CN115594764A

CN115594764A - Hybridoma cell strain 1C2B8-2 and application thereof to antibody secretion

Info

Publication number: CN115594764A
Application number: CN202111082848.XA
Authority: CN
Inventors: 詹红; 石广举; 田莎; 叶賽
Original assignee: Shanghai Tellgen Life Science Co ltd; Shanghai Tellgen Diagnosis Technology Co ltd
Current assignee: Shanghai Tellgen Life Science Co ltd; Shanghai Tellgen Diagnosis Technology Co ltd
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2023-01-13

Abstract

The invention provides an anti-calmodulin (Calponin 1, CNN1) monoclonal antibody, which can be specifically combined with CNN1 protein and 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, and a CNN1 serum detection kit developed on the basis of a chemiluminescence detection platform can be used for screening CNN1 in patient serum, so that the accuracy of early diagnosis of acute aortic dissection is greatly improved, the prognosis condition of a patient can be preliminarily judged, the death rate of the aortic dissection is reduced, and the kit has the advantages of high sensitivity, good specificity, accurate quantification and the like.

Description

Hybridoma cell strain 1C2B8-2 and application thereof to antibody secretion

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to a hybridoma cell strain 1C2B8-2 and application of a secreted antibody thereof.

Background

With the modernization of the life style of people and the high incidence of diseases such as hypertension, diabetes, atherosclerosis and the like, the incidence rate of aorta diseases in China is in a rapid rising trend. Aortic disease is one of the cardiovascular diseases that seriously threaten human health, and 15000 die every year from aortic aneurysm and aortic dissection, even in the united states where medical conditions and technology are developed. Aortic Dissection (AD) is a very dangerous acute and severe cardiovascular disease that can cause multiple system serious complications (e.g., rupture of aorta can cause pericardial stuffing, pleural hemorrhage, retroperitoneal hematoma, aortic insufficiency leading to left heart enlargement, heart failure, and myocardial infarction, cerebral apoplexy, paraplegia, and abdominal organ ischemia due to blood supply disorder of important organs), and has extremely high mortality rate and high misdiagnosis rate. AD is one of the three major critical diseases with chest pain as the main manifestation, except myocardial infarction and pulmonary embolism, especially Acute Aortic Dissection (AAD) has extremely high lethality rate. Untreated AAD patients die approximately 1% per hour, more than half within 1 week, 70% within 2 weeks, 90% within 1 year, with significant increases in survival and 18% to 27% mortality after timely appropriate drug and surgical treatment. The aortic dissection has multiple etiologies and has multiple possible disease manifestations, which causes high misdiagnosis rate. Early diagnosis of aortic dissection is critical to reducing mortality, as mortality increases as a direct consequence of delayed diagnosis or improper treatment due to misdiagnosis.

At present, the aortic dissection is mainly diagnosed according to a single mode of an imaging examination result, the CT angiography and the magnetic resonance imaging can evaluate the whole aortic dissection, the sensitivity and the specificity are high, but the diagnosis is also obviously insufficient, the examination cost is high, a patient needs to be transferred to a radiology department with a special instrument for examination, the time is long, the possibility of disease aggravation is caused, the treatment is easily delayed, in addition, for patients with renal insufficiency and contrast agent allergy, life-threatening severe diseases such as contrast agent kidney and anaphylactic shock can be caused, and the method is not suitable for early rapid diagnosis and elimination of the aortic dissection.

Therefore, there is an urgent need to find a single diagnosis mode that breaks through the existing aortic dissection iconography, provide a simple, fast and highly specific diagnosis mode for aortic dissection peripheral blood for diagnosis and elimination, prognosis evaluation and the like of AAD, and provide a new tool for diagnosis, death early warning and the like of AAD so as to reduce the fatality rate of patients.

Disclosure of Invention

The invention aims to provide a technical means for diagnosing and eliminating AAD, evaluating prognosis and the like more simply, conveniently, quickly and specifically.

In the first aspect of the invention, the anti-calmodulin (Calponin 1, CNN1) monoclonal antibody is provided, the antibody can be specifically combined with CNN1 protein, and the antibody is generated by a hybridoma cell strain 1C2B8-2 with the preservation number of CCTCC NO: C2021118.

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

In another preferred embodiment, the CNN1 protein is of human origin or recombinant.

In another preferred example, the amino acid sequence of the CNN1 protein is shown as SEQ ID NO. 1.

In another preferred embodiment, the EC50 of the antibody secreted by the hybridoma cell strain 1C2B8-2 (with the preservation number of CCTCC NO: C2021118) and the CNN1 protein is 0.76-2.05ng/mL, preferably 1.05-1.50ng/mL.

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

In another preferred embodiment, the titer of the antibody is greater than or equal to 1.

In another preferred example, the antibody specifically binds to the CNN1 protein.

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

In another preferred example, the antibody does not bind to a MYO protein, a CTNI protein and/or a SMMHC protein.

In another preferred embodiment, the antibody is conjugated to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In the second aspect of the invention, the invention provides a hybridoma cell strain with a preservation number of CCTCC NO of C2021118; the hybridoma cell strain can produce the anti-CNN 1 monoclonal antibody of the first aspect of the invention.

In a third aspect of the present invention, there is provided a recombinant protein having:

(i) A monoclonal antibody according to the first aspect of the invention;

(ii) Optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the expression and/or purification assisting tag 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.

In a fourth aspect of the present invention, there is provided an assay system for detecting CNN1 protein, the assay system comprising:

(i) A solid support Z0;

(ii) A first binding protein a which is a monoclonal antibody according to the first aspect of the invention or a recombinant protein according to the third aspect of the invention, coated onto the solid support Z0; and

(iii) A second binding protein B, which specifically binds to the CNN1 protein and which is coupled to or detectably labeled;

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

In another preferred example, when the detection system contains the CNN1 protein to be detected, the detection system may form a complex represented by formula I:

Z0-(A-C-B)n (I)

wherein the content of the first and second substances,

z0 is a solid phase carrier;

a is a first binding protein, said A is a monoclonal antibody according to the first aspect of the invention or a recombinant protein according to the third aspect of the invention, coated on a solid support surface;

b is a second binding protein, which is specifically bound with the CNN1 protein to be detected and is coupled or carries a detectable label; wherein the binding between said B and CNN1 proteins is non-competitive with the binding between said a and CNN1 proteins;

c is CNN1 protein to be detected;

n is a positive integer greater than or equal to 1; and is provided with

"-" is a bond or a linking group.

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

In another preferred embodiment, the solid phase carrier material comprises: a homopolymer, a copolymer, or a combination thereof.

In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.

In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: microspheres, microplates, slats, tubes, or combinations thereof.

In another preferred embodiment, the solid phase carrier is a magnetic particle.

In another preferred embodiment, the concentration of the magnetic particles is in the range of 0.1-10mg/mL, preferably 0.3-3mg/mL, more preferably 0.5-2mg/mL.

In another preferred embodiment, the concentration of the first binding protein A is in the range of 0.5-50. Mu.g/mL, preferably 1-20. Mu.g/mL, more preferably 5-10. Mu.g/mL.

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 sheep anti-human CNN1 polyclonal antibody.

In another preferred embodiment, the second binding protein B is a murine anti-human CNN1 mab IgG type.

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

In another preferred embodiment, the concentration of the second binding protein B is 0.05-5. Mu.g/mL, preferably 0.1-2. Mu.g/mL, more preferably 0.5-1. Mu.g/mL.

In a fifth aspect of the present invention, there is provided a kit comprising: a container and a reagent for forming a raw material for a detecting system according to the fourth aspect of the present invention, the reagent being located in the container.

In another preferred embodiment, the kit comprises:

(a) A first container and a solid phase carrier Z0 in the detection system in the first container;

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

(c) A third container and a second binding protein B in said detection system in the third container;

(d) Optionally a fourth container and a buffer for the reaction system located in the fourth container;

(e) Optionally a fifth container and a sample diluent in the fifth container; and

(f) Optionally a sixth container and a wash liquor located in the sixth container.

In another preferred example, the first container, the second container and the third container can be the same or different containers.

In another preferred embodiment, the second binding protein B in the detection system in the third container may be unlabeled for detection during long-term storage, and may be labeled within a certain time before use according to the use requirement.

In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is for (a) detecting a CNN1 protein, and/or (b) detecting or diagnosing aortic dissection.

In another preferred embodiment, the aortic dissection is detected by a plasma or serum assay, preferably a serum assay, more preferably a human serum assay.

In another preferred embodiment, the label or instructions states the following:

(i) If the CNN1 concentration of the sample from the test subject is higher than 2.43ng/ml, the subject has a greater probability of aortic dissection than the normal population.

In another preferred example, the detection object is a human.

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

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

In another preferred embodiment, the kit is a kit according to the fifth aspect of the invention.

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

In another preferred embodiment, the aortic dissection comprises: acute aortic dissection or/and chronic aortic dissection.

In another preferred embodiment, the aortic dissection is acute aortic dissection.

In a seventh aspect of the present invention, there is provided a method for detecting whether a CNN1 protein is contained in a sample, the method comprising:

providing a detection system according to the fourth aspect of the present invention, adding the sample to the detection system to perform a sufficient reaction, and detecting the obtained reaction solution.

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

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.

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

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

In another preferred embodiment, in said method, if a signal of a detectable label coupled or carried in said second binding protein B is detected, it is indicative that the CNN1 protein is contained in said sample; if no detectable signal is detected for the detectable label coupled or carried in the second binding protein B, this indicates that the sample does not contain CNN1 protein.

In another preferred embodiment, the method is diagnostic.

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

In another preferred embodiment, the method further comprises a chemiluminescence detection method.

In an eighth aspect of the present invention, there is provided a method for detecting or judging aortic dissection, comprising the steps of:

(a) Providing a test sample from a test subject ex vivo;

(b) Detecting the expression level of the CNN1 protein in the test sample; and

(c) Comparing the expression level of CNN1 determined in (b) with a control reference value; wherein the control is a control of the animal,

if the expression level of CNN1 in the test sample of the test subject is higher than the reference value A1,

it is suggested that the subject may be preliminarily judged that the probability of aortic dissection is higher than that of normal healthy people.

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

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

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

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

In another preferred example, the expression level of CNN1 protein in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.

In another preferred example, the expression level of the CNN1 protein in the sample is detected using the kit according to the fifth aspect of the present invention.

In another preferred example, the reference value A1 is a cut-off value (cut-off value).

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the antibody titers in the mouse serum after the fourth immunization. After dilution by two fold, the anti-CNN 1 monoclonal antibody was finally assayed for potency of 1.

FIG. 2 shows the SDS-PAGE protein electrophoresis test report of the CNN1 recombinant antigen after purification. According to the test report, the molecular weight of the CNN1 recombinant antigen is about 38 KDa.

FIG. 3 shows the SDS-PAGE protein electrophoresis test report of the CNN1 monoclonal antibody after purification. According to the test report, the heavy chain and the light chain of the anti-CNN 1 monoclonal antibody respectively have molecular weights of about 50kDa and about 26kDa.

Fig. 4 shows the reactivity of CNN1 monoclonal antibody to CNN1. As can be seen, the CNN1 antibody has good reactivity to the CNN1 antigen, and the potency of the antibody is 1.

Fig. 5 shows the specificity of CNN1 monoclonal antibody to MYO. As can be seen from the figure, the CNN1 monoclonal antibody is non-reactive to MYO, which indicates that the CNN1 monoclonal antibody has good specificity to MYO.

Fig. 6 shows the specificity of CNN1 monoclonal antibody to CTNI. As can be seen, the CNN1 monoclonal antibody is non-reactive to CTNI, which indicates that the CNN1 monoclonal antibody has good specificity to CTNI.

Fig. 7 shows the specificity of CNN1 monoclonal antibody for SMMHC. As can be seen, the CNN1 monoclonal antibody is non-reactive to SMMHC, which indicates that the CNN1 monoclonal antibody has good specificity to CNN1.

FIG. 8 shows the ROC curve determined for the CNN1 reference. The ROC had an area of 0.993 (95% CI, 0.986-1.000) and p-values of 0.000-0.05, which was found to be statistically significant for the model simulations.

FIG. 9 shows the comparison of CNN1 content in serum of healthy and aortic dissection patients. Comparison of serum CNN1 levels between healthy and aortic dissection patients was statistically significant (p < 0.05).

Detailed Description

The inventor of the invention develops a monoclonal antibody for efficiently detecting CNN1 protein and a kit thereof for the first time through extensive and intensive research and a large amount of screening. Specifically, the invention provides a hybridoma cell strain 1C2B8-2 capable of secreting the anti-CNN 1 monoclonal antibody. The results show that the presence or absence of CNN1 protein in a sample can be efficiently detected by the detection principle of the antigen sandwich method using the monoclonal antibody of the present invention as a primary antibody and antibody a (a commercially available detection antibody recommended for labeling) as a secondary antibody. The method of the invention has great clinical application value. The present invention has been completed based on this finding.

The invention relates to a hybridoma cell strain 1C2B8-2, a preparation method thereof, an anti-calcinin 1 monoclonal antibody secreted by the hybridoma cell strain and application of the monoclonal antibody in aortic dissection serological detection. Specifically, the invention provides a hybridoma cell strain 1C2B8-2 which is preserved in China Center for Type Culture Collection (CCTCC) at 27 months 4 and 2021 with the preservation number of CCTCC NO: C2021118.

The invention also provides a preparation method of the hybridoma cell line 1C2B8-2, which is obtained by immunizing a mouse by taking mature CNN1 recombinant protein with the sequence number of SEQ ID No. 1 as an antigen.

The invention also provides an anti-CNN 1 monoclonal antibody, which is secreted and generated by the hybridoma cell strain 1C2B8-2.

The anti-CNN 1 monoclonal antibody secreted and generated based on the hybridoma cell strain is IgG1, and has good specificity and high titer. Through inspection, the titer is as high as 1.

The invention also provides an anti-CNN 1 monoclonal antibody for capturing.

The present invention also provides a kit for detecting aortic dissection, comprising: the anti-CNN 1 monoclonal antibody secreted by the hybridoma cell strain 1C2B8-2 can be used as a capture antibody in a kit. The anti-CNN 1 monoclonal antibody and another anti-CNN 1 monoclonal antibody form a novel kit, which takes a direct chemiluminescence detection method as a platform and is used for detecting CNN1 in serum.

Term(s) for

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of or" consisting of 823030A ".

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Calmodulin (Calponin 1, CNN1)

Calmodulin is released from the inside of smooth muscle cells when the vascular wall is damaged, so that the serum concentration is increased, the blood serum concentration is stable after the increase, the window period duration is long, and the calmodulin can be used for the early rapid diagnosis or elimination and prognosis evaluation of AAD. However, no suitable antibodies are currently on the market that can be used in the development of commercial kits. Therefore, the invention aims to provide a hybridoma cell 1C2B8-2 and a preparation method thereof, an anti-CNN 1 monoclonal antibody secreted by the hybridoma cell strain and a CNN1 kit which is developed by using the antibody and is used for AAD diagnosis exclusion and prognosis evaluation based on an immunochemiluminescence platform.

As used herein, the terms "antigenic protein of the invention", "CNN1 protein", "CNN1 polypeptide" or "calmodulin CNN1" are used interchangeably and all refer to proteins or polypeptides having the amino acid sequence of calmodulin 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 provided by the invention comprises a complete amino acid sequence, a secreted protein, a mutant and a functionally active fragment thereof.

When the amino acid fragment of CNN1 is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the CNN1 nucleotide sequence disclosed in the present 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 by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, the sequence of interest can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments, derivatives thereof) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the present invention may be used to express or produce recombinant CNN1 polypeptides by conventional recombinant DNA techniques. Generally, the following steps are provided:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the present invention encoding a CNN1 polypeptide, or with a recombinant expression vector containing the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying protein from culture medium or cell.

In the present invention, the CNN1 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing CNN 1-encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; an insect cell; animal cells, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods of treatment, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the physical, chemical and other properties of the recombinant protein can be utilized for isolation and purification of the recombinant protein by various separation methods. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Specific antibodies

As used herein, the terms "antibody of the present invention", "anti-CNN 1 monoclonal antibody" and "anti-CNN 1 specific antibody" are used interchangeably.

The invention also includes monoclonal antibodies specific for the CNN1 protein. Here, "specificity" means that the antibody can bind to the CNN1 gene product or fragment. Preferably, those antibodies that bind to CNN1 gene products or fragments but do not recognize and bind to other non-relevant antigenic molecules. The antibodies of the present invention include those molecules capable of binding to CNN1 protein. The invention also includes those antibodies that bind to the CNN1 gene product in modified or unmodified form.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ A fragment; an antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but retain portions of the antibody from a human.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the purified CNN1 gene product, or antigenic fragment thereof, can be administered to an animal 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 the invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al,Nature256 of; 495,1975; in the case of the plant of Kohler et al,Eur.J.Immunol.6:511,1976; in the case of the plant of Kohler et al,Eur.J.Immunol.6:292,1976; in Hammerling et al,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the CNN1 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human CNN1 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

The anti-CNN 1 antibody can be used in immunohistochemical technique to detect CNN1 protein in specimen (especially serum specimen).

Affinity is a characteristic parameter of the relative state between antigen, antibody and antigen-antibody complex during a reversible reaction, which is more professionally and formally named dissociation equilibrium constant KD. In the present invention, the EC50 (half maximal effect concentration (EC 50) of the antibody secreted by the hybridoma cell line 1C2B8-2 and the CNN1 protein, which means the concentration causing 50% of the maximal effect) is equivalent to the KD value of the antibody and the CNN1 protein. The smaller the EC50 value is, the stronger the affinity is, and the strength of the affinity determines the relative amount of each component of the reversible reaction when the reaction is completed.

The antibody secreted by the hybridoma cell strain 1C2B8-2 (the preservation number is CCTCC NO: C2021118) has good affinity. In the specific embodiment of the invention, the EC50 of the antibody secreted by the hybridoma cell strain 1C2B8-2 (with the preservation number of CCTCC NO: C2021118) and the CNN1 protein is 0.76-2.05ng/mL, preferably 1.05-1.50ng/mL.

Coated antibodies

As used herein, "coated antibody," "capture antibody," or "solid phase antibody" are used interchangeably and refer to an antibody coated on a solid phase support, i.e., the first binding protein in the detection system of the invention.

The coated antibody can be nonspecifically adsorbed or physically adsorbed on polystyrene (ELISA plate), nitrocellulose membrane, etc., to form 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. And (3) adding a sample to be detected, and if the sample contains the target antigen, capturing the target antigen by the coated antibody. The antigen (target antigen) is CNN1 protein.

In a preferred embodiment of the invention, the coated antibody is capable of binding to the CNN1 protein with high affinity and high specificity. In another preferred embodiment, the coating antibody is produced by hybridoma cell line CCTCC NO: C2021118.

Detection of antibodies

As used herein, "detection antibody" or "labeled antibody" are used interchangeably to refer to an antibody that is used to detect whether a sample contains an antigen of interest, i.e., a second binding protein of the invention.

The detection antibody typically carries a detectable label, including a chromophore, chemiluminescent group, fluorophore, isotope, or 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. The presence and quantity of the antigen is determined by the detection of the label by the property of the detection antibody to bind specifically to the antigen of interest. In a preferred embodiment of the present invention, the detection antibody can bind to the CNN1 protein with high affinity and high specificity.

In a preferred embodiment of the invention, the detection antibody is antibody A (CNN 1 antibody (J9231-S1, 0.1 mg) purchased from ABNOVA), which is the detection antibody recommended for labeling.

Detection system and detection kit

The invention provides a detection system for detecting CNN1 protein, which comprises:

(i) A solid support Z0;

(ii) A first binding protein a, which is a monoclonal antibody according to the first aspect of the invention or a recombinant protein according to the third aspect of the invention, which is coated on the solid support Z0; and

(iii) A second binding protein B, which can be specifically bound to the CNN1 protein and is coupled to or carries a detectable label;

In another preferred example, when the detection system contains the CNN1 protein to be detected, a complex represented by formula I may be formed in the detection system:

Z0-(A-C-B)n (I)

wherein, the first and the second end of the pipe are connected with each other,

z0 is a solid phase carrier;

a is a first binding protein, said a being a monoclonal antibody according to the first aspect of the invention or a recombinant protein according to the third aspect of the invention, coated on a solid support surface;

b is a second binding protein, and the B is specifically bound with the CNN1 protein to be detected and is coupled or carries a detectable label; wherein the binding between the B and CNN1 proteins is non-competitive with the binding between the A and CNN1 proteins;

c is CNN1 protein to be detected;

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

"-" is a bond or a linking group.

The invention also provides a kit for detecting aortic dissection. The kit contains the detection system.

Specifically, the kit comprises:

(e) Optionally a fifth container and a sample diluent located in the fifth container; and

(f) Optionally a sixth container and a wash liquid located in the sixth container.

In the kit provided by the invention, the solid phase carrier contains a magnetic particle solid phase conjugate, wherein the concentration range of the magnetic particles is 0.3-3mg/mL, and the concentration range of the antibody is 1-20 mug/mL; the antibody concentration in the luminescent conjugate is 0.1-2. Mu.g/mL.

The kit of the present invention further contains a calibrator. Usually, CNN1 antigen is diluted into a series of antigen samples with a certain concentration by an antigen dilution solution, and the dilution is usually performed to 6 concentrations: 0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, as CAL1, CAL2, CAL3, CAL4, CAL5, CAL6.

The kit provided by the invention has good sensitivity and specificity, when the john index is 0.960, the sensitivity and the specificity are 100% and 96% respectively, and the reference value (cutoff value) of CNN1 is 2.43ng/ml. Wherein, if the CNN1 concentration of the sample from the test object is higher than 2.43ng/ml, the probability of aortic dissection of the object is higher than that of the normal population.

Detection method

The invention also provides methods for detecting or determining aortic dissection, in particular serological detection methods. The aortic dissection comprises: acute aortic dissection or/and chronic aortic dissection. Preferably, the aortic dissection is acute aortic dissection.

In a preferred embodiment of the present invention, the CNN1 chemiluminescence detection kit of the present invention is used in combination with a chemiluminescence analyzer for detecting CNN1 in a serum sample.

Application of the invention

The kit is used for accurately detecting the content of CNN1 in human plasma and detecting the variable quantity of CNN1 in human serum, and can be used for diagnosis and prognosis evaluation of aortic dissection.

The main advantages of the invention include:

(1) The hybridoma provided by the invention has high secretion output, and the secreted CNN1 monoclonal antibody has the advantages of high titer, good specificity and the like, and can be widely applied to the CNN1 detection field, such as the preparation field of detection reagents or detection equipment;

(2) The hybridoma cell strain provided by the invention secretes the monoclonal antibody CNN1 with IgG type, and the monoclonal antibody CNN1 is combined with CNN1 protein to have extremely strong specificity and sensitivity;

(3) The antibody secreted by the hybridoma cell strain can be applied to methodologies such as immunohistochemistry, immunoblotting, ELISA, chemiluminescence and the like, and has wide application scenes;

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

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

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1: screening and preparation of hybridoma cell strain

1.1 animal immunization

BlAB/C mice (only) were immunized after coupling the prepared calcinin 1 protein to Immunoplus for a total of 5 immunizations (20 ug each using a multiple spot immunization) with one immunization cycle of 2 weeks.

1.2 cell fusion and selection

Calponin1 protein was coated on the plate 3 days before the fusion, the mouse tail serum titer was detected by indirect Elisa method, and finally high titer BLAB/C mice (1. After 3 days, spleens of immunized mice were removed and crushed, and fused with an SP2/0 mouse myeloma cell line by the action of PEG (Sigma, P7181).

1.3 screening of hybridoma-Positive cell lines

Adding the fused cells into a 96-well plate with the laid feeder cells in advance, screening the supernatant of the fused cells by an indirect ELISA method after 7-10 days, and selecting the supernatant which is positive to the target protein Calponin 1. And screening all positive clones obtained in the primary screening stage by adopting an indirect ELISA method, and performing reverse screening by using His tag protein to show negative. The positive clones obtained at this stage were: 1C2B8, 1E10B4, 2G6B2, 8H8F4, 9A8D7, 9B4a10.

1.4 cloning screening by limiting dilution method

And c, subcloning the positive clone cells obtained in the step c by a limiting dilution method, and visually observing the monoclonal cells at least 3 times until a hybridoma cell line capable of stably secreting the anti-Calponin 1 protein is screened. The subclones obtained at this stage were: 1C2B8-1, 1C2B8-2, 1E10B4-1, 1E10B4-2, 2G6B2-1, 2G6B2-2, 8H8F4-1, 8H8F4-2, 9A8D7-1, 9A8D7-2, 9B4A10-1, 9B4A10-2.

1.5 subcloned cell supernatant titer determination

And D, performing titer determination on the subclones obtained in the step D by adopting an ELISA indirect method. The specific ELISA indirect method steps are as follows:

1) Antigen coating: recombinant CNN1 antigen is selected, diluted to 1 mu g/mL by coating liquid, added into a polystyrene 96 reaction well plate at 100 mu L/well and placed at 4 ℃ overnight.

2) Washing: the next day, the liquid in the wells was decanted and washed 3 times with the wash solution.

3) And (3) sealing: add 100. Mu.L/well blocking solution and let stand at room temperature for 0.5h.

4) Washing: washed 3 times with wash solution.

5) Add test sample (cell supernatant): cell supernatants containing monoclonal antibodies were serially diluted with PBS according to a gradient (1. And (4) covering the mixture, and incubating the mixture for 1 to 2 hours at a constant temperature of 37 ℃.

6) Washing: washed 3 times with wash solution.

7) Adding an enzyme-labeled secondary antibody: rabbit anti-mouse IgG-HRP, diluted with blocking solution 1, 8000, 100. Mu.L/well, covered with 37 ℃ and incubated for 1h at constant temperature.

8) Washing: washed 5 times with washing solution.

9) Color development: adding substrate 100 μ L/well, standing at room temperature in dark for 5-30min to show blue

10 Termination reaction, color comparison: add 50. Mu.L/well stop solution. The color turned yellow, and the absorbance of each well at 450nm was measured with a microplate reader. The maximum dilution of the positive reaction (at this time, the absorbance of the positive sample/the absorbance of the negative sample is more than or equal to 2.1) is the titer of the sample to be detected.

The specific measurement results are shown in table 1:

TABLE 1 hybridoma cell supernatant antibody titer determination

As shown in table 1, the following conclusions were drawn by potency measurements:

(1) The 12 subclones of this example were all determined to have a certain titer, and were all effective cell lines.

(2) Combining an ELISA indirect method to measure a potency determination standard (OD sample absorbance/OD control sample absorbance is more than or equal to 2.1, the data is effective, and the data can be continuously diluted), selecting subcloned cells with higher OD ratio under the same dilution degree, namely the subcloned cells with higher potency, and finally screening one subcloned cell for each positive cell, wherein the values are respectively as follows: 1C2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1, 9B4A10-1.

Example 2: preparation, purification and screening of monoclonal antibodies

2.1 antibody preparation

According to example 1, the present invention was carried out by producing an antibody by a method using an antibody cultured from a cell supernatant, and affinity chromatography purification was carried out using a Protein A pre-packed column. Specifically, the affinity column was equilibrated with 1 × PBS, the cell supernatant after centrifugation at 10000rpm (adjusted to pH 8.0 with Tris) was applied to the column, and after washing 5 column volumes with 1 × PBS, the cell supernatant was finally eluted with pH 3.5.1 MGly (adjusted to pH7.4 with Tris pH 8.8), and finally dialyzed against 1 × PBS buffer for Lowery quantification.

After the subcloned cells in example 1 were purified, they were processed with commercially available anti-CNN 1 monoclonal antibodies by the techniques of coating carboxyl magnetic particles and labeling acridinium ester, respectively, and antibody-paired on a chemiluminescence platform, and the final paired antibodies were screened.

Specific screening pairing results are shown in table 2:

TABLE 2 antibody Cross-pairing test results

Note: in the table a is a commercially available detection antibody recommended for labeling; f is a commercially available capture antibody recommended for coating.

As shown in table 2, the following conclusions were obtained from the cross-pairing experiments on the chemiluminescent platform:

(1) The selected 6 subcloned antibodies have certain reactivity, and the following pairs have good reactivity: 1C2B8-2 (coating) -9A8D7-1 (label); 1E10B4-1 (coating) -1C2B8-2 (marker); 2G6B2-1 (coated) -1C2B8-2 (labeled); 8H8F4-2 (coating) -1C2B8-2 (label); 9A8D7-1 (coating) -1C2B8-2 (marker); 9B4A10-1 (coating) -1C2B8-2 (marker);

(2) Cross-pairing of purchased antibody F (recommended for coating) and antibody A (recommended for labeling) was unresponsive.

(3) The purchased antibody F (recommended for coating) and the 6 selected antibodies (1C 2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1, 9B4A 10-1) were not reactive in pairing.

(4) The pairing results of the purchased antibody A (recommended for labeling) and the 6 selected antibodies (1C 2B8-2, 1E10B4-1, 2G6B2-1, 8H8F4-2, 9A8D7-1, 9B4A 10-1) were only relatively good in reactivity with 1C2B8-2, and the remaining pairs were non-reactive.

The above 4 conclusions are combined: the following pairs of 1C2B8-2 (coating) -9A8D7-1 (marker), 1E10B4-1 (coating) -1C2B8-2 (marker), 2G6B2-1 (coating) -1C2B8-2 (marker), 8H8F4-2 (coating) -1C2B8-2 (marker), 9A8D7-1 (coating) -1C2B8-2 (marker), 9B4A10-1 (coating) -1C2B8-2 (marker) and 1C2B8-2 (coating) -A (marker) are screened out preliminarily.

In view of the concentration proportion relation of the CAL1-CAL6 antigen samples used in the pairing test, the CAL1 is a blank sample, the CAL2-CAL5 optimal luminescence values are sequentially in a 2-time increasing relation, and the CAL6 antigen sample luminescence value is about 5 times of the CAL5 antigen sample luminescence value.

Thus, the binding assay results initially exclude the 1E10B4-1 (coated) -1C2B8-2 (labeled), 2G6B2-1 (coated) -1C2B8-2 (labeled), 8H8F4-2 (coated) -1C2B8-2 (labeled), 9A8D7-1 (coated) -1C2B8-2 (labeled), 9B4A10-1 (coated) -1C2B8-2 (labeled) pairings.

Further, according to the principle of priority of luminescence value, that is, the higher the luminescence value of the high-concentration sample is, the higher the sensitivity of the reagent is, the better the performance of the reagent is reflected, so that 1C2B8-2 (coated) -A (labeled) is finally selected as the final paired antibody. The 1C2B8-2 antibody secreting cells are the cell strains provided by the invention.

The evaluation of the antibody titer secreted by 1C2B8-2 in the serum after four immunizations of the mice is shown in figure 1, and the antibody titer in the serum after four immunizations is 1. The purified CNN1 recombinant antigen and the SDS-PAGE protein electrophoresis test report of the monoclonal antibody are respectively shown in fig. 2 and fig. 3, and the molecular weight of the CNN1 recombinant antigen is 38kDa, and the molecular weights of the heavy chain and the light chain of the anti-CNN 1 monoclonal antibody are respectively 50kDa and 26kDa.

2.2 Activity assay

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

1. preparation of CNN 1-coated ELISA plate

Dissolving CNN1 antigen with concentration of 100 mu g/mL at room temperature, uniformly mixing, diluting the mixture with coating solution to 0.2 mu g/mL,0.5 mu g/mL and 1 mu g/mL according to the following steps, adding the mixture into 100 mu L/well according to a loading layout table, respectively adding the mixture into an enzyme label strip, adding the coating solution as blank control of a coated antibody, and standing overnight at 2-8 ℃;

2. washing the plate with washing solution for 3 times, drying, adding 300 μ L/hole sealing solution, and sealing at room temperature for 1 hr; washing the plate with the washing solution for 3 times, and patting the plate dry for later use;

3. diluting the antibody with 3 times of initial gradient of 100000ng/mL according to the layout, and adding 100 mu L/hole into a micro-porous plate according to the layout;

4. placing the mixture into a micropore plate oscillator, and oscillating for 1 hour at 37 ℃ and 600 rpm;

5. washing the plate with washing solution for 3 times, and drying;

6. diluting the enzyme-linked antibody to a proper concentration, and uniformly mixing on a vortex mixer, wherein the concentration is 100 mu L/hole;

7. placing into a microplate oscillator, and oscillating at 37 deg.C and 600rpm for 1 hr;

8. washing the plate with washing solution for 4 times, and patting to dry;

9. taking out the color developing solution corresponding to the enzyme-linked antibody, balancing to room temperature within 20 minutes before use, and uniformly mixing on a vortex mixer;

10. adding color developing solution into the mixture at a rate of 100 mu L/hole by using 8 rows of guns;

11. the color developing solution is placed for 10-30 minutes at room temperature in a dark place;

12. stopping the reaction by adding 100 mu L of stop solution into the reaction vessel by using an 8-channel discharging gun; (this step may not be required depending on the substrate);

13. measuring a signal value by using an enzyme-labeling instrument;

14. analysis of results

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

Example 3: the specificity detection of the anti-CNN 1 monoclonal antibody secreted by the hybridoma cell 1C2B8-2 provided by the invention

In this example, CNN1 was detected by indirect method using the CNN1 recombinant antigen and MYO, CTNI, SMMHC in example 1 as coating antigens and the monoclonal antibody prepared in example 2 as a recognition antibody, respectively.

3.1 coating of enzyme-labeled plate

Coating with coating liquid (CBS: na) ₂ CO ₃ 0.8g，NaHCO ₃ 1.46g of distilled water, pH 9.0 and constant volume to 1L. ) The coating antigen was diluted to 1. Mu.g/mL, added to a 96-well plate at 0.1mL per well, and the plate was sealed in a 4 ℃ freezer overnight.

3.2 blocking of enzyme plates

The ELISA plate blocked overnight at 4 ℃ was removed, the supernatant discarded, the plate blotted dry, 300. Mu.L/well blocking solution (3% BSA + PBS) was added, and incubated for 2h at 37 ℃ in a thermostat.

3.3 Elisa plate

The microplate incubated at 37 ℃ for 2h was taken out of the incubator, the supernatant was discarded, the microplate was taken out, and a washing solution (PBST: KH) was used ₂ PO ₄ 0.2g，Na ₂ HPO ₄ ·12H ₂ O2.9g, naCl 8.0g, KCl 0.2g and Tween-200.5mL, adding water to a constant volume of 1L), and washing the plate washer for 5 times.

3.4 detection of anti-CNN 1 antibody by Indirect ELISA

After washing and drying the ELISA plate, respectively adding 100 mu L/hole of anti-CNN 1 monoclonal antibody secreted by hybridoma cell strains (1C 2B 8-2) into the ELISA plate with different envelope antigens, adding physiological saline into a control test, incubating in a 37 ℃ incubator for 2h, washing the plate washer for 5 times, adding HRP-labeled goat anti-mouse IgG secondary antibody, incubating in a 37 ℃ incubator for 1h, washing the plate washer for 5 times, adding 100 mu L/hole of TMB substrate (purchased from Sigma), incubating in a 37 ℃ incubator for 10min, displaying blue, adding 2M concentrated sulfuric acid to terminate the reaction, changing the color into yellow, measuring OD450nm by using an ELISA reader, and comparing and analyzing with the control test.

The final result is: as can be derived from fig. 4, the anti-CNN 1 monoclonal antibody in example 2 can detect only the CNN1 protein antigen and the antibody detection titer is 1; as can be arbitrarily derived from fig. 5, the anti-CNN 1 monoclonal antibody is non-reactive to MYO protein (myoglobin); from fig. 6, the anti-CNN 1 monoclonal antibody was non-reactive to CTNI protein (cardiac troponin); from fig. 7, the anti-CNN 1 monoclonal antibody was non-reactive to SMMHC protein (smooth muscle myosin heavy chain).

From the above, compared with other cardiac muscle proteins and smooth muscle proteins, the anti-CNN 1 monoclonal antibody provided by the invention has better specificity to CNN1.

Example 4: the invention provides a preparation component refinement of a kit

The invention provides a detection kit for detecting CNN1 based on a chemiluminescence platform, which mainly comprises the following components: solid phase conjugate, luminescent conjugate and matched calibrator and quality control product. The magnetic particle solid-phase conjugate mainly comprises a magnetic particle solid-phase conjugate and a luminescent conjugate. Wherein, the first and the second end of the pipe are connected with each other,

the magnetic particle solid phase conjugate is prepared by coating magnetic particles with the anti-CNN 1 monoclonal antibody in the embodiment 2, the concentration range of the magnetic particles in the solid phase conjugate system is 0.3-3mg/mL, and the concentration range of the antibody is 1-20 mug/mL;

the luminous conjugate is a commercially available anti-CNN 1 monoclonal antibody (J9231-S1, abnova) for labeling, and is an anti-CNN 1 antibody with mature and stable performances.

The anti-CNN 1 monoclonal antibody is marked by acridinium ester to prepare a luminescent conjugate, wherein the antibody concentration in the luminescent conjugate is 0.1-2 mug/mL; in addition, a calibrator (6-point calibration) and a quality control material (2-point quality control) are matched.

The kit provided by the invention is matched with a direct chemiluminescence detection immunoassay analyzer, and has the advantages of high sensitivity, high specificity, high accuracy, wide linear range, high-throughput rapid detection and the like.

Example 5: the invention provides a method for using a kit

5.1 calibration curve preparation:

1) Putting the reagent loaded with the solid-phase conjugate and the luminescent conjugate into a reagent disk of a full-automatic chemiluminescence analyzer (Kesimei Smart 6500/Touching i 100) to detect the reagent loading;

2) Placing the calibrator into a sample rack, conveying the calibrator to a sample channel of a full-automatic chemiluminescence immunoassay instrument, and obtaining a sample

The sample adding amount is 50 mu L;

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

cleaning for two steps, wherein each step is performed for 3 times;

4) A light emitting result of a full-automatic chemiluminescence immunoassay analyzer;

5) And (4) making a corresponding calibration curve according to the luminous value of the calibrator.

5.2 test procedure calibration:

1) Placing the quality control product into a sample rack, and conveying the sample rack to a sample channel of a full-automatic chemiluminescence immunoassay instrument, wherein the sample adding amount is 50 mu L;

2) Reaction mode: a two-step method, wherein the first step of reaction incubation is 6min, the second step of reaction incubation is 6min, the incubation is at 37 ℃, and the two steps of washing are carried out for 3 times;

3) A light emitting result of a full-automatic chemiluminescence immunoassay analyzer;

analyzing by combining the calibration curve and the test result of the quality control product, and if the quality control product is in the quality control range, indicating that the measurement program is in the quality control range and the test result is credible; otherwise, the reagents and instruments need to be re-checked and validated.

5.3 sample testing:

from the above, if the quality control product is within the controllable range, the sample test can be performed, and the specific steps are as follows:

1) Placing the sample into a sample rack, and conveying the sample to a sample channel of a full-automatic chemiluminescence immunoassay instrument, wherein the sample adding amount of a serum sample is 50 mu L;

2) Reaction mode: a two-step method, wherein the first step of reaction incubation is 6min, the second step of reaction incubation is 6min, the incubation is at 37 ℃, and the two steps of washing are carried out, wherein each step of washing is carried out for 3 times;

and combining the calibration curve and the sample test luminescence value to obtain the final test result of the sample.

Example 6: the kit performance provided by the invention

6Blank detection Limit (LOB)

Detecting with zero concentration calibrator or sample diluent as sample, repeating the measurement for 20 times to obtain RLU value (relative luminescence value) of 20 measurement results, calculating average value (M) and Standard Deviation (SD) to obtain RLU value corresponding to M +2SD, and substituting the RLU value corresponding to M +2SD into the above equation according to calibration curve equation of calibrator used in kit to obtain corresponding concentration value, which is blank detection limit. According to the test result, fitting a four-parameter Logistic curve to obtain a calibration curve:

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

A＝5.72720

B＝-0.57635

C＝44.58894

D＝2.63110

r^2＝0.99990

The blank detection limit is: 0.3ng/mL.

6.2 accuracy

The sample (A) is added into the sample (B), the volume ratio between the added sample (A) and the added sample (B) is 1.

In the formula:

r-recovery rate;

v-volume of liquid A;

v0-volume of liquid B;

c-the detection concentration of the solution B after being added into the solution A;

c0-detection concentration of solution B;

cs-concentration of liquid A.

The concentration values of the samples A and B and the mixed sample are respectively measured as follows: 107.0ng/ml, 13.7ng/ml and 21.7ng/ml, the recovery rate of the test is 87.1 percent, and the test meets the requirement.

Example 7 detection of clinical serum samples by the CNN1 chemiluminescence assay kit provided by the invention

45 aortic dissection patient samples and 150 health examination samples were collected from a certain Shanghai hospital and tested by using the kit provided in example 5, and the test results of CNN1 of aortic dissection patients and healthy persons are shown in Table 3.

TABLE 3 serum test results for healthy and aortic dissection patients

From the test results of healthy persons and aortic dissection patients shown in table 3, the ROC curve method was used in the present invention to fit the measurement values of CNN1 detected by the kit, and the ROC curve graph (see fig. 8) and the area under the ROC curve (see table 4) were obtained.

TABLE 4 area under the line

Testing result variables: CNN1 test results

a. Under the nonparametric assumption

b. Zero hypothesis: real area =0.5

As can be seen from Table 4, the area of the ROC curve was 0.993 (95% CI, 0.986-1.000), the p-values were 0.000-0.05, and it was found that the model simulation was statistically significant, and when the Jordan index was 0.960, the sensitivity and specificity were 100% and 96%, respectively, and the reference value for CNN1 was preliminarily found to be 2.43ng/mL. The reference value is combined, so that the detection kit provided by the invention has higher accuracy in clinical comparison.

As can be seen from FIG. 9, the mean values of healthy persons and patients are (0.43. + -. 0.22) ng/mL and (18.68. + -. 4.38) ng/mL respectively, and the serum level of CNN1 patient is 40 times of that of healthy persons (p < 0.05), again indicating that the kit provided by the invention has very high specificity and sensitivity.

Example 8: clinical cross-sample validation

10 myocardial injury clinical samples with the high-sensitivity troponin T (hs-cTnT) concentration level of 0.014-0.05ng/mL (99% percentile of apparent healthy people of hs-cTnT of Roche company is 0.014 ng/mL) are taken and detected by using the kit provided by the invention, and the test result is shown in Table 5.

TABLE 5 myocardial injury sample test results

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

As can be seen from Table 5, the test results of 10 samples of clinical myocardial damage tested by the kit provided by the invention are less than 2.43ng/ml and are all negative (the cutoff value of the kit provided by the invention is 2.43 ng/ml). Therefore, the kit provided by the invention can be used for testing that the aortic dissection sample and the myocardial injury sample are not crossed.

Strain preservation

The hybridoma cell strain 1C2B8-2 for producing the anti-calmodulin (Calponin 1, CNN1) monoclonal antibody is preserved in China center for type culture Collection (CCTCC, china, wuhan) at 27 months 4 in 2021, and the preservation number is CCTCC NO: C2021118.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Shanghai perspective diagnosis science and technology Limited

Shanghai Jingjing Life science and technology corporation

<120> hybridoma cell strain 1C2B8-2 and application thereof in antibody secretion

<130> P2020-2193

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 303

<212> PRT

<213> Artificial Sequence (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 Gln Met Gly Thr Asn Lys Phe Ala Ser Gln

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 Gln Pro Leu Asp Gln Ala Thr Ile Ser Leu Gln Met

195 200 205

Gly Thr Asn Lys Gly Ala Ser Gln Ala Gly Met Thr Ala Pro Gly Thr

210 215 220

Lys Arg Gln Ile Phe Glu Pro Gly Leu Gly Met Glu His Cys Asp Thr

225 230 235 240

Leu Asn Val Ser Leu Gln Met Gly Ser Asn Lys Gly Ala Ser Gln Arg

245 250 255

Gly Met Thr Val Tyr Gly Leu Pro Arg Gln Val Tyr Asp Pro Lys Tyr

260 265 270

Cys Leu Thr Pro Glu Tyr Pro Glu Leu Gly Glu Pro Ala His Asn His

275 280 285

His Ala His Asn Tyr Tyr Asn Ser Ala His His His His His His

290 295 300

Claims

1. The anti-calmodulin (Calponin 1, CNN 1) monoclonal antibody is characterized in that the antibody can be specifically combined with CNN1 protein, and the antibody is generated by a hybridoma cell strain 1C2B8-2 with the preservation number of CCTCC NO: C2021118.

2. The antibody of claim 1, wherein the EC50 of the antibody secreted by the hybridoma cell line 1C2B8-2 (CCTCC NO: C2021118) and the CNN1 protein is 0.76-2.05ng/mL, preferably 1.05-1.50ng/mL.

3. A hybridoma cell strain, which is characterized in that the preservation number is CCTCC NO of C2021118; the hybridoma cell strain can produce the anti-CNN 1 monoclonal antibody of claim 1.

4. A recombinant protein, said recombinant protein having:

(i) The monoclonal antibody of claim 1;

(ii) Optionally a tag sequence to assist expression and/or purification.

5. A detection system for detecting CNN1 protein, characterized in that the detection system comprises:

(i) A solid support Z0;

(ii) A first binding protein a which is the monoclonal antibody of claim 1 or the recombinant protein of claim 4, coated on the solid support Z0; and

6. The detection system according to claim 5, wherein when the detection system contains the CNN1 protein to be detected, a complex represented by formula I can be formed in the detection system:

Z0-(A-C-B)n(I)

z0 is a solid phase carrier;

a is a first binding protein, said A is the monoclonal antibody of claim 1 or the recombinant protein of claim 4, coated on a solid support surface;

c is CNN1 protein to be detected;

n is a positive integer greater than or equal to 1; and is provided with

"-" is a bond or a linking group.

7. A kit, comprising: a container and a raw reagent located within the container for forming a detection system as claimed in claim 5.

8. The use of the monoclonal antibody according to claim 1, the hybridoma cell strain according to claim 3, or the recombinant protein according to claim 4 for preparing a reagent or a kit for detecting CNN1 protein.

9. A method for detecting whether a CNN1 protein is contained in a sample, the method comprising:

providing a detection system as claimed in claim 5, adding the sample to the detection system to perform a sufficient reaction, and detecting the obtained reaction solution.

10. A method for detecting or determining aortic dissection comprising the steps of:

(a) Providing a test sample ex vivo from a test subject;

(b) Detecting the expression level of the CNN1 protein in the test sample; and