Hybridoma cell line 1c2b8-2 and its secreted antibody

Abstract

The application provides an anti-calponin (Calponin 1, CNN1) monoclonal antibody, the antibody can be specifically combined with the CNN1 protein, and the antibody is generated by a hybridoma cell strain 1C2B8-2 (the preservation number is CCTCC NO: C2021118). In addition, the monoclonal antibody secreted by the hybridoma cell strain 1C2B8-2 has good specificity, a CNN1 serum detection kit developed on the basis of the chemical luminescence detection platform can be used for screening CNN1 in patient serum, can greatly improve the accuracy of early diagnosis of acute aortic dissection, can preliminarily judge the prognosis of patients, can reduce the mortality of aortic dissection, and has the advantages of high sensitivity, good specificity, accurate quantification and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine, and particularly relates to a hybridoma cell strain 1C2B8-2 and application of an antibody secreted by the hybridoma cell strain. BACKGROUND

[0002] With the modernization of people's lifestyle, the high incidence of diseases such as hypertension, diabetes, and atherosclerosis, the incidence of aortic diseases in China is rapidly increasing. Aortic disease is one of the cardiovascular diseases that seriously threaten human health. Even in the United States with advanced medical conditions and technology, 15,000 people die from aortic aneurysm and aortic dissection every year. Aortic dissection (AD) is a very dangerous and severe cardiovascular disease that can cause multiple system complications (such as aortic rupture, which can cause pericardial tamponade, hematothorax, retroperitoneal hematoma; aortic valve insufficiency leading to left heart enlargement and heart failure; and important organ blood supply obstruction causing myocardial infarction, stroke, paraplegia, and abdominal organ ischemia, etc.), with a very high mortality rate and a high misdiagnosis rate. AD is one of the three major critical diseases with chest pain as the main manifestation, in addition to myocardial infarction and pulmonary embolism, especially acute aortic dissection (AAD) has a very high mortality rate. The mortality rate of untreated AAD patients is about 1% per hour within 24 hours of onset, more than half of the patients die within 1 week, 70% of the patients die within 2 weeks, and 90% of the patients die within 1 year. If appropriate drug and surgical treatment is given in time, the survival rate can be greatly improved, and the mortality rate can be reduced to 18% to 27%. Aortic dissection has multiple causes and a variety of possible disease symptoms, resulting in a high misdiagnosis rate. The direct consequence of delayed diagnosis or inappropriate treatment due to incorrect diagnosis is an increase in mortality rate, so early diagnosis of aortic dissection is the key to reducing mortality rate.

[0003] Currently, the diagnosis of aortic dissection is mainly based on a single mode of imaging examination results. CT angiography and magnetic resonance imaging can evaluate the entire aortic dissection, with high sensitivity and specificity, but also have obvious shortcomings. The examination is expensive, requires the patient to be transferred to a radiology department with special equipment for examination, takes a long time, and has the possibility of causing the disease to worsen, is prone to delay treatment, and for patients with renal dysfunction and contrast agent allergy, may cause contrast agent nephropathy and anaphylactic shock, which are life-threatening severe conditions, and is not suitable for early and rapid diagnosis and exclusion of aortic dissection.

[0004] Therefore, it is urgent to find a breakthrough in the existing single diagnosis mode of aortic dissection imaging, to provide a simple, fast, and highly specific peripheral blood diagnosis mode for AAD diagnosis and exclusion, prognosis evaluation, and other purposes, to provide a new tool for AAD diagnosis and death warning, and to reduce the mortality rate of patients. SUMMARY

[0005] The present application aims to provide a more simple, fast and high-specificity technical means for diagnosing and excluding AAD, evaluating prognosis, etc.

[0006] In a first aspect of the present application, an anti-calponin (Calponin 1, CNN1) monoclonal antibody is provided, which can specifically bind to the CNN1 protein, and is produced by a hybridoma cell strain 1C2B8-2, the hybridoma cell strain having a preservation number of CCTCC NO: C2021118.

[0007] In another preferred embodiment, the antibody is used for detecting the CNN1 protein.

[0008] In another preferred embodiment, the CNN1 protein is human-derived or recombinant.

[0009] In another preferred embodiment, the amino acid sequence of the CNN1 protein is shown as SEQ ID NO: 1.

[0010] In another preferred embodiment, the antibody secreted by the hybridoma cell strain 1C2B8-2 (having a preservation number of CCTCC NO: C2021118) has an EC50 of 0.76-2.05 ng / mL, preferably 1.05-1.50 ng / mL, for the CNN1 protein.

[0011] In another preferred embodiment, the antibody is of the IgG1 type.

[0012] In another preferred embodiment, the antibody has a titer of ≥1:512000.

[0013] In another preferred embodiment, the antibody specifically binds to the CNN1 protein.

[0014] In another preferred embodiment, the antibody does not bind to proteins in cardiac muscle and smooth muscle other than the CNN1 protein.

[0015] In another preferred embodiment, the antibody does not bind to MYO protein, CTNI protein and / or SMMHC protein.

[0016] In another preferred embodiment, the antibody is coupled with or carries a detectable label.

[0017] In another preferred embodiment, the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0018] In another preferred embodiment, the antibody is used for detecting the CNN1 protein.

[0019] In a second aspect of the present application, a hybridoma cell line is provided, with the preservation number of CCTCC NO: C2021118; the hybridoma cell line is capable of producing the anti-CNN1 monoclonal antibody as described in the first aspect of the present application.

[0020] In a third aspect of the present application, a recombinant protein is provided, the recombinant protein has:

[0021] (i) the monoclonal antibody as described in the first aspect of the present application;

[0022] (ii) an optional tag sequence for assisting expression and / or purification.

[0023] 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.

[0024] In a fourth aspect of the present application, a detection system for detecting CNN1 protein is provided, the detection system comprises:

[0025] (i) a solid phase carrier Z0;

[0026] (ii) a first binding protein A, the first binding protein A is the monoclonal antibody as described in the first aspect of the present application or the recombinant protein as described in the third aspect of the present application, the first binding protein A is coated on the solid phase carrier Z0; and

[0027] (iii) a second binding protein B, the second binding protein specifically binds to CNN1 protein, and the second binding protein is coupled with or carries a detectable label;

[0028] wherein the binding between the second binding protein B and CNN1 protein is non-competitive with the binding between the first binding protein A and CNN1 protein.

[0029] In another preferred embodiment, when the detection system contains the CNN1 protein to be detected, the detection system can form a complex as shown in Formula I:

[0030] Z0-(A-C-B)n (I)

[0031] wherein,

[0032] Z0 is a solid phase carrier;

[0033] A is a first binding protein, the A is the monoclonal antibody as described in the first aspect of the present application or the recombinant protein as described in the third aspect of the present application, which is coated on the surface of the solid phase carrier;

[0034] B is a second binding protein, which specifically binds to the CNN1 protein to be detected, and is coupled with or carries a detectable label; wherein the binding between the B and the CNN1 protein is not competitive with the binding between the A and the CNN1 protein;

[0035] C is the CNN1 protein to be detected;

[0036] n is a positive integer greater than or equal to 1; and

[0037] "-" is a bond or a linking group.

[0038] In another preferred embodiment, the solid support material is selected from the group consisting of metal, glass, gel, plastic, or a combination thereof.

[0039] In another preferred embodiment, the solid support material comprises a homopolymer, a copolymer, or a combination thereof.

[0040] In another preferred embodiment, the solid support material is selected from the group consisting of polystyrene, polyethylene, polypropylene, or a combination thereof.

[0041] In another preferred embodiment, the solid support material is selected from the group consisting of microspheres, microwell plates, strips, test tubes, or a combination thereof.

[0042] In another preferred embodiment, the solid support is a magnetic micro-particle.

[0043] In another preferred embodiment, the concentration of the magnetic micro-particle is in the range of 0.1-10 mg / mL, preferably 0.3-3 mg / mL, and more preferably 0.5-2 mg / mL.

[0044] In another preferred embodiment, the concentration of the first binding protein A is in the range of 0.5-50 μg / mL, preferably 1-20 μg / mL, and more preferably 5-10 μg / mL.

[0045] In another preferred embodiment, the second binding protein B is selected from the group consisting of rabbit anti-human CNN1 monoclonal antibody, rabbit anti-human CNN1 polyclonal antibody, mouse anti-human CNN1 monoclonal antibody, mouse anti-human CNN1 polyclonal antibody, and goat anti-human CNN1 polyclonal antibody.

[0046] In another preferred embodiment, the second binding protein B is mouse anti-human CNN1 monoclonal antibody IgG type.

[0047] In another preferred embodiment, the detectable label is selected from the group consisting of fluorescent substance, radioactive element, enzyme, chemiluminescent agent, colloidal gold, or a combination thereof.

[0048] In another preferred embodiment, the concentration of the second binding protein B is in the range of 0.05-5 μg / mL, preferably 0.1-2 μg / mL, and more preferably 0.5-1 μg / mL.

[0049] In a fifth aspect of the present application, a kit is provided, which comprises: a container and reagents for forming the detection system as described in the fourth aspect of the present application in the container.

[0050] In another preferred embodiment, the kit comprises:

[0051] (a) a first container and the solid support Z0 in the detection system in the first container;

[0052] (b) a second container and the first binding protein A in the detection system in the second container;

[0053] (c) a third container and the second binding protein B in the detection system in the third container;

[0054] (d) optionally a fourth container and a buffer for the reaction system in the fourth container;

[0055] (e) optionally a fifth container and a sample diluent in the fifth container; and

[0056] (f) optionally a sixth container and a washing solution in the sixth container.

[0057] In another preferred embodiment, the first container, the second container and the third container can be the same or different containers.

[0058] In another preferred embodiment, the second binding protein B in the detection system in the third container can be unlabeled during long-term storage, and is labeled within a certain time before use according to the need of use.

[0059] In another preferred embodiment, the kit further comprises a label or an instruction, which indicates that the kit is used for (a) detecting the CNN1 protein, and / or (b) detecting or diagnosing aortic dissection.

[0060] In another preferred embodiment, the detection of aortic dissection is a plasma or serum detection, preferably a serum detection, more preferably a human serum detection.

[0061] In another preferred embodiment, the label or the instruction indicates the following:

[0062] (i) if the CNN1 concentration in the sample from the detection subject is higher than 2.43 ng / ml, the subject has a higher probability of aortic dissection than the normal population.

[0063] In another preferred embodiment, the detection subject is a human.

[0064] In another preferred embodiment, the sample is a serum or plasma sample.

[0065] In a sixth aspect of the present application, there is provided use of the monoclonal antibody according to the first aspect of the present application, the hybridoma cell strain according to the second aspect of the present application, or the recombinant protein according to the third aspect of the present application, for the preparation of a reagent or a kit for detecting CNN1 protein.

[0066] In another preferred embodiment, the kit is the kit according to the fifth aspect of the present application.

[0067] In another preferred embodiment, the reagent or the kit is a diagnostic reagent or a kit for detecting aortic dissection.

[0068] In another preferred embodiment, the aortic dissection comprises acute aortic dissection or / and chronic aortic dissection.

[0069] In another preferred embodiment, the aortic dissection is acute aortic dissection.

[0070] In a seventh aspect of the present application, there is provided a method for detecting whether a sample contains CNN1 protein, the method comprising:

[0071] providing a detection system according to the fourth aspect of the present application, adding the sample to the detection system for sufficient reaction, and detecting the obtained reaction solution.

[0072] In another preferred embodiment, the sample is an ex vivo sample or an in vitro sample.

[0073] In another preferred embodiment, the sample is selected from the group consisting of a serum sample, a plasma sample, a urine sample, a tissue sample, or a combination thereof.

[0074] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0075] In another preferred embodiment, the detection comprises qualitative detection and quantitative detection.

[0076] In another preferred embodiment, in the method, if the signal of the detectable label coupled to or carried by the second binding protein B is detected, it indicates that the sample contains CNN1 protein; if the signal of the detectable label coupled to or carried by the second binding protein B is not detected, it indicates that the sample does not contain CNN1 protein.

[0077] In another preferred embodiment, the method is diagnostic.

[0078] In another preferred embodiment, the method is a method for diagnosis and prognosis evaluation of aortic dissection.

[0079] In another preferred embodiment, the method is a chemiluminescent detection method.

[0080] In an eighth aspect of the present application, there is provided a method for detecting or determining aortic dissection, comprising the steps of:

[0081] (a) providing an ex vivo test sample from a test subject;

[0082] (b) detecting the expression level of CNN1 protein in the test sample; and

[0083] (c) comparing the expression level of CNN1 determined in (b) with a control reference value; wherein compared with the control,

[0084] if the expression level of CNN1 in the test sample of the test subject is higher than the reference value Al,

[0085] the test subject is preliminarily determined to have a higher possibility of aortic dissection than a normal healthy population.

[0086] In another preferred embodiment, the sample is from a test subject.

[0087] In another preferred embodiment, the test subject is a human or a non-human mammal.

[0088] In another preferred embodiment, the sample is a serum sample, a plasma sample, a urine sample, a tissue sample, or a combination thereof.

[0089] In another preferred embodiment, the aortic dissection is acute aortic dissection.

[0090] In another preferred embodiment, the detecting step (b) comprises detecting the amount of CNN1 mRNA; and / or detecting the amount of CNN1 protein.

[0091] In another preferred embodiment, the expression level of CNN1 protein in the sample is detected by fluorescence quantitative PCR or immunohistochemistry.

[0092] In another preferred embodiment, the expression level of CNN1 protein in the sample is detected using the kit as described in the fifth aspect of the present application.

[0093] In another preferred embodiment, the reference value Al is a cut-off value.

[0094] In another preferred embodiment, the cut-off value is 2.43 ng / ml.

[0095] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0096] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0097] Figure 1 The antibody titer in the serum of the mice after the fourth immunization is shown. After the anti-CNN1 monoclonal antibody is diluted by several times, the final determination titer is 1:512000.

[0098] Figure 2 The SDS-PAGE protein electrophoresis test report of the purified CNN1 recombinant antigen is shown. According to the test report, the molecular weight of the CNN1 recombinant antigen is about 38KDa.

[0099] Figure 3 The SDS-PAGE protein electrophoresis test report of the purified CNN1 monoclonal antibody is shown. According to the test report, the molecular weights of the heavy chain and the light chain of the anti-CNN1 monoclonal antibody are about 50kDa and about 26kDa, respectively.

[0100] Figure 4 The reactivity of the CNN1 monoclonal antibody to CNN1 is shown. According to the figure, the CNN1 antibody has good reactivity to the CNN1 antigen, and the titer of the antibody is 1:512000.

[0101] Figure 5 The specificity of the CNN1 monoclonal antibody to MYO is shown. According to the figure, the CNN1 monoclonal antibody has no reactivity to MYO, indicating that the CNN1 monoclonal antibody has good specificity to MYO.

[0102] Figure 6 The specificity of the CNN1 monoclonal antibody to CTNI is shown. According to the figure, the CNN1 monoclonal antibody has no reactivity to CTNI, indicating that the CNN1 monoclonal antibody has good specificity to CTNI.

[0103] Figure 7 The specificity of the CNN1 monoclonal antibody to SMMHC is shown. According to the figure, the CNN1 monoclonal antibody has no reactivity to SMMHC, indicating that the CNN1 monoclonal antibody has good specificity to CNN1.

[0104] Figure 8 The ROC curve for determining the reference value of CNN1 is shown. The area of the ROC is 0.993 (95% CI, 0.986-1.000), and the p value is 0.000<0.05, indicating that the model simulation has statistical significance.

[0105] Figure 9 The content of CNN1 in the serum of healthy people and aortic dissection patients is shown. The content of CNN1 in the serum of healthy people and aortic dissection patients has statistical significance (p<0.05). DETAILED DESCRIPTION

[0106] The present inventors have developed, for the first time, a monoclonal antibody and a kit for efficiently detecting CNN1 protein through extensive and in-depth research and a large number of screenings. Specifically, the present application provides a hybridoma cell strain 1C2B8-2 capable of secreting the anti-CNN1 monoclonal antibody of the present application. The results show that the monoclonal antibody of the present application as a primary antibody, the antibody A (a commercially available recommended detection antibody for labeling) as a secondary antibody, and the detection principle of the antigen sandwich method can efficiently detect whether CNN1 protein exists in the sample. The method of the present application has great clinical application value. On this basis, the present application is completed.

[0107] The present application relates to a hybridoma cell strain 1C2B8-2 and a preparation method thereof, and an anti-Calponin 1 monoclonal antibody secreted by the hybridoma cell strain and the application thereof in serological detection of aortic dissection. Specifically, the present application provides a hybridoma cell strain 1C2B8-2, which was deposited at the China Center for Type Culture Collection on April 27, 2021, and the deposit number is CCTCC NO: C2021118.

[0108] The present application also provides a preparation method of the above-mentioned hybridoma strain 1C2B8-2, which is obtained by immunizing mice with the mature CNN1 recombinant protein having the sequence of SEQ ID No: 1 as an antigen.

[0109] The present application also provides an anti-CNN1 monoclonal antibody, which is secreted by the above-mentioned hybridoma cell strain 1C2B8-2.

[0110] The anti-CNN1 monoclonal antibody secreted by the above-mentioned hybridoma cell strain is of the IgG1 type, has good specificity and high titer. The titer is as high as 1:512000 after inspection.

[0111] The present application also provides an anti-CNN1 monoclonal antibody for capture.

[0112] The present application also provides a kit for detecting aortic dissection, which comprises the anti-CNN1 monoclonal antibody secreted by the hybridoma cell strain 1C2B8-2 provided by the present application, and the monoclonal antibody can be used as a capture antibody in the kit. The anti-CNN1 monoclonal antibody and another anti-CNN1 monoclonal antibody constitute a new type of kit, which takes the direct chemiluminescence detection method as a platform and is used for detecting CNN1 in serum.

[0113] Terminology

[0114] As used herein, the terms "containing" or "including" can be open, semi-closed and closed. In other words, the terms also include "consisting essentially of" or "consisting of".

[0115] As used herein, the term "about" when used in the context of a numerically recited value means that the value can vary from the recited value by not more than 1%. 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.).

[0116] Calponin (Calponin 1, CNN1)

[0117] Calponin is released from the inside of smooth muscle cells when the blood vessel wall is damaged, resulting in an increase in serum concentration, and the increase is very stable, with a long window period duration, which can be used for early rapid diagnosis or exclusion, prognosis evaluation of AAD. However, there is no suitable antibody on the market that can be used for the development of commercial kits. Therefore, the purpose of the present application is to provide a hybridoma cell 1C2B8-2 and a preparation method thereof, an anti-CNN1 monoclonal antibody secreted by the hybridoma cell strain, and a CNN1 kit based on an immunochemical luminescence platform for AAD diagnosis, exclusion and prognosis evaluation using the antibody.

[0118] As used herein, the terms "antigen protein of the present application", "CNN1 protein", "CNN1 polypeptide" or "calponin CNN1" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence of calponin CNN1 (SEQ ID NO: 1). They include CNN1 with or without the initial methionine. In addition, the term also includes full-length CNN1 and fragments thereof. The CNN1 protein referred to in the present application includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragment.

[0119] In the case of obtaining an amino acid fragment of CNN1, a nucleic acid sequence encoding it can be constructed according to it, and a specific probe can be designed according to the nucleotide sequence. The nucleotide full-length sequence or its fragment can be obtained by PCR amplification method, recombination method or artificial synthesis method. For PCR amplification method, primers can be designed according to the disclosed CNN1 nucleotide sequence, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template for amplification to obtain the relevant sequence. When the sequence is long, it is often necessary to carry out two or more times of PCR amplification, and then the fragments amplified in each time are spliced together in the correct order.

[0120] Once the relevant sequence has been obtained, it can be produced in large quantities using recombinant methods. This is usually achieved by cloning the sequence into a vector, which is then transferred into cells, and the sequence is isolated from the propagated host cells by conventional methods.

[0121] Alternatively, the relevant sequence can be synthesized using artificial synthesis methods, particularly when the length of the fragment is short. Usually, a long sequence is obtained by synthesizing a plurality of small fragments and then ligating them together.

[0122] At present, it is possible to obtain a DNA sequence encoding the protein (or fragment, derivative) of the present application by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

[0123] The polynucleotide sequence of the present application can be used to express or produce a recombinant CNN1 polypeptide by conventional recombinant DNA techniques. In general, the following steps are involved:

[0124] (1) transforming or transducing a suitable host cell with a polynucleotide of the present application (or variant) encoding a CNN1 polypeptide, or with a recombinant expression vector containing the polynucleotide;

[0125] (2) culturing the host cell in a suitable medium;

[0126] (3) isolating and purifying the protein from the medium or the cell.

[0127] In the present application, the CNN1 polynucleotide sequence can be inserted into a recombinant expression vector. In general, any plasmid or vector that can replicate and be stably maintained in a host cell can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element.

[0128] Methods well known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding CNN1 and suitable transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operably linked to a suitable promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0129] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance in eukaryotic cells, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance in E. coli.

[0130] Vectors comprising appropriate DNA sequences as described above, together with appropriate promoters or control sequences, can be used to transform appropriate host cells to enable them to express the protein.

[0131] The host cell can be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples of useful host cells are: bacterial cells of the genera E. coli, Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells; and the like.

[0132] 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, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation, followed by transformation of competent cells. Alternatively, other methods, such as the use of MgCh, can be used. If necessary, electroporation can be used to transform the host cell. When the host cell is a eukaryote, such as a yeast or a mammalian cell, the transformation of the host cell can be effected by the use of techniques such as calcium phosphate precipitation, electroporation, lipofection, and the like.

[0133] The resulting transformant can be cultured in conventional nutrient media to express the polypeptide encoded by the genes of the application. The culture conditions, such as temperature, pH and the like, are those previously determined to be appropriate for the host cell. When the host cell has reached an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0134] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0135] Specific antibodies

[0136] As used herein, the terms "antibodies of the application", "anti-CNN1 monoclonal antibodies" and "antibodies specific for CNN1" are used interchangeably.

[0137] This invention also includes monoclonal antibodies specific to the CNN1 protein. Here, "specific" means that the antibody can bind to the CNN1 gene product or fragment. Preferably, it refers to antibodies that can bind to the CNN1 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 the CNN1 protein. This invention also includes antibodies capable of binding to modified or unmodified forms of the CNN1 gene product.

[0138] 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.

[0139] The antibodies of this invention can be prepared using various techniques known to those skilled in the art. For example, purified CNN1 gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing the CNN1 protein 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 Cell Hybridomas (Elsevier, NY, 1981). The various antibodies of this invention can be obtained using fragments or functional regions of the CNN1 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 CNN1 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).

[0140] Anti-CNN1 protein antibodies can be used in immunohistochemistry to detect CNN1 protein in specimens (especially serum samples).

[0141] Affinity is a characteristic parameter of the relative state among antigen, antibody and antigen-antibody complex in the process of reversible reaction, and its more professional and terminological name is dissociation equilibrium constant KD. In the present application, the EC50 (concentration for 50% of maximum effect, EC50, refers to the concentration capable of causing 50% of maximum effect) of the antibody secreted by hybridoma cell strain 1C2B8-2 to the CNN1 protein is equivalent to the KD value of the antibody to the CNN1 protein. The smaller the EC50 value is, the stronger the affinity is, and the strength of the affinity determines how much each component of the reversible reaction is relative when the reaction is completed.

[0142] The antibody secreted by the hybridoma cell strain 1C2B8-2 (CCTCC NO: C2021118) of the present application has good affinity. In the specific embodiments of the present application, the EC50 of the antibody secreted by the hybridoma cell strain 1C2B8-2 (CCTCC NO: C2021118) to the CNN1 protein is 0.76-2.05 ng / mL, preferably 1.05-1.50 ng / mL.

[0143] Coating antibody

[0144] As used herein, "coating antibody", "capture antibody" or "solid-phase antibody" can be used interchangeably, which refers to an antibody coated on a solid-phase carrier, i.e., the first binding protein in the detection system of the present application.

[0145] The coating antibody can be adsorbed or physically adsorbed on polystyrene (enzyme-labeled plate), nitrocellulose membrane and other materials in a non-specific manner to become a solid-phase antibody, and still maintain its immunological activity. The coating antibody has high affinity and high specificity to the antigen, but does not affect the binding of the antigen to the detection antibody. After adding the sample to be detected, if the sample contains the target antigen, it will be captured by the coating antibody. The antigen (target antigen) described in the present application is CNN1 protein.

[0146] In the preferred embodiments of the present application, the coating antibody can bind to the CNN1 protein with high affinity and high specificity. In another preferred embodiment, the coating antibody is produced by hybridoma cell strain CCTCC NO: C2021118.

[0147] Detection antibody

[0148] As used herein, "detection antibody" or "labeled antibody" can be used interchangeably, which refers to an antibody used to detect whether the sample contains the target antigen, i.e., the second binding protein in the present application.

[0149] The detection antibody is usually labeled with a detectable label, including a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme. The detection antibody has high affinity and high specificity to the antigen, but does not affect the binding of the antigen to the coating antibody. By taking advantage of the specific binding of the detection antibody to the target antigen, the presence and quantity of the antigen are determined by detecting the label. In a preferred embodiment of the present application, the detection antibody can bind to the CNN1 protein with high affinity and high specificity.

[0150] In a preferred embodiment of the present application, the detection antibody is antibody A (CNN1 antibody purchased from ABNOVA (J9231-S1, 0.1 mg), which is a recommended detection antibody for labeling.

[0151] Detection system and detection kit

[0152] The present application provides a detection system for detecting CNN1 protein, which comprises:

[0153] (i) a solid phase carrier Z0;

[0154] (ii) a first binding protein A, which is a monoclonal antibody according to the first aspect of the present application or a recombinant protein according to the third aspect of the present application, and the first binding protein A is coated on the solid phase carrier Z0; and

[0155] (iii) a second binding protein B, which can specifically bind to the CNN1 protein, and the second binding protein is coupled with or labeled with a detectable label;

[0156] Wherein, the binding between the second binding protein B and the CNN1 protein is not competitive with the binding between the first binding protein A and the CNN1 protein.

[0157] In another preferred embodiment, when the detection system contains the CNN1 protein to be detected, the detection system can form a complex as shown in formula I:

[0158] Z0-(A-C-B)n (I)

[0159] Wherein,

[0160] Z0 is a solid phase carrier;

[0161] A is a first binding protein, which is a monoclonal antibody according to the first aspect of the present application or a recombinant protein according to the third aspect of the present application, and the first binding protein is coated on the surface of the solid phase carrier;

[0162] B is a second binding protein, which specifically binds to the CNN1 protein to be detected, and is coupled or carries a detectable label; wherein the binding between B and the CNN1 protein is not competitive with the binding between A and the CNN1 protein;

[0163] C is the CNN1 protein to be detected;

[0164] n is a positive integer greater than or equal to 1; and

[0165] "-" is a bond or a linking group.

[0166] The present application also provides a kit for detecting aortic dissection. The kit contains the detection system as described above.

[0167] Specifically, the kit comprises:

[0168] (a) a first container and the solid carrier Z0 in the detection system in the first container;

[0169] (b) a second container and the first binding protein A in the detection system in the second container;

[0170] (c) a third container and the second binding protein B in the detection system in the third container;

[0171] (d) optionally a fourth container and a buffer for the reaction system in the fourth container;

[0172] (e) optionally a fifth container and a sample diluent in the fifth container; and

[0173] (f) optionally a sixth container and a washing solution in the sixth container.

[0174] In the kit provided by the present application, the solid carrier contains magnetic microparticle solid combination, wherein the concentration of magnetic microparticles ranges from 0.3 to 3 mg / mL, and the concentration of antibodies ranges from 1 to 20 μg / mL; the concentration of antibodies in the luminescent combination is 0.1-2 μg / mL.

[0175] The kit of the present application also contains a calibrator. Generally, it refers to diluting a certain concentration of CNN1 antigen mother night antigen diluent into a series of antigen samples with different concentrations, usually into 6 concentrations of 0 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, respectively, CAL1, CAL2, CAL3, CAL4, CAL5, CAL6.

[0176] The kit has good sensitivity and specificity, when the Youden index is 0.960, the sensitivity and specificity are 100% and 96% respectively, and the reference value (cutoff value) of the CNN1 is 2.43 ng / ml. If the sample CNN1 concentration from the detection object is higher than 2.43 ng / ml, the probability of the object suffering from aortic dissection is greater than that of the normal population.

[0177] Detection method

[0178] The application also provides a method for detecting or judging aortic dissection, in particular a serological detection method. The aortic dissection includes acute aortic dissection or / and chronic aortic dissection. Preferably, the aortic dissection is acute aortic dissection.

[0179] In a preferred embodiment of the application, the CNN1 chemiluminescence detection kit is used with a chemiluminescence analyzer to detect CNN1 in a serum sample.

[0180] Use of the application

[0181] The kit is used for accurately detecting the content of CNN1 in human plasma and detecting the change amount of CNN1 in human serum, and can be used for diagnosis and prognosis evaluation of aortic dissection.

[0182] The main advantages of the application include:

[0183] (1) The hybridoma cell provided by the application has high secretion yield, and the secreted CNN1 monoclonal antibody has the advantages of high titer and good specificity, and can be widely used in the field of CNN1 detection, such as the preparation field of detection reagents or detection equipment, etc.

[0184] (2) The hybridoma cell strain provided by the application secretes a monoclonal anti-CNN1 antibody of IgG type, which has strong specificity and sensitivity in combination with the CNN1 protein;

[0185] (3) The antibody secreted by the hybridoma cell strain of the application can be applied to immunohistochemistry, immunoblotting, ELISA method, chemiluminescence method and other methodologies, and has a wide application scenario;

[0186] (4) The antibody secreted by the hybridoma cell strain of the application has the advantages of stable titer and low production cost, and has wide application value;

[0187] (5) The antibody secreted by the hybridoma cell strain of the application applied to the CNN1 detection kit has the advantages of high sensitivity, good specificity and short detection time, and can better assist the clinical diagnosis of aortic dissection.

[0188] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0189] Example 1: Screening and preparation of hybridoma cell lines

[0190] 1.1 Animal Immunization

[0191] The prepared Calponin 1 protein was coupled with ImmunoPlus to immunize BLAB / C mice (female only), and a total of 5 immunizations were performed (using multi-point immunization, 20 ug per mouse per time), with 2 weeks as an immunization cycle.

[0192] 1.2 Cell Fusion and Screening

[0193] Three days before fusion, the Calponin 1 protein was coated on plates, and the titer of the mouse tail serum was detected by indirect Elisa method. Finally, the BLAB / C mice with high immune titer (1:8000) were selected for booster immunization. Three days later, the spleen of the immunized mice was removed and crushed, and was fused with SP2 / 0 mouse myeloma cell lines by the action of PEG (Sigma, P7181).

[0194] 1.3 Screening of Hybridoma Positive Cell Lines

[0195] The fused cells were added to the 96-well plates in which the feeder cells were pre-plated. After 7-10 days, the supernatant of the fused cells was screened by indirect ELISA method, and the supernatant positive to the target protein Calponin 1 was selected. All the positive clones obtained in the primary screening stage were screened by indirect ELISA method, and the His-tag protein was used for negative screening. The positive clones obtained in this stage were 1C2B8, 1E10B4, 2G6B2, 8H8F4, 9A8D7, and 9B4A10.

[0196] 1.4 Clonal Screening by Limiting Dilution

[0197] The positive clone cells obtained in step c are subcloned by limiting dilution method, and the single clone cells are visually observed at least 3 times until the hybridoma cell strain stably secreting anti-Calponin 1 protein is screened. The subclones obtained in this stage are: 1C2B8-1, 1C2B8-2, 1E10B4-1, 1E10B4-2, 2G6B2-1, 2G6B2-2, 8H8F4-1, 8H8F4-2, 9A8D7-1, 9A8D7-2, 9B4A10-1, and 9B4A10-2.

[0198] 1.5 Subcloning Cell Supernatant Titer Determination

[0199] The subclones obtained in step d are determined for titer by ELISA indirect method. The specific steps of the ELISA indirect method are as follows:

[0200] 1) Antigen coating: select recombinant CNN1 antigen, dilute to 1 μg / mL with coating solution, and add 100 μL / well to polystyrene 96 reaction well plates, and place at 4°C overnight.

[0201] 2) Washing: the next day, pour out the liquid in the concave hole, and wash with washing solution for 3 times.

[0202] 3) Blocking: add 100 μL / well of blocking solution, and place at room temperature for 0.5 h.

[0203] 4) Washing: wash with washing solution for 3 times.

[0204] 5) Add the sample to be tested (cell supernatant): dilute the cell supernatant containing the monoclonal antibody with PBS according to a certain gradient (1:10, 1:30, 1:90, 1:270, 1:810, 1:2430), and add 100 μL / well to the coated plate as the experimental group, and each gradient is tested in duplicate, and add PBS wells as the control group. Cover and incubate at 37°C for 1-2 h.

[0205] 6) Washing: wash with washing solution for 3 times.

[0206] 7) Add enzyme-labeled secondary antibody: rabbit anti-mouse IgG-HRP, dilute with blocking solution at 1:8000, add 100 μL / well, cover and incubate at 37°C for 1 h.

[0207] 8) Washing: wash with washing solution for 5 times.

[0208] 9) Color development: add 100 μL / well of substrate, and place at room temperature in the dark for 5-30 min to show blue color

[0209] 10) Termination reaction, colorimetric: add 50 μL / well termination solution. Color turns yellow, use enzyme marker to measure absorbance of each well at 450 nm. The maximum dilution of positive reaction (at this time, the absorbance of positive sample / absorbance of negative sample ≥ 2.1) is the titer of the sample to be tested.

[0210] The specific determination results are shown in Table 1:

[0211] Table 1 Antibody titer determination of hybridoma cell supernatant

[0212]

[0213]

[0214] As shown in Table 1, the following conclusions are drawn through titer determination:

[0215] (1) It is determined that the 12 subclones of the present embodiment have certain titers, and are all effective cell strains.

[0216] (2) According to the determination standard of ELISA indirect method titer (OD sample absorbance / OD control sample absorbance ≥ 2.1, data is valid, can continue to dilute), select the subclone cell with higher OD ratio under the same dilution, that is, the subclone cell with higher titer, and finally select one subclone cell for each positive cell, which are 1C2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1 and 9B4A10-1.

[0217] Example 2: Preparation, purification and screening of monoclonal antibody

[0218] 2.1 Antibody preparation

[0219] According to Example 1, the antibody production method of cell supernatant culture is used in the present application, and affinity chromatography purification is carried out by using Protein A pre-packed column. Specifically, first, the affinity column is balanced with 1 × PBS, then the cell supernatant (pH adjusted to 8.0 by Tris) after centrifugation at 10000 rpm and filtration is hung on the column, 1 × PBS is used to wash the column for 5 times the column volume, finally eluted with pH 3.5 0.1 M Gly (pH 7.4 adjusted by Tris pH 8.8), finally dialyzed in 1 × PBS buffer, and quantified by Lowery.

[0220] After the subclone cells in Example 1 are produced and purified, they are respectively coated with carboxyl magnetic microparticles and labeled with acridinium ester process, and the final paired antibodies are screened on a chemiluminescence platform.

[0221] The specific screening and pairing results are shown in Table 2:

[0222] Table 2 Antibody cross-pairing test results

[0223]

[0224]

[0225] Note: In the table, A is a commercially purchased detection antibody recommended for labeling; F is a commercially purchased capture antibody recommended for coating.

[0226] As shown in Table 2, after cross-pairing experiments on a chemiluminescence platform, the following conclusions were drawn:

[0227] (1) There is a certain reactivity between the selected 6 subcloned antibodies, and the following pairs have good reactivity: 1C2B8-2 (coating) - 9A8D7-1 (labeling); 1E10B4-1 (coating) - 1C2B8-2 (labeling); 2G6B2-1 (coating) - 1C2B8-2 (labeling); 8H8F4-2 (coating) - 1C2B8-2 (labeling); 9A8D7-1 (coating) - 1C2B8-2 (labeling); 9B4A10-1 (coating) - 1C2B8-2 (labeling);

[0228] (2) The purchased antibody F (recommended for coating) and antibody A (recommended for labeling) have no reactivity when cross-paired.

[0229] (3) The purchased antibody F (recommended for coating) and the 6 screened antibodies (1C2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1, 9B4A10-1) have no reactivity when paired.

[0230] (4) Among the pairing results of the purchased antibody A (recommended for labeling) and the 6 screened antibodies (1C2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1, 9B4A10-1), only 1C2B8-2 has good reactivity, and the rest have no reactivity.

[0231] Based on the above four conclusions: the following pairs were preliminarily screened: 1C2B8-2 (coating) - 9A8D7-1 (labeling), 1E10B4-1 (coating) - 1C2B8-2 (labeling), 2G6B2-1 (coating) - 1C2B8-2 (labeling), 8H8F4-2 (coating) - 1C2B8-2 (labeling), 9A8D7-1 (coating) - 1C2B8-2 (labeling), 9B4A10-1 (coating) - 1C2B8-2 (labeling), 1C2B8-2 (coating) - A (labeling).

[0232] In view of the concentration ratio of the antigen samples CAL1-CAL6 used in the pairing test, CAL1 is a blank sample, the optimal luminescence values of CAL2-CAL5 are in a 2-fold increasing relationship in turn, and the luminescence value of the CAL6 antigen sample is about 5 times that of the CAL5 antigen sample.

[0233] Therefore, in combination with the test results, the following pairs are preliminarily excluded: 1E10B4-1 (coating)-1C2B8-2 (labeling), 2G6B2-1 (coating)-1C2B8-2 (labeling), 8H8F4-2 (coating)-1C2B8-2 (labeling), 9A8D7-1 (coating)-1C2B8-2 (labeling), and 9B4A10-1 (coating)-1C2B8-2 (labeling).

[0234] Further, according to the luminescence value priority principle, that is, the higher the luminescence value of a high-concentration sample, the greater the sensitivity of the reagent, and the better the performance of the reagent, therefore, 1C2B8-2 (coating)-A (labeling) is finally selected as the final paired antibody. The 1C2B8-2 antibody-secreting cell is the cell strain provided by the present application.

[0235] The evaluation of the antibody titer secreted by 1C2B8-2 in the serum of the mouse after four immunizations is shown in Table 1. Figure 1 The antibody titer in the serum after four immunizations is 1:512000, and the antibody subtype reagent Elisa detection identifies it as IgG1 type. The purified CNN1 recombinant antigen and the SDS-PAGE protein electrophoresis test report of the monoclonal antibody are shown in Figure 2 and Figure 3 It can be concluded that the molecular weight of the CNN1 recombinant antigen is 38 kDa, and the molecular weights of the heavy chain and the light chain of the anti-CNN1 monoclonal antibody are 50 kDa and 26 kDa, respectively.

[0236] 2.2 Activity determination

[0237] The experimental steps for determining the EC50 of the antibody secreted by the hybridoma cell strain 1C2B8-2 (preservation number: CCTCC NO: C2021118) and the CNN1 protein are as follows:

[0238] 1. Preparation of CNN1-coated enzyme-labeled plate

[0239] Take CNN1 antigen with a concentration of 100 μg / mL, dissolve at room temperature, mix well, dilute to 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL with coating solution according to the following steps, add 100 μL / well according to the sample layout table, and add to the enzyme-labeled plate, among which add coating solution as a blank control for coating antibodies, and incubate overnight at 2-8°C;

[0240] 2. Wash the plate with washing solution 3 times, pat dry, add 300 μL / well of blocking solution, and incubate at room temperature for 1 hour; wash the plate with washing solution 3 times, pat dry, and wait for use;

[0241] 3. Dilute the antibody according to the layout with a 3-fold gradient starting at 100,000 ng / mL, and add 100 μL / well to the microplate according to the layout;

[0242] 4. Place the microplate in a microplate shaker, set at 37°C, 600 rpm, and shake for 1 hour;

[0243] 5. Wash the plate 3 times with the washing solution and pat dry;

[0244] 6. Dilute the enzyme-linked antibody to the appropriate concentration, mix on a vortex mixer, and add 100 μL / well;

[0245] 7. Place the microplate in a microplate shaker, set at 37°C, 600 rpm, and shake for 1 hour;

[0246] 8. Wash the plate 4 times with the washing solution and pat dry;

[0247] 9. Take the color developing solution corresponding to the enzyme-linked antibody, and take it out of the room temperature 20 minutes before use, mix on a vortex mixer, and add 100 μL / well;

[0248] 10. Use an 8-channel row gun to add the color developing solution at 100 μL / well;

[0249] 11. Place the color developing solution at room temperature in the dark for 10-30 minutes;

[0250] 12. Use an 8-channel row gun to add the stop solution to stop the reaction at 100 μL / well; (This step may not be needed depending on the substrate)

[0251] 13. Measure the signal value with an enzyme label instrument;

[0252] 14. Result analysis

[0253] The experimental results show that the EC50 of the antibody secreted by the hybridoma cell strain 1C2B8-2 (preservation number CCTCC NO: C2021118) and the CNN1 protein is 1.05-1.50 ng / mL.

[0254] Example 3: Specificity detection of the anti-CNN1 monoclonal antibody secreted by the hybridoma cell 1C2B8-2 provided by the application

[0255] In this example, the CNN1 recombinant antigen in Example 1 and MYO, CTNI, and SMMHC are used as coating antigens, the monoclonal antibody prepared in Example 2 is used as a recognition antibody, and the indirect method is used to detect CNN1.

[0256] 3.1 Coating of the ELISA Plate

[0257] Coat the antigen with coating solution (CBS: Na2CO3 0.8 g, NaHCO3 1.46 g, distilled water, pH 9.0, and dilute to 1 L). Dilute the coated antigen to 1 μg / mL with coating solution, add 0.1 mL to each well of a 96-well plate, and seal the plate in a 4°C refrigerator overnight.

[0258] 3.2 Blocking of the ELISA Plate

[0259] Take out the enzyme-labeled plate that was blocked overnight at 4°C, discard the supernatant, pat the enzyme-labeled plate dry, add 300 μL / well of blocking solution (3% BSA + PBS), and incubate in a 37°C incubator for 2 h.

[0260] 3.3 Washing of the ELISA Plate

[0261] Take out the enzyme-labeled plate that was incubated at 37°C for 2 h, discard the supernatant, take out the enzyme-labeled plate, and wash it 5 times with a washing solution (PBST: KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-20 0.5 mL, and dilute to 1 L with water) using a plate washer.

[0262] 3.4 Detection of Anti-CNN1 Antibodies by Indirect ELISA

[0263] After washing and patting the enzyme-labeled plate dry, add 100 μL / well of the anti-CNN1 monoclonal antibody secreted by the hybridoma cell line (1C2B8-2) to the enzyme-labeled plate coated with different antigens, and add physiological saline to the control test. Incubate in a 37°C incubator for 2 h, wash 5 times using a plate washer, add 100 μL / well of HRP-labeled goat anti-mouse IgG secondary antibody, incubate in a 37°C incubator for 1 h, wash 5 times using a plate washer, add 100 μL / well of TMB substrate (purchased from Sigma), incubate in a 37°C incubator for 10 min, show blue color, add 2 M concentrated sulfuric acid to stop the reaction, change the color to yellow, and determine OD450nm using an enzyme-labeled instrument. Compare and analyze the results with the control test.

[0264] The final result is: Figure 4 It can be concluded that the anti-CNN1 monoclonal antibody in Example 2 can only detect the CNN1 protein antigen and the antibody detection titer is 1:512000; from Figure 5 It can be arbitrarily concluded that the anti-CNN1 monoclonal antibody has no reactivity to the MYO protein (myoglobin); from Figure 6 It can be arbitrarily concluded that the anti-CNN1 monoclonal antibody has no reactivity to the CTNI protein (cardiac troponin); from Figure 7 It can be arbitrarily concluded that the anti-CNN1 monoclonal antibody has no reactivity to the SMMHC protein (smooth muscle myosin heavy chain).

[0265] From the above, compared with other myocardin and smooth muscle proteins, the anti-CNN1 monoclonal antibody provided by the application has better specificity for CNN1.

[0266] Example 4: The preparation of the kit is refined

[0267] The kit for detecting CNN1 provided by the application is based on a chemiluminescence platform, and the main components of the kit include: a solid phase conjugate, a luminescent conjugate, and matching calibrators and quality control products. The kit mainly consists of a magnetic particle solid phase conjugate and a luminescent conjugate. Among them,

[0268] The magnetic particle solid phase conjugate is made of the anti-CNN1 monoclonal antibody coated magnetic particles in Example 2, and the concentration of the magnetic particles in the solid phase conjugate system ranges from 0.3 to 3 mg / mL, and the concentration of the antibody ranges from 1 to 20 μg / mL;

[0269] The luminescent conjugate is a commercially available anti-CNN1 monoclonal antibody (J9231-S1, Abnova) for labeling, which is a mature and stable anti-CNN1 antibody in various performance comparisons.

[0270] The anti-CNN1 monoclonal antibody is labeled with acridinium ester to make a luminescent conjugate, and the antibody concentration in the luminescent conjugate is 0.1-2 μg / mL; in addition, matching calibrators (6-point calibration) and quality control products (2-point quality control).

[0271] The kit provided by the application is matched with a direct chemiluminescence detection immunoassay analyzer, which has the advantages of high sensitivity, high specificity, high accuracy, wide linear range, high throughput and rapid detection.

[0272] Example 5: The use method of the kit provided by the application

[0273] 5.1 Calibration Curve Preparation:

[0274] 1) Put the reagents loaded with the solid phase conjugate and the luminescent conjugate into the reagent disc of the full-automatic chemiluminescence analyzer (Kosmo Smart 6500 / Transgenic i100) for reagent loading detection;

[0275] 2) Place the calibrators into the sample holder and transport them to the sample channel of the full-automatic chemiluminescence immunoassay analyzer, and the sample

[0276] The sample amount is 50 μL;

[0277] 3) Reaction mode: two-step method, the first step reaction incubation is 6 min, the second step reaction incubation is 6 min, 37℃ incubation,

[0278] Two-step washing, 3 times for each step;

[0279] 4) The light emission result of the automatic chemiluminescence immunoassay analyzer;

[0280] 5) According to the light emission value of the calibrator, the corresponding calibration curve is prepared.

[0281] 5.2 Test Procedure Calibration:

[0282] 1) The quality control sample is placed in the sample holder and transported to the sample channel of the automatic chemiluminescence immunoassay analyzer, and the sample loading amount is 50 μL;

[0283] 2) Reaction mode: two-step method, first-step reaction incubation for 6 min, second-step reaction incubation for 6 min, 37°C incubation, two-step washing, and each step is washed for 3 times;

[0284] 3) The light emission result of the automatic chemiluminescence immunoassay analyzer;

[0285] The test results of the calibrator are analyzed in combination with the calibration curve, and if the calibrator is within the control range, it indicates that the measurement procedure is within the control range, and the test result is reliable; otherwise, the reagent and instrument need to be rechecked and verified to determine.

[0286] 5.3 Sample Testing:

[0287] From the above, if the calibrator is within the controllable range, the sample test can be performed, and the specific steps are as follows:

[0288] 1) The sample is placed in the sample holder and transported to the sample channel of the automatic chemiluminescence immunoassay analyzer, and the serum sample loading amount is 50 μL;

[0289] 2) Reaction mode: two-step method, first-step reaction incubation for 6 min, second-step reaction incubation for 6 min, 37°C incubation, two-step washing, and each step is washed for 3 times;

[0290] 3) The light emission result of the automatic chemiluminescence immunoassay analyzer;

[0291] The final test result of the sample is obtained in combination with the calibration curve and the light emission value of the sample test.

[0292] Example 6: Performance of the kit provided by the application

[0293] 6 .1 Limit of Blank (LOB)

[0294] The RLU value (relative luminescence value) of 20 measurement results is obtained by detecting 20 times with zero concentration calibrator or sample diluent as sample, the average value (M) and standard deviation (SD) are calculated, the RLU value corresponding to M+2SD is obtained, the RLU value corresponding to M+2SD is brought into the above equation according to the calibration curve equation of the calibrator used in the kit, and the corresponding concentration value is obtained, that is, the blank detection limit. According to the test results, four-parameter Logistic curve fitting is adopted to obtain the calibration curve as follows:

[0295] Equation: y=(A-D) / [1+(x / C)^B]+D

[0296] A=5.72720

[0297] B=-0.57635

[0298] C=44.58894

[0299] D=2.63110

[0300] r^2=0.99990

[0301] The blank detection limit is 0.3 ng / mL.

[0302] 6.2 Accuracy

[0303] The sample (A) is added to the sample (B), the volume ratio between the added sample (A) and the sample (B) is 1:9, and the recovery rate should be 85%-115%.

[0304]

[0305] In the formula:

[0306] R-recovery rate;

[0307] V-volume of A liquid;

[0308] V0-volume of B liquid;

[0309] C-detection concentration of B liquid after adding A liquid;

[0310] C0-detection concentration of B liquid;

[0311] Cs-concentration of A liquid.

[0312] The concentration values of samples A, B and mixed samples are 107.0 ng / ml, 13.7 ng / ml and 21.7 ng / ml respectively, and the recovery rate of this test is 87.1%, which meets the requirements.

[0313] Example 7 The CNN1 chemiluminescence detection kit provided by the application was used to detect clinical serum samples

[0314] 45 samples of aortic dissection patients and 150 samples of healthy people were collected from a hospital in Shanghai, and the kit provided in Example 5 was used for detection. The CNN1 test results of aortic dissection patients and healthy people are shown in Table 3.

[0315] Table 3 Serum test results of healthy people and aortic dissection patients

[0316]

[0317]

[0318] From the test results of healthy people and aortic dissection patients shown in Table 3, the measured values of the kit for detecting CNN1 were fitted using the ROC curve method, and the ROC curve graph (see Figure 8 ) and the area under the ROC curve (see Table 4) were obtained.

[0319] Table 4 Area under the curve

[0320] Test result variable: CNN1 test result

[0321]

[0322]

[0323] a. Under the non-parametric hypothesis

[0324] b. Zero hypothesis: actual area = 0.5

[0325] From Table 4, the area of the ROC curve is 0.993 (95% CI, 0.986-1.000), the p value is 0.000<0.05, it is concluded that the simulation of the model has statistical significance, when the Youden index is 0.960, the sensitivity and specificity are 100% and 96% respectively, and the reference value of CNN1 is preliminarily obtained as 2.43 ng / mL. Combined with this reference value, it is indicated that the detection kit provided by the application has high accuracy compared with the clinic.

[0326] From Figure 9 It can be seen that the average values of healthy people and patients are (0.43±0.22) ng / mL and (18.68±4.38) ng / mL respectively, and the serum level of CNN1 patients is 40 times that of healthy people (p<0.05), which again indicates that the kit provided by the application has high specificity and sensitivity.

[0327] Example 8: Clinical cross-sample verification

[0328] Take the high sensitivity troponin T (hs-cTnT) concentration level in 0.014-0.05ng / mL 10 cases of myocardial injury clinical samples (Roche hs-cTnT apparent healthy population 99% percentile is 0.014ng / mL), using the kit provided by the application, the test results are shown in table 5.

[0329] Table 5 test results of myocardial injury samples

[0330] CTNT Sample Concentration (ng / mL) CNN1 Test Concentration (ng / mL) 0.0216 1.94 0.0316 0.55 0.0319 0.63 0.0356 0.66 0.0407 0.86 0.0422 0.32 0.0467 0.35 0.0489 0.93 0.0495 0.78 0.0499 0.62

[0331] As can be seen from table 5, the test results of 10 cases of clinical myocardial injury samples tested by the kit provided by the application are all less than 2.43ng / ml, which are negative (the cutoff value of the kit provided by the application is 2.43ng / ml). Therefore, the kit provided by the application has no cross between aortic dissection sample and myocardial injury sample.

[0332] Strain preservation

[0333] The hybridoma cell strain 1C2B8-2 of the application for producing anti-calponin (Calponin 1, CNN1) monoclonal antibody was preserved in China Center for Type Culture Collection (CCTCC, China, Wuhan) on April 27, 2021, and the preservation number was CCTCC NO: C2021118.

[0334] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference. In addition, it should be understood that those skilled in the art can make various modifications or modifications to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application. SEQUENCE LISTING <110> Shanghai Transgenic Diagnostics Technology Co., Ltd. Shanghai Transgenic Life Science Technology Co., Ltd. <120> Hybridoma cell strain 1C2B8-2 and application of antibody secreted by the same <130> P2020-2193 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 303 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Ser Ala His Phe Asn Arg Gly Pro Ala Tyr Gly Leu Ser Ala 1 5 10 15 Glu Val Lys Asn Lys Leu Ala Gln Lys Tyr Asp His Gln Arg Glu Gln 20 25 30 Glu Leu Arg Glu Trp Ile Glu Gly Val Thr Gly Arg Arg Ile Gly Asn 35 40 45 Asn Phe Met Asp Gly Leu Lys Asp Gly Ile Ile Leu Cys Glu Phe Ile 50 55 60 Asn Lys Leu Gln Pro Gly Ser Val Lys Lys Ile Asn Glu Ser Thr Gln 65 70 75 80 Asn Trp His Gln Leu Glu Asn Ile Gly Asn Phe Ile Lys Ala Ile Thr 85 90 95 Lys Tyr Gly Val Lys Pro His Asp Ile Phe Glu Ala Asn Asp Leu Phe 100 105 110 Glu Asn Thr Asn His Thr Gln Val Gln Ser Thr Leu Leu Ala Leu Ala 115 120 125 Ser Met Ala Lys Thr Lys Gly Asn Lys Val Asn Val Gly Val Lys Tyr 130 135 140 Ala Glu Lys Gln Glu Arg Lys Phe Glu Pro Gly Lys Leu Arg Glu Gly 145 150 155 160 Arg Asn Ile Ile Gly Leu Gin Met Gly Thr Asn Lys Phe Ala Ser Gin 165 170 175 Gln Gly Met Thr Ala Tyr Gly Thr Arg Arg His Leu Tyr Asp Pro Lys 180 185 190 Leu Gly Thr Asp Gin Pro Leu Asp Gin Ala Thr Ile Ser Leu Gin Met 195 200 205 Gly Thr Asn Lys Gly Ala Ser Gin Ala Gly Met Thr Ala Pro Gly Thr 210 215 220 Lys Arg Gin Ile Phe Gin Pro Gly Leu Gly Met Gin His Cys Asp Thr 225 230 235 240 Leu Asn Val Ser Leu Gin Met Gly Ser Asn Lys Gly Ala Ser Gin Arg 245 250 255 Gly Met Thr Val Tyr Gly Leu Pro Arg Gin Val Tyr Asp Pro Lys Tyr 260 265 270 Cys Leu Thr Pro Gin Tyr Pro Gin Leu Gly Gin Pro Gin His Asn His 275 280 285 His Ala His Asn Tyr Tyr Asn Ser Ala His His His His His His His 290 295 300

Claims

1. An anti-Calponin 1 (CNN1) monoclonal antibody, characterized in that, The antibody can specifically bind to the CNN1 protein, and the antibody is produced by a hybridoma cell strain 1C2B8-2, and the hybridoma cell strain has a preservation number of CCTCC NO: C2021118.

2. A hybridoma cell line, characterized in that, The hybridoma cell strain has a preservation number of CCTCC NO: C2021118, and the hybridoma cell strain can produce the anti-CNN1 monoclonal antibody according to claim 1.

3. A recombinant protein, characterized in that, The recombinant protein consists of (i) and (ii): (i) the monoclonal antibody according to claim 1; (ii) a tag sequence for assisting expression and / or purification.

4. An assay system for detecting a CNN1 protein, characterized by, The detection system comprises: (i) a solid phase carrier Z0; (ii) a first binding protein A, which is the monoclonal antibody according to claim 1 or the recombinant protein according to claim 3, and the first binding protein A is coated on the solid phase carrier Z0; and (iii) a second binding protein B, which specifically binds to the CNN1 protein, and the second binding protein B is labeled with a detectable label; wherein the binding between the second binding protein B and the CNN1 protein is not competitive with the binding between the first binding protein A and the CNN1 protein.

5. The detection system as described in claim 4, characterized in that, The concentration of the first binding protein A ranges from 0.5 to 50 μg / mL.

6. The detection system as described in claim 4, characterized in that, The concentration of the first binding protein A ranges from 1 to 20 μg / mL.

7. The detection system as described in claim 4, characterized in that, The concentration of the first binding protein A ranges from 5 to 10 μg / mL.

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

9. A kit characterized in that, The kit comprises a container and raw reagents for forming the detection system according to claim 4 in the container.

10. The kit of claim 9, wherein The kit further comprises a label or instruction, which indicates that the kit is used for (a) detecting the CNN1 protein, and / or (b) detecting or diagnosing aortic dissection.

11. The kit of claim 10, wherein The label or instruction indicates the following: (i) if the CNN1 concentration in a sample from a detection object is higher than 2.43 ng / ml, the object has a higher probability of aortic dissection than a normal population.

12. The kit of claim 11, wherein The sample is a serum or plasma sample.

13. Use of the monoclonal antibody according to claim 1, the hybridoma cell strain according to claim 2 or the recombinant protein according to claim 3, characterized in that, A reagent or kit for detecting the CNN1 protein. A reagent or kit for detecting the CNN1 protein.

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