, this invention provides hybridoma cell lines 10B9E3-1 and 2D5C12-1, which were deposited at the China Center for Type Culture Collection (CCTCC) on April 27, 2021. The accession number for hybridoma cell line 10B9E3-1 is CCTCC NO: C2021117; and the accession number for hybridoma cell line 2D5C12-1 is CCTCC NO: C2021119.

[0117] The present invention also provides a method for preparing the above-mentioned hybridoma cell lines 10B9E3-1 and 2D5C12-1, which are obtained by immunizing mice with mature SMMHC recombinant protein with SEQ ID No:1 as an antigen.

[0118] The present invention also provides two anti-SMMHC monoclonal antibodies, which are secreted by the hybridoma cell lines 10B9E3-1 and 2D5C12-1 described above.

[0119] The anti-SMMHC monoclonal antibodies secreted by the above hybridoma cell lines are all IgG1, exhibiting good specificity and high titers. Tests showed their titers to be as high as 1:512000.

[0120] This invention also provides a kit for detecting aortic dissection, comprising: an anti-SMMHC monoclonal antibody secreted by the hybridoma cell line 10B9E3-1 provided by this invention, which can be used as a capture antibody in the kit; and an anti-SMMHC monoclonal antibody secreted by the hybridoma cell line 2D5C12-1 provided by this invention, which can be used as a detection antibody in the kit.

[0121] The two antibodies together form a novel reagent kit that uses direct chemiluminescence detection as a platform to detect SMMHC in serum.

[0122] the term

[0123] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0124] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0125] Smooth muscle myosin heavy chain (SMMHC)

[0126] Smooth muscle myosin heavy chain is released from smooth muscle cells when the blood vessel wall is damaged, leading to an increase in serum concentration. This concentration returns to normal levels in healthy individuals after 24 hours, exhibiting a short window period, making it suitable for early and rapid diagnosis or exclusion of acute arterial disease (AAD) and for prognostic assessment. However, currently, there are no suitable antibodies available for the development of commercial kits. Therefore, the purpose of this invention is to provide hybridoma cells 10B9E3-1 and 2D5C12-1, their preparation methods, anti-SMMHC monoclonal antibodies secreted by these two hybridoma cell lines, and an SMMHC kit developed using this antibody based on an immunochemiluminescence platform for the diagnosis, exclusion, and prognostic assessment of AAD.

[0127] As used herein, the terms "antigen protein of the present invention," "SMMHC protein," "SMMHC polypeptide," or "smooth muscle myosin heavy chain SMMHC" are used interchangeably and all refer to proteins or polypeptides having the amino acid sequence (SEQ ID NO:1) of smooth muscle myosin heavy chain SMMHC. These include SMMHC with or without a starting methionine. Furthermore, the term also includes full-length SMMHC and fragments thereof. The SMMHC protein referred to in this invention includes its complete amino acid sequence, its secreted protein, its mutants, and its functionally active fragments.

[0128] Once the amino acid fragment of SMMHC is obtained, the nucleic acid sequence encoding it can be constructed, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the ECM1 nucleotide sequence disclosed in this invention, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0129] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0130] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0131] Currently, the DNA sequence encoding the protein of this invention (or its fragments, derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0132] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant ECM1 peptides. Generally, the following steps are involved:

[0133] (1) Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the SMMHC polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0134] (2) Host cells cultured in a suitable culture medium;

[0135] (3) Isolate and purify proteins from culture media or cells.

[0136] In this invention, the SMMHC polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0137] Methods well known to those skilled in the art can be used to construct expression vectors containing SMMHC-coding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0138] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0139] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0140] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces bacteria; fungal cells such as yeast; plant cells; insect cells; and animal cells.

[0141] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0142] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0143] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0144] Specific antibodies

[0145] As used herein, the terms “antibody of the present invention,” “anti-SMMHC monoclonal antibody,” and “anti-SMMHC specific antibody” are used interchangeably.

[0146] This invention also includes monoclonal antibodies specific to SMMHC proteins. Here, "specific" means that the antibody can bind to the SMMHC gene product or fragment. Preferably, it refers to antibodies that can bind to the SMMHC gene product or fragment but do not recognize or bind to other unrelated antigen molecules. Antibodies in this invention include molecules capable of binding to SMMHC proteins. This invention also includes antibodies capable of binding to modified or unmodified forms of the SMMHC gene product.

[0147] This invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have mouse antibody binding specificity but still retain the antibody portion derived from humans.

[0148] The antibodies of this invention can be prepared using various techniques known to those skilled in the art. For example, purified SMMHC gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing SMMHC proteins or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibodies of this invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al.). Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol. 6:511, 1976; Kohler et al., Eur.J.Immunol .6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cells Hybridomas (Elsevier, NY, 1981). The various antibodies of this invention can be obtained using fragments or functional regions of the SMMHC gene product through conventional immunoassay techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies binding to the unmodified form of the human ECM1 gene product can be produced by immunizing animals with the gene product generated in prokaryotic cells (e.g., E. coli); antibodies binding to the post-translational modified form (such as glycosylated or phosphorylated proteins or peptides) can be obtained by immunizing animals with the gene product generated in eukaryotic cells (e.g., yeast or insect cells).

[0149] Antibodies against SMMHC protein can be used in immunohistochemistry to detect SMMHC protein in specimens (especially plasma samples).

[0150] Affinity is a characteristic parameter of the relative state between antigens, antibodies, and antigen-antibody complexes in a reversible reaction; its more technical and terminological name is the dissociation equilibrium constant KD. In this invention, the EC50 (concentration for 50% of maximal effect, or EC50) of the antibody secreted by hybridoma cell line 10B9E3-1 or hybridoma cell line 2D5C12-1 with SMMHC protein is equivalent to the KD value of the antibody with SMMHC protein. The smaller the EC50 value, the stronger the affinity. The strength of the affinity determines the relative amounts of each component in the reversible reaction at the end of the reaction.

[0151] The antibodies secreted by the hybridoma cell line 10B9E3-1 (accession number CCTCC NO: C2021117) or hybridoma cell line 2D5C12-1 (accession number CCTCC NO: C2021119) of the present invention have good affinity.

[0152] In a specific embodiment of the present invention, the EC50 of the antibody secreted by hybridoma cell line 10B9E3-1 (accession number CCTCC NO: C2021117) with SMMHC protein is 0.50-2.00 ng / mL; preferably 0.60-1.85 ng / mL; the EC50 of the antibody secreted by hybridoma cell line 2D5C12-1 (accession number CCTCC NO: C2021119) with SMMHC protein is 0.55-2.10 ng / mL, preferably 0.70-1.80 ng / mL.

[0153] Coated antibody

[0154] As used herein, “coated antibody,” “capture antibody,” or “solid-phase antibody” are used interchangeably and refer to antibodies coated on a solid-phase carrier, i.e., the first binding protein in the detection system of this invention.

[0155] Coated antibodies can be non-specifically adsorbed or physically adsorbed onto materials such as polystyrene (ELISA plates) and nitrocellulose membranes, becoming solid-phase antibodies while retaining their immunological activity. The coated antibodies exhibit high affinity and specificity for the antigen, but do not affect the binding of the antigen to the detection antibody. When a sample to be tested is added, if the sample contains the target antigen, it will be captured by the coated antibody. The antigen (target antigen) described in this invention is an SMMHC protein.

[0156] In a preferred embodiment of the invention, the coated antibody binds to the SMMHC protein with high affinity and high specificity. In another preferred embodiment, the coated antibody is produced by the hybridoma cell line CCTCC NO:C2021117.

[0157] Antibody detection

[0158] As used herein, "detection antibody" or "labeled antibody" are used interchangeably to refer to an antibody used to detect whether a sample contains a target antigen, namely the second binding protein of this invention.

[0159] Detection antibodies typically carry detectable labels, including chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes. Detection antibodies exhibit high affinity and high specificity for antigens without affecting the binding of the antigen to the coated antibody. By utilizing the specific binding property of the detection antibody to the target antigen, the presence and quantity of the antigen are determined through the detection of the label. In a preferred embodiment of the invention, the detection antibody binds to SMMHC proteins with high affinity and high specificity.

[0160] In a preferred embodiment of the present invention, the detection antibody is produced by the hybridoma cell line CCTCC NO:C2021119.

[0161] Detection system and test kit

[0162] This invention provides a detection system for detecting SMMHC protein, the detection system comprising:

[0163] (i) Solid support Z0;

[0164] (ii) a first binding protein A, wherein the first binding protein A is a monoclonal antibody as described in the first aspect of the present invention or a recombinant protein as described in the third aspect of the present invention, and the first binding protein A is coated on the solid support Z0; and

[0165] (iii) Second binding protein B, which can specifically bind to SMMHC protein and is coupled to or carries a detectable tag.

[0166] The binding between the second binding protein B and the SMMHC protein is not competitive with the binding between the first binding protein A and the SMMHC protein.

[0167] In another preferred embodiment, when the detection system contains the SMMHC protein to be detected, a complex as shown in Formula I can be formed in the detection system:

[0168] Z0-(ACB)n (I)

[0169] in,

[0170] Z0 is a solid support;

[0171] A is the first binding protein, wherein A is a monoclonal antibody as described in the first aspect of the present invention or a recombinant protein as described in the third aspect of the present invention, and is coated on the surface of a solid support.

[0172] B is a second binding protein that specifically binds to the SMMHC protein to be detected and is coupled with or carries a detectable tag; wherein the binding between B and the SMMHC protein is not competitive with the binding between A and the SMMHC protein.

[0173] C represents the SMMHC protein to be detected;

[0174] n is a positive integer greater than or equal to 1; and

[0175] "-" indicates a bond or linking group.

[0176] The present invention also provides a kit for detecting aortic dissection. The kit contains the detection system described above.

[0177] Specifically, the kit includes:

[0178] (a) The first container and the solid support Z0 in the detection system located in the first container;

[0179] (b) The second container and the first binding protein A in the detection system located within the second container;

[0180] (c) The third container and the second binding protein B in the detection system located in the third container;

[0181] (d) Optionally, a fourth container and a buffer solution for the reaction system located in the fourth container;

[0182] (e) Optionally, a fifth container and the sample diluent contained in the fifth container; and

[0183] (f) Optionally, a sixth container and the washing liquid located in the sixth container.

[0184] In the kit provided by this invention, the solid-phase carrier contains a magnetic microparticle solid-phase conjugate, wherein the concentration of the magnetic microparticles ranges from 0.2 to 4 mg / mL, and the concentration of the antibody ranges from 0.5 to 25 μg / mL; the concentration of the antibody in the luminescent conjugate is 0.2 to 1.8 μg / mL.

[0185] The kit of this invention also contains calibrators. This typically refers to diluting a certain concentration of SMMHC antigen maternal antigen diluent into a series of antigen samples, usually diluted to six concentrations: 0 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL, denoted as CAL1, CAL2, CAL3, CAL4, CAL5, and CAL6.

[0186] The kit provided by this invention exhibits good sensitivity and specificity. With a Youden index of 0.755, the sensitivity and specificity are 80.5% and 95.0%, respectively, and the preliminary reference value (cutoff value) for SMMHC is determined to be 1.20 ng / mL. Furthermore, if the SMMHC concentration in a sample from a test subject is higher than 1.20 ng / mL, that subject has a higher probability of developing aortic dissection than the normal population.

[0187] Detection methods

[0188] This invention also provides a method for detecting or diagnosing aortic dissection, particularly a serological detection method. The aortic dissection includes acute aortic dissection and / or chronic aortic dissection. Preferably, the aortic dissection is acute aortic dissection.

[0189] In a preferred embodiment of the present invention, the SMMHC chemiluminescence detection kit of the present invention is used in conjunction with a chemiluminescence analyzer to detect SMMHC in serum samples.

[0190] Uses of the present invention

[0191] This kit is used to accurately detect the content of SMMHC in human serum and to detect changes in the amount of SMMHC in human serum. This invention can be used for the diagnosis and prognostic assessment of aortic dissection.

[0192] The main advantages of this invention include:

[0193] (1) The hybridoma cells provided by the present invention have high secretion yield, and the SMMHC monoclonal antibodies they secrete have advantages such as high titer and good specificity. They can be widely used in the field of SMMHC detection, such as the preparation of detection reagents or detection equipment.

[0194] (2) The hybridoma cell line provided by the present invention secretes IgG monoclonal anti-SMMHC antibody, which has extremely high specificity and sensitivity in binding to SMMHC protein.

[0195] (3) The antibodies secreted by the hybridoma cell line of the present invention can be applied to immunohistochemistry, immunoblotting, ELISA, chemiluminescence and other methodologies, and have a wide range of applications.

[0196] (4) The antibodies secreted by the hybridoma cell line of the present invention have the advantages of stable titer and low production cost, and have wide application value.

[0197] (5) The antibodies secreted by the hybridoma cell line of the present invention are applied to the SMMHC detection kit, which has the advantages of high sensitivity, good specificity and short detection time, and can better assist in the clinical diagnosis of aortic dissection.

[0198] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0199] Example 1: Screening and preparation of hybridoma cell lines

[0200] 1.1 Animal Immunization

[0201] The prepared myosin-11isoform SM1A protein was conjugated with ImmunoPlus and then used to immunize BLAB / C mice (n=1). A total of 5 immunizations were performed (multi-site immunization, 20ug per mouse each time), with 2 weeks as one immunization cycle.

[0202] 1.2 Cell Fusion and Screening

[0203] Three days prior to fusion, myosin-11isoform SM1A protein was plated, and the titer of mouse tail serum was detected using an indirect ELISA method. Finally, BLAB / C mice with high immunogenicity (1:8000) were selected for booster immunization. Three days later, the spleens of the immunized mice were removed, crushed, and fused with SP2 / 0 mouse myeloma cell lines using PEG (Sigma, P7181).

[0204] 1.3 Screening of hybridoma-positive cell lines

[0205] Fusion cells were added to 96-well plates pre-coated with feeder cells. After 7-10 days, the supernatant of the fusion cells was screened using an indirect ELISA method, selecting the supernatant that was positive for the target protein myosin-11 isoform SM1A. For all positive clones obtained in the initial screening stage, the supernatant of all positive maternal clones was screened using an indirect ELISA method, while a reverse screening using His-tagged proteins showed negative results. The positive clones obtained in this stage were: 2D5C12, 7F11C4, 8C8E11, 10B9E3, 13D7E5, and 15D5A3.

[0206] 1.4 Limiting dilution method for cloning and screening

[0207] Subcloning was performed on the positive clones obtained in step c using a limiting dilution method. Each single clone was visually inspected at least three times until a hybridoma cell line stably secreting anti-myosin-11isoform SM1A protein was selected. The subclones obtained at this stage are: 2D5C12-1, 2D5C12-2, 7F11C4-1, 7F11C4-2, 8C8E11-1, 8C8E11-2, 10B9E3-1, 10B9E3-2, 13D7E5-1, 13D7E5-2, 15D5A3-1, and 15D5A3-2.

[0208] 1.5 Determination of titer of subclonal cell supernatant

[0209] The potency of the subclones obtained in step d was determined using an indirect ELISA method. The specific steps of the indirect ELISA method are as follows:

[0210] 1) Antigen coating: Recombinant SMMHC antigen was selected, diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to a polystyrene 96 reaction plate and incubated overnight at 4°C.

[0211] 2) Washing: The next day, pour out the liquid from the concave hole and wash 3 times with washing solution.

[0212] 3) Blocking: Add 100 μL / well blocking solution and let stand at room temperature for 0.5 h.

[0213] 4) Washing: Wash 3 times with detergent.

[0214] 5) Add test samples (cell supernatant): Continuously dilute the cell supernatant containing monoclonal antibodies with PBS in a specific gradient (1:10, 1:30, 1:90, 1:270, 1:810, 1:2430), adding 100 μL / well to the coated plate. Perform two parallel tests for each gradient. Use PBS as a negative control. Cover and incubate at 37°C for 1-2 hours.

[0215] 6) Washing: Wash 3 times with detergent.

[0216] 7) Add enzyme-labeled secondary antibody: rabbit anti-mouse IgG-HRP, diluted 1:8000 with blocking buffer, 100 μL / well, capped and incubated at 37℃ for 1 h.

[0217] 8) Washing: Wash 5 times with detergent.

[0218] 9) Color development: Add 100 μL of substrate per well and incubate at room temperature in the dark for 5-30 min until a blue color is developed.

[0219] 10) Terminate the reaction and perform colorimetric analysis: Add 50 μL of stop solution per well. The color will turn yellow. Measure the absorbance of each well at 450 nm using a microplate reader. The maximum dilution of the positive reaction (where the absorbance of the positive sample / absorbance of the negative sample is ≥2.1) is the titer of the sample to be tested.

[0220] The specific measurement results are shown in Table 1:

[0221] Table 1. Antibody titer determination in hybridoma cell supernatant.

[0222]

[0223]

[0224]

[0225] As shown in Table 1 above, the following conclusions were drawn from the potency determination:

[0226] 1) It was determined that all 12 subclones in this experiment had a certain titer and were all effective cell lines.

[0227] 2) Based on the ELISA indirect method titer determination criteria (OD sample absorbance / OD control sample absorbance ≥ 2.1, the data is valid and can be further diluted), subclones with higher OD ratios at the same dilution, i.e., subclones with higher titers, were selected as follows: 2D5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1, and 15D5A3-1.

[0228] Example 2: Preparation, purification, and screening of monoclonal antibodies

[0229] According to Example 1, this invention uses a method of antibody production from cell supernatant culture, and employs affinity chromatography purification using a Protein A pre-packed column. Specifically, the affinity column is first equilibrated with 1×PBS, then the cell supernatant (adjusted to pH 8.0 with Tris) after centrifugation at 10,000 rpm is loaded onto the column. After washing with 5 column volumes of 1×PBS, elution is finally performed with pH 3.50 1 MJly (adjusted to pH 7.4 with Tris pH 8.8), followed by dialyzing in 1×PBS buffer for Lowery quantification.

[0230] The specific screening and matching results are shown in Table 2:

[0231] Table 2 Results of antibody cross-pairing test

[0232]

[0233]

[0234] Note: B is a commercially available antibody recommended for labeling; P is a commercially available antibody.

[0235] As shown in Table 2, the following conclusions were drawn from the cross-pairing experiments on the chemiluminescence platform:

[0236] 1) The selected 6 subclonal antibodies showed some reactivity with each other, and the following pairs showed good reactivity: 2D5 C12-1 (coated)-13D7E5-1 (labeled); 7F11C4-2 (coated)-2D5 C12-1 (labeled); 8C8E11-2 (coated)-15D5A3-1 (labeled); 10B9E3-1 (coated)--2D5C12-1 (labeled); 13D7E5-1 (coated)-15D5A3-1 (labeled); 15D5A3-1 (coated)-7F11C4-2 (labeled).

[0237] 2) The purchased antibody P (coated) and antibody B (recommended for labeling) have a certain reactivity when cross-paired.

[0238] 3) The purchased antibody B (recommended for labeling) and the 6 selected antibodies (2D5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1, 15D5A3-1) showed no reactivity when cross-paired.

[0239] 4) The purchased antibody P (used for labeling) and the 6 selected antibodies (2D5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1, 15D5A3-1) were cross-paired, and 10B9E3-1 (coated) -- P (labeled) showed the best reactivity.

[0240] Based on the above four conclusions, the following pairs were initially selected: 2D5 C12-1 (coated)-13D7E5-1 (labeled); 7F11C4-2 (coated)-2D5 C12-1 (labeled); 8C8E11-2 (coated)-15D5A3-1 (labeled); 10B9E3-1 (coated)--2D5C12-1 (labeled); 13D7E5-1 (coated)-15D5A3-1 (labeled); 15D5A3-1 (coated)-7F11C4-2 (labeled); P (coated)-B (labeled); 10B9E3-1 (coated)--P (labeled).

[0241] Given the concentration ratio of antigen samples CAL1-CAL6 used in the paired assay, the optimal luminescence values ​​of CAL2-CAL5 increase sequentially by a factor of 2, with the luminescence value of the CAL6 antigen sample being approximately five times that of the CAL5 antigen sample. Simultaneously, considering the principle of prioritizing luminescence value—that is, higher concentration samples with higher luminescence values ​​indicate greater sensitivity and better reagent performance—10B9E3-1 (coated) – 2D5C12-1 (labeled) were ultimately selected as the final paired antibodies. The secretory cells corresponding to the 10B9E3-1 and 2D5C12-1 antibodies are the cell lines provided in this invention.

[0242] The serum titers of 10B9E3-1 and 2D5C12-1 and the secreted antibody levels in mice after four immunizations are shown in the figures below. Figure 1 and Figure 2 After four immunizations, the serum antibody titer was consistently 1:512000, and ELISA testing identified it as IgG1. The SDS-PAGE protein electrophoresis reports for the purified SMMHC recombinant antigen and monoclonal antibody are shown below. Figure 3 , Figure 4 and Figure 5 It can be concluded that the molecular weight of the SMMHC recombinant antigen is 90kDa, and the molecular weights of the heavy chain and light chain of the anti-SMMHC monoclonal antibody are 50kDa and 26kDa, respectively.

[0243] The experimental procedure for reacting antibodies secreted by hybridoma cell lines 10B9E3-1 (accession number CCTCC NO: C2021117) and 2D5C12-1 (accession number CCTCC NO: C2021119) with the EC50 of SMMHC protein is as follows:

[0244] 1. Preparation of SMMHC-coated microplates

[0245] Take 100 μg / mL SMMHC antigen, dissolve it at room temperature, mix well, and dilute it with coating buffer to 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL as follows. Add 100 μL / well according to the sample loading layout table and add it to the enzyme-labeled strips respectively. Add coating buffer as a blank control for coating antibody. Incubate overnight at 2-8℃.

[0246] 2. Wash the plate three times with washing solution, pat dry, add 300 μL / well blocking solution and block at room temperature for 1 hour; wash the plate three times with washing solution, pat dry and set aside for use.

[0247] 3. Dilute the antibody serially 3-fold starting at 100,000 ng / mL according to the layout, and add 100 μL / well to the microplate according to the layout;

[0248] 4. Place it in a microplate shaker and set it to 37°C and 600 rpm for 1 hour;

[0249] 5. Wash the plate three times with detergent and pat dry;

[0250] 6. Dilute the enzyme-linked antibody to an appropriate concentration and mix well on a vortex mixer, 100 μL / well;

[0251] 7. Place it in a microplate shaker and set it to 37°C and 600 rpm for 1 hour;

[0252] 8. Wash the plate 4 times with detergent and pat dry;

[0253] 9. Take the colorimetric solution corresponding to the enzyme-linked antibody, take it out 20 minutes before use and bring it to room temperature, then mix it on a vortex mixer.

[0254] 10. Add the developing solution at a rate of 100 μL / well using an 8-channel gun;

[0255] 11. Let the colorimetric solution stand at room temperature away from light for 10-30 minutes;

[0256] 12. Use an 8-channel pipette to add stop solution at 100 μL / well to terminate the reaction; (this step may not be necessary depending on the substrate);

[0257] 13. Measure the signal value using an ELISA reader;

[0258] 14. Results Analysis

[0259] Affinity is a characteristic parameter of the relative state between antigens, antibodies, and antigen-antibody complexes in a reversible reaction. Its more technical and technical name is the dissociation equilibrium constant, KD. EC50 (the concentration for 50% of the maximal effect, which is the concentration at which a 50% maximal effect is achieved) is equivalent to the KD value. A smaller EC50 value indicates stronger affinity, and the strength of the affinity ultimately determines the relative proportions of each component in the reversible reaction at its completion.

[0260] The EC50 values ​​of antibodies secreted by hybridoma cell lines 10B9E3-1 (accession number CCTCC NO: C2021117) and 2D5C12-1 (accession number CCTCC NO: C2021119) and SMMHC protein were 0.60-1.85 ng / mL and 0.70-1.80 ng / mL, respectively.

[0261] Example 3: Specificity detection of anti-SMMHC monoclonal antibodies secreted by hybridoma cells 10B9E3-1 and 2D5C12 provided by the present invention.

[0262] In this embodiment, the SMMHC recombinant antigen and MYO, CTNI, and CNN1 from Example 1 were used as coating antigens, and the monoclonal antibody prepared in Example 2 was used as the recognition antibody. The indirect method was used to detect SMMHC.

[0263] 3.1 Coating of ELISA plates

[0264] Dilute the coating antigen to 1 μg / mL with coating buffer (CBS: Na2CO3 0.8g, NaHCO3 1.46g, Procling 300 1mL, distilled water, pH 9.0, bring the volume to 1L). Add 0.1mL to each well of a 96-well plate and seal the plate overnight at 4°C.

[0265] 3.2 Blocking of ELISA plates

[0266] Remove the ELISA plate that has been sealed overnight at 4°C, discard the supernatant, pat the plate dry, add 300 μL / well blocking buffer (3% BSA + PBS), and incubate at 37°C for 2 hours.

[0267] 3.3 Washing the ELISA plate

[0268] Remove the ELISA plate from the incubator after incubating at 37℃ for 2 hours, discard the supernatant, and remove the plate. Wash the plate 5 times with washing buffer (PBST: KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5mL, diluted with water to 1L).

[0269] 3.4 Detection of anti-SMMHC antibodies by indirect ELISA.

[0270] After washing and drying the ELISA plates, add 100 μL / well of anti-SMMHC monoclonal antibody secreted by hybridoma cell lines 10B9E3-1 and 2D5C12 to ELISA plates coated with different antigens. For the control assay, add physiological saline. Incubate at 37°C for 2 hours, wash 5 times, add 100 μL / well of HRP-labeled goat anti-mouse IgG secondary antibody, incubate at 37°C for 1 hour, wash 5 times, add 100 μL / well of TMB substrate (purchased from Sigma), incubate at 37°C for 10 minutes. When a blue color appears, stop the reaction by adding 2M concentrated sulfuric acid; the color turns yellow. Measure the OD using an ELISA reader. 450nm The results were compared and analyzed with a control experiment. Final results: Figure 6 , 7 It can be concluded that the anti-SMMHC monoclonal antibody in Example 2 can only detect SMMHC protein antigen and the antibody detection titer is 1:512000; Figure 8 , 9It can be concluded that anti-SMMHC monoclonal antibodies are unresponsive to MYO protein; by Figure 10 , 11 Anti-SMMHC monoclonal antibodies are not reactive to CTNI protein; Figure 12 , 13 The anti-SMMHC monoclonal antibody showed no reactivity to CNN1 protein. Therefore, it can be concluded that the anti-SMMHC monoclonal antibody provided by this invention has good specificity.

[0271] Example 4: Refinement of the preparation components of the reagent kit provided by the present invention.

[0272] The chemiluminescence-based detection kit for SMMHC provided by this invention mainly consists of two parts: a magnetic microparticle solid-state conjugate and a luminescent conjugate. The magnetic microparticle solid-state conjugate is made by coating magnetic microparticles with anti-SMMHC monoclonal antibody secreted by hybridoma cells 10B9E3-1 (Example 2). The concentration of magnetic microparticles in the solid-state conjugate system ranges from 0.2-4 mg / mL, and the antibody concentration ranges from 0.5-25 μg / mL. The luminescent conjugate is made by labeling acrid ester with anti-SMMHC monoclonal antibody secreted by hybridoma cells 2D5C12-1 (Example 2). The antibody concentration in the luminescent conjugate is 0.2-1.8 μg / mL. Additionally, calibrators (6-point calibration) and quality control samples (2-point quality control) are included.

[0273] The reagent kit provided by this invention, when used with a direct chemiluminescence immunoassay analyzer, has advantages such as high sensitivity, high specificity, high accuracy, wide linear range, and high-throughput rapid detection.

[0274] Example 5: Method of using the reagent kit provided by the present invention

[0275] 5.1 Calibration Curve Creation:

[0276] 1) Place the reagents containing the solid conjugate and the luminescent conjugate into the reagent tray of the fully automated chemiluminescence immunoassay analyzer (Cosmax Smart 6500, or TESMI i 100) and detect the reagent volume.

[0277] 2) Place the calibrator into the sample rack and transfer it to the sample channel of the fully automated chemiluminescence immunoassay analyzer. The sample volume is 50 μL.

[0278] 3) Reaction mode: Two-step method, the first step reaction is incubated for 6 min, the second step reaction is incubated for 6 min, incubated at 37℃, two-step washing, each step washing 3 times;

[0279] 4) The fully automated chemiluminescence immunoassay analyzer outputs chemiluminescence results;

[0280] 5) Based on the luminescence value of the calibrator, create the corresponding calibration curve.

[0281] 5.2 Test procedure calibration:

[0282] 1) Place the quality control sample into the sample rack and transfer it to the sample channel of the fully automated chemiluminescence immunoassay analyzer. The sample volume is 50 μL.

[0283] 2) Reaction mode: Two-step method, the first step reaction is incubated for 6 min, the second step reaction is incubated for 6 min, incubated at 37℃, two-step washing, each step washing 3 times;

[0284] 3) The fully automated chemiluminescence immunoassay analyzer outputs chemiluminescence results;

[0285] By combining the test results of the calibration curve and the quality control sample, if the quality control sample is within the quality control range, it indicates that the measurement procedure is within the quality control range and the test results are reliable; otherwise, the reagents and instruments need to be re-examined and verified.

[0286] 5.3 Sample Testing:

[0287] Based on the above, if the quality control sample is within a controllable range, sample testing can be performed. The specific steps are as follows:

[0288] 1) Place the sample into the sample rack and transport it to the sample channel of the fully automated chemiluminescence immunoassay analyzer. The sample volume is 50 μL (the same as for serum samples for plasma samples);

[0289] 2) Reaction mode: Two-step method, the first step reaction is incubated for 6 min, the second step reaction is incubated for 6 min, incubated at 37℃, two-step washing, each step washing 3 times;

[0290] 3) The fully automated chemiluminescence immunoassay analyzer outputs chemiluminescence results;

[0291] By combining the calibration curve and the sample luminescence value, the final test result of the sample is obtained.

[0292] Example 6: Performance of the reagent kit provided by the present invention

[0293] 6.1 Limit of Detection (LOD)

[0294] The test was performed using a zero-concentration calibrator or sample diluent as the sample, and the measurement was repeated 20 times. The RLU values ​​(relative luminescence values) of the 20 measurements were obtained, and their mean (M) and standard deviation (SD) were calculated. The RLU value corresponding to M+2SD was then obtained. According to the calibration curve equation of the calibrator used in the kit, the RLU value corresponding to M+2SD was substituted into the above equation to find the corresponding concentration value, which is the limit of detection.

[0295] Based on the test results, a four-parameter Logistic curve was fitted to obtain the calibration curve as follows:

[0296] Equation: y = (AD) / [1 + (x / C)^B] + D

[0297] A = 6.04309

[0298] B = -0.57649

[0299] C = 35.77110

[0300] D = 2.58059

[0301] r^2 = 0.99986

[0302] The limit of detection is 0.13 ng / mL.

[0303] 6.2 Accuracy

[0304] Sample (A) was added to sample (B) at a volume ratio of 1:9.

[0305] The recovery rate should be between 85% and 115%.

[0306]

[0307] In the formula:

[0308] R – Recovery rate;

[0309] V – Volume of liquid A;

[0310] V0 — Volume of liquid B;

[0311] C – The concentration of solution B after adding solution A;

[0312] C0—Detection concentration of solution B;

[0313] Cs — Concentration of solution A.

[0314] The concentrations of samples A, B, and the mixed sample were measured to be 88.27 ng / ml, 12.22 ng / ml, and 21.03 ng / ml, respectively. The recovery rate of this test was 113.6%, which meets the requirements.

[0315] Example 7: Detection of clinical serum samples using the SMMHC chemiluminescence detection kit provided by the present invention.

[0316] 1. Collection of clinical serum samples

[0317] Samples from 41 patients with aortic dissection and 180 healthy individuals were collected from a hospital in Shanghai. The kits provided in Example 5 were used for testing. The SMMHC test results of the patients and healthy individuals are shown in Table 3.

[0318] Table 3. Serum test results of healthy individuals and patients with aortic dissection.

[0319]

[0320]

[0321]

[0322] Based on the data from Table 3 for healthy individuals and patients with aortic dissection, the ROC curve method of this invention was used to fit the reference values ​​for SMMHC detection using this kit, resulting in the ROC curve diagram, as shown below. Figure 14 The area under the ROC curve is shown in Table 4.

[0323] Table 4. Area under the curve

[0324] Test result variable: SMMHC test

[0325]

[0326] a. Under nonparametric assumptions

[0327] b. Null hypothesis: Real area = 0.5

[0328] The area under the ROC curve in the table above is 0.964 (95% CI, 0.940-0.989), and the p-value is 0.000 < 0.05, indicating that the model simulation is statistically significant. With a Youden index of 0.755, the sensitivity and specificity are 80.5% and 95.0%, respectively, leading to a preliminary reference value of 1.20 ng / mL for SMMHC. Based on this reference value, the detection kit provided by this invention demonstrates high accuracy compared to clinical settings. Figure 15 As can be seen, the average values ​​for healthy individuals and patients were (0.41±0.19) ng / mL and (7.55±2.88) ng / mL, respectively. The serum level of SMMHC patients was 18 times that of healthy individuals (p<0.05), which further demonstrates that the kit provided by this invention has very high specificity and sensitivity.

[0329] Example 8: Clinical Cross-Sample Validation

[0330] Ten clinical samples of myocardial injury with CTNT concentration levels ranging from 0.014 to 0.06 ng / ml were collected and tested using the kit provided in this invention. The test results are shown in Table 5.

[0331] Table 5. Test results of myocardial injury samples

[0332]

[0333]

[0334] As shown in Table 5, the test results of the kit provided by this invention for 10 clinical myocardial injury samples were all less than 1.20 ng / ml, and all were negative (the cutoff value of the kit provided by this invention is 1.20 ng / ml). Therefore, there is no overlap between the aortic dissection samples and the myocardial injury samples tested by the kit provided by this invention.

[0335] strain preservation

[0336] The hybridoma cell line 10B9E3-1 for producing anti-smooth muscle myosin heavy chain (SMMHC) monoclonal antibody of the present invention was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan, China) on April 27, 2021, with accession number CCTCC NO: C2021117.

[0337] The hybridoma cell line 2D5C12-1 for producing anti-smooth muscle myosin heavy chain (SMMHC) monoclonal antibody of the present invention was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan, China) on April 27, 2021, with accession number CCTCC NO:C2021119.

[0338] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai TransGen Diagnostics Technology Co., Ltd. Shanghai TransGen Biotech Co., Ltd. <120> Applications of hybridoma cell lines 10B9E3-1 and 2D5C12-1 and their secreted antibodies <130> P2021-1160 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 798 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Gln Lys Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val 1 5 10 15 Asp Lys Asn Phe Ile Asn Ser Pro Val Ala Gln Ala Asp Trp Ala Ala 20 25 30 Lys Arg Leu Val Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala 35 40 45 Ser Ile Lys Glu Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu 50 55 60 Asn Gly Lys Lys Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn 65 70 75 80 Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu 85 90 95 Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly 100 105 110 Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr 115 120 125 Lys His Leu Pro Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly 130 135 140 Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr 145 150 155 160 Ala Tyr Arg Ser Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys 165 170 175 Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile 180 185 190 Gln Tyr Leu Ala Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Thr 195 200 205 Ser Ile Thr Gly Glu Leu Glu Lys Gln Leu Leu Gln Ala Asn Pro Ile 210 215 220 Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser 225 230 235 240 Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile 245 250 255 Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Ile 260 265 270 Arg Gln Ala Arg Asp Glu Arg Thr Phe His Ile Phe Tyr Tyr Met Ile 275 280 285 Ala Gly Ala Lys Glu Lys Met Arg Ser Asp Leu Leu Leu Glu Gly Phe 290 295 300 Asn Asn Tyr Thr Phe Leu Ser Asn Gly Phe Val Pro Ile Pro Ala Ala 305 310 315 320 Gln Asp Asp Glu Met Phe Gln Glu Thr Val Glu Ala Met Ala Ile Met 325 330 335 Gly Phe Ser Glu Glu Glu Gln Leu Ser Ile Leu Lys Val Val Ser Ser 340 345 350 Val Leu Gln Leu Gly Asn Ile Val Phe Lys Lys Glu Arg Asn Thr Asp 355 360 365 Gln Ala Ser Met Pro Asp Asn Thr Ala Ala Gln Lys Val Cys His Leu 370 375 380 Met Gly Ile Asn Val Thr Asp Phe Thr Arg Ser Ile Leu Thr Pro Arg 385 390 395 400 Ile Lys Val Gly Arg Asp Val Val Gln Lys Ala Gln Thr Lys Glu Gln 405 410 415 Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu 420 425 430 Phe Arg Trp Ile Leu Thr Arg Val Asn Lys Ala Leu Asp Lys Thr His 435 440 445 Arg Gln Gly Ala Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu 450 455 460 Ile Phe Glu Val Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn 465 470 475 480 Glu Lys Leu Gln Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln 485 490 495 Glu Glu Tyr Gln Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly 500 505 510 Leu Asp Leu Gln Pro Cys Ile Glu Leu Ile Glu Arg Pro Asn Asn Pro 515 520 525 Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala 530 535 540 Thr Asp Lys Ser Phe Val Glu Lys Leu Cys Thr Glu Gln Gly Ser His 545 550 555 560 Pro Lys Phe Gln Lys Pro Lys Gln Leu Lys Asp Lys Thr Glu Phe Ser 565 570 575 Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Asn Ala Ser Ala Trp Leu 580 585 590 Thr Lys Asn Met Asp Pro Leu Asn Asp Asn Val Thr Ser Leu Leu Asn 595 600 605 Ala Ser Ser Asp Lys Phe Val Ala Asp Leu Trp Lys Asp Val Asp Arg 610 615 620 Ile Val Gly Leu Asp Gln Met Ala Lys Met Thr Glu Ser Ser Leu Pro 625 630 635 640 Ser Ala Ser Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu 645 650 655 Tyr Lys Glu Gln Leu Gly Lys Leu Met Thr Thr Leu Arg Asn Thr Thr 660 665 670 Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ser Gly 675 680 685 Lys Leu Asp Ala Phe Leu Val Leu Glu Gln Leu Arg Cys Asn Gly Val 690 695 700 Leu Glu Gly Ile Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val 705 710 715 720 Phe Gln Glu Phe Arg Gln Arg Tyr Glu Ile Leu Ala Ala Asn Ala Ile 725 730 735 Pro Lys Gly Phe Met Asp Gly Lys Gln Ala Cys Ile Leu Met Ile Lys 740 745 750 Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile 755 760 765 Phe Phe Arg Thr Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu 770 775 780 Lys Ile Thr Asp Val Ile His His His His His His His His 785 790 795

Claims

1. A monoclonal antibody against smooth muscle myosin heavy chain (SMMHC), characterized in that, The antibody can specifically bind to the SMMHC protein, and the antibody is produced by hybridoma cell line 10B9E3-1, the hybridoma cell line preservation number is CCTCC NO:C2021117; or the antibody is produced by hybridoma cell line 2D5C12-1, the hybridoma cell line preservation number is CCTCC NO:C2021119.

2. A hybridoma cell line, characterized in that, The accession number is CCTCC NO:C2021117; or the accession number is CCTCC NO:C2021119; the hybridoma cell line described herein is capable of producing the anti-SMMHC monoclonal antibody as described in claim 1.

3. A recombinant protein, characterized in that, The recombinant protein consists of (i) and (ii): (i) the monoclonal antibody as described in claim 1; (ii) Tag sequences that assist in expression and / or purification.

4. A detection system for detecting SMMHC protein, characterized in that, The detection system includes: (i) Solid support Z0; (ii) a first binding protein A, wherein the first binding protein A is a monoclonal antibody as described in claim 1 or a recombinant protein as described in claim 3, and the first binding protein A is coated on the solid-phase support Z0; and (iii) Second binding protein B, which specifically binds to SMMHC protein and has a detectable label on it; The binding between the second binding protein B and the SMMHC protein is not competitive with the binding between the first binding protein A and the SMMHC protein.

5. The detection system as described in claim 4, characterized in that, The first binding protein A was produced by hybridoma cell line CCTCC NO:C2021117.

6. The detection system as described in claim 4, characterized in that, The antibody titer is ≥1:512000.

7. A reagent kit, characterized in that, The kit includes: a container and raw material reagents located within the container for forming the detection system as described in claim 4.

8. The kit according to claim 7, characterized in that, The kit also includes a label or instruction manual indicating that the kit is used for (a) detecting SMMHC protein and / or (b) detecting or diagnosing aortic dissection.

9. The reagent kit as described in claim 8, characterized in that, The label or instruction manual shall state the following: (i) If the SMMHC concentration in a sample from a test subject is higher than 1.20 ng / ml, the subject has a greater chance of developing aortic dissection than the normal population.

10. The kit according to claim 9, characterized in that, The sample is a serum or plasma sample.

11. The use of the monoclonal antibody as claimed in claim 1, the hybridoma cell line as claimed in claim 2, or the recombinant protein as claimed in claim 3, characterized in that, Used to prepare reagents or kits for detecting SMMHC proteins.

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