CN115594763A - Hybridoma cell strains 10B9E3-1 and 2D5C12-1 and application thereof in antibody secretion - Google Patents

Abstract

The invention provides a pair of anti-Smooth Muscle Myosin Heavy Chain (SMMHC) monoclonal antibodies, wherein the antibodies can be specifically combined with SMMHC, and are respectively generated by a hybridoma cell strain 10B9E3-1 (the preservation number is CCTCC NO: C2021117) and a hybridoma cell strain 2D5C12-1 (the preservation number is CCTCC NO: C2021119). In addition, the monoclonal antibodies secreted by the hybridoma cell strains 10B9E3-1 and 2D5C12-1 have good specificity, and the SMMHC serum detection kit developed on the basis of the chemiluminescence detection platform can be used for screening the SMMHC in the serum of a patient, so that the accuracy of early diagnosis of the acute aortic dissection is greatly improved, the prognosis condition of the 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 strains 10B9E3-1 and 2D5C12-1 and application thereof in antibody secretion

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to hybridoma cell strains 10B9E3-1 and 2D5C12-1 and application of the hybridoma cell strains to antibody secretion.

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 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. The Aortic Dissection (AD) is a very dangerous acute and severe cardiovascular disease (such as rupture of aorta can cause pericardial stuffing, pleural hemorrhage and retroperitoneal hematoma; aortic valve insufficiency causes left heart enlargement and heart failure; and also causes myocardial infarction, cerebral apoplexy, paraplegia, abdominal organ ischemia and the like due to blood supply disturbance of important organs) which can cause multiple system serious complications, and has extremely high fatality rate and 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 about 1% per hour, half or more than 1 week, 70% within 2 weeks, 90% within 1 year, and if appropriate medications and surgical treatments are given in time, survival rates can be greatly increased and fatality rates reduced to 18% to 27%. The aortic dissection has multiple etiologies and has multiple possible disease manifestations, which results in 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, it is urgently needed to find a single diagnosis mode which breaks through the existing aortic dissection imaging, provide a simple, convenient and high-specificity diagnosis mode of peripheral blood of the aortic dissection for diagnosis elimination, prognosis evaluation and the like of the AAD, and provide a new tool for diagnosis, death early warning and the like of the AAD so as to reduce the fatality rate of patients.

Disclosure of Invention

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

In a first aspect of the invention, an anti-Smooth Muscle Myosin Heavy Chain (SMMHC) monoclonal antibody is provided, wherein the antibody can be specifically combined with SMMHC protein, and is produced by a hybridoma cell strain 10B9E3-1 with the preservation number of CCTCC NO: C2021117; or the antibody is generated by a hybridoma cell strain 2D5C12-1 with the preservation number of CCTCC NO: C2021119.

In another preferred embodiment, the antibody is used for detection of SMMHC proteins.

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

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

In another preferred embodiment, the EC50 of the antibody secreted by the hybridoma cell strain 10B9E3-1 (CCTCC NO: C2021117) and SMMHC protein is 0.50-2.00ng/mL, preferably 0.60-1.85ng/mL.

In another preferred embodiment, the EC50 of the antibody secreted by the hybridoma cell strain 2D5C12-1 (CCTCC NO: C2021119) and SMMHC protein is 0.55-2.10ng/mL, preferably 0.70-1.80ng/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 embodiment, the antibody specifically binds to the SMMHC protein.

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

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

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: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

In another preferred embodiment, the antibody is used for detection of a SMMHC protein.

In a second aspect of the invention, the invention provides a hybridoma cell strain with a preservation number of CCTCC NO of C2021117; or the preservation number is CCTCC NO of C2021119; the hybridoma cell line can produce the anti-SMMHC 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 invention, there is provided an assay system for the detection of SMMHC proteins, 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, which is coated on the solid support Z0; and

(iii) A second binding protein B that specifically binds to an SMMHC protein and is coupled to or detectably labeled;

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

In another preferred embodiment, when the detection system contains the SMMHC protein to be detected, a complex represented by formula I can be formed in the detection system:

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 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 that specifically binds to an SMMHC protein to be detected and is coupled to or carries a detectable label; wherein the binding between the B and SMMHC proteins is non-competitive with the binding between the A and SMMHC proteins;

c is SMMHC protein to be detected;

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

"-" 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 first binding protein A is produced by hybridoma cell line CCTCC NO: C2021117.

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 SMMHC monoclonal antibody, rabbit anti-human SMMHC polyclonal antibody, mouse anti-human SMMHC monoclonal antibody, mouse anti-human SMMHC polyclonal antibody, and sheep anti-human SMMHC polyclonal antibody.

In another preferred embodiment, the second binding protein B is a murine anti-human SMMHC monoclonal antibody IgG class.

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

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 of a raw material for forming a detecting system according to the fourth aspect of the present invention, which is 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 located 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 long-term storage, and the labeling reaction may be carried out for a certain period of time before use, depending on the requirements of use.

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

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.

In another preferred embodiment, the label or instructions may indicate the following:

(i) If the SMMHC concentration of the sample from the subject is higher than 1.20ng/ml, the subject has a greater chance 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 a 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, for preparing a reagent or a kit for detecting SMMHC protein.

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

In another preferred embodiment, the reagent or kit is a diagnostic reagent or 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 sample contains SMMHC proteins, 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 comprises qualitative detection and quantitative detection.

In another preferred embodiment, in the method, a signal from the detectable label coupled or carried by the second binding protein B is detected, indicating that the sample contains SMMHC protein; if no signal is detected from the detectable label coupled or carried by the second binding protein B, it is an indication that the sample does not contain SMMHC 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 is 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 ex vivo from a test subject;

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

(c) Comparing the expression level of SMMHC determined in said (b) with a control reference value; wherein the control is a control of the plurality of control cells,

if the expression level of SMMHC 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 subject.

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 embodiment, the aortic dissection is acute aortic dissection.

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

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

In another preferred embodiment, the expression level of SMMHC protein in a sample is detected using a kit according to the fifth aspect of the 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 1.20ng/ml.

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

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 repeated herein, depending on the space.

Drawings

Figure 1 shows the antibody titers in the mouse serum after the fourth immunization. The anti-SMMHC monoclonal antibody (10B 9E 3-1) was diluted in two-fold and the final titer was determined to be 1.

Figure 2 shows the antibody titers in the mouse serum after the fourth immunization. The anti-SMMHC monoclonal antibody (2D 5C 12-1) was diluted in two-fold and the final titer was determined to be 1.

FIG. 3 shows a purified SMMHC recombinant antigen SDS-PAGE protein electrophoresis test report. The molecular weight of the SMMHC recombinant antigen is about 90KDa according to the test report.

FIG. 4 shows a report of SDS-PAGE protein electrophoresis test of purified SMMHC monoclonal antibody (10B 9E 3-1). The molecular weights of the heavy chain and the light chain of the anti-SMMHC monoclonal antibody are about 50kDa and about 26kDa respectively according to test reports.

FIG. 5 SDS-PAGE protein electrophoresis test report of purified SMMHC monoclonal antibody (2D 5C 12-1). The molecular weights of the heavy chain and the light chain of the anti-SMMHC monoclonal antibody are about 50kDa and about 26kDa respectively according to test reports.

FIG. 6 shows the response of SMMHC monoclonal antibody (10B 9E 3-1) to SMMHC. As can be seen, the SMMHC antibody was very reactive against the SMMHC antigen and the antibody titer was 1.

FIG. 7 shows the response of SMMHC monoclonal antibody (2D 5C 12-1) to SMMHC. As can be seen, the SMMHC antibody was very reactive against the SMMHC antigen and the antibody titer was 1.

FIG. 8 shows the reactivity of SMMHC monoclonal antibody (10B 9E 3-1) to MYO. As can be seen, the SMMHC antibody has no reactivity to MYO antigen, which indicates that the SMMHC monoclonal antibody has good specificity to MYO.

FIG. 9 shows the reactivity of SMMHC monoclonal antibody (2D 5C 12-1) to MYO. As can be seen, the SMMHC antibody is non-reactive to MYO antigen, indicating that the SMMHC monoclonal antibody has good specificity to MYO.

FIG. 10 shows the reactivity of SMMHC monoclonal antibody (10B 9E 3-1) to CTNI. As can be seen, the SMMHC antibody was non-reactive to CTNI antigen, indicating that the SMMHC monoclonal antibody has good specificity for CTNI.

FIG. 11 shows the reactivity of SMMHC monoclonal antibody (2D 5C 12-1) to CTNI. As can be seen, the SMMHC antibody was non-reactive to CTNI antigen, indicating that the SMMHC monoclonal antibody has good specificity for CTNI.

FIG. 12 shows the reactivity of SMMHC monoclonal antibody (10B 9E 3-1) to CNN 1. As can be seen, the SMMHC antibody was non-reactive to CNN1 antigen, indicating that the SMMHC monoclonal antibody has good specificity for CNN 1.

FIG. 13 shows the reactivity of SMMHC monoclonal antibody (2D 5C 12-1) to CNN 1. As can be seen, the SMMHC antibody was non-reactive to CNN1 antigen, indicating that the SMMHC monoclonal antibody has good specificity for CNN 1.

Figure 14 shows ROC curves determined for SMMHC reference values. The ROC area was 0.964 (95% CI, 0.940-0.989), and the p-values were 0.000-0.05, indicating that the model simulations were statistically significant.

FIG. 15 shows comparison of SMMHC levels in serum of healthy and aortic dissection patients. Comparison of serum SMMHC levels between healthy and aortic dissectors was statistically significant (p < 0.05).

Detailed Description

The inventor develops a monoclonal antibody pair for efficiently detecting SMMHC 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 10B9E3-1 and 2D5C12-1 capable of secreting the anti-SMMHC monoclonal antibody. The result shows that the monoclonal antibody secreted by the hybridoma cell strain 10B9E3-1 is used as a primary antibody, the monoclonal antibody secreted by the hybridoma cell strain 2D5C12-1 is used as a secondary antibody, and the detection principle of an antigen sandwich method is utilized to efficiently detect whether SMMHC protein exists in a sample. The method of the invention has great clinical application value. The present invention has been completed based on this finding.

The invention relates to hybridoma cell strains 10B9E3-1 and 2D5C12-1, a preparation method thereof, an SMMHC (small molecule antigen binding domain) resistant monoclonal antibody secreted by the hybridoma cell strains and application of the SMMHC resistant monoclonal antibody in aortic dissection serological detection. Specifically, the invention provides hybridoma cell strains 10B9E3-1 and 2D5C12-1 which are preserved in China center for type culture Collection at 27.4.2021, wherein the preservation number of the hybridoma cell strains 10B9E3-1 is CCTCC NO: C2021117; the preservation number of the hybridoma cell strain 2D5C12-1 is CCTCC NO: C2021119.

The invention also provides a preparation method of the hybridoma strains 10B9E3-1 and 2D5C12-1, which is obtained by immunizing a mouse by taking mature SMMHC recombinant protein with the sequence number of SEQ ID No:1 as an antigen.

The invention also provides two anti-SMMHC monoclonal antibodies, wherein the monoclonal antibodies are secreted and generated by the hybridoma cell strain 10B9E3-1 and 2D5C12-1.

The SMMHC monoclonal antibodies secreted and generated based on the hybridoma cell strain are all IgG1, and have good specificity and high titer. Through inspection, the titer is as high as 1.

The present invention also provides a kit for detecting aortic dissection, comprising: the anti-SMMHC monoclonal antibody secreted by the hybridoma cell strain 10B9E3-1 can be used as a capture antibody in a kit, the anti-SMMHC monoclonal antibody secreted by the hybridoma cell strain 2D5C12-1 can be used as a detection antibody in the kit,

the two antibodies constitute a novel kit, which takes a direct chemiluminescence detection method as a platform and is used for detecting SMMHC 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 ….

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.).

Smooth Muscle Myosin Heavy Chain (SMMHC)

The heavy chain of the myosin of the smooth muscle is released from the inside of smooth muscle cells when the blood vessel wall is damaged, so that the concentration of serum is increased, the concentration of the myosin of the smooth muscle is restored to the concentration level of normal healthy people after 24 hours, the duration of a window period is short, and the myosin of the smooth muscle can be used for 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 hybridoma cells 10B9E3-1 and 2D5C12-1, a preparation method thereof, anti-SMMHC monoclonal antibodies secreted by the two hybridoma cell strains, and an SMMHC kit which is developed by using the antibodies 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", "SMMHC protein", "SMMHC polypeptide" or "smooth muscle myosin heavy chain SMMHC" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of smooth muscle myosin heavy chain SMMHC (SEQ ID NO: 1). They include SMMHC with or without the initial methionine. In addition, the term also includes full-length SMMHC and fragments thereof. The SMMHC proteins of the present invention include the complete amino acid sequence, secreted proteins, mutants and functionally active fragments thereof.

When an amino acid fragment of SMMHC 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 ECM1 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, two or more PCR amplifications are often required, and then the amplified fragments are spliced together 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 a cell, and isolating the relevant sequence from the propagated host cell 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 ECM1 polypeptides by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a SMMHC polypeptide, or with a recombinant expression vector comprising 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 SMMHC 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 SMMHC-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.

Furthermore, the expression vector preferably comprises 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 encapsulation, 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 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 invention", "anti-SMMHC monoclonal antibody" and "anti-SMMHC specific antibody" are used interchangeably.

The invention also includes monoclonal antibodies specific for SMMHC proteins. Herein, "specificity" refers to the ability of an antibody to bind to a SMMHC gene product or fragment. Preferably, those antibodies that bind to SMMHC gene products or fragments thereof but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that are capable of binding to SMMHC proteins. The invention also includes those antibodies that bind to the SMMHC 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 the antibody portion 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, a purified SMMHC gene product, or antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing SMMHC proteins or antigenic fragments thereof can be used to immunize animals to produce antibodies. Book (I)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; the result of Kohler et al,Eur.J.Immunol.6:511,1976; the result of Kohler et al,Eur.J.Immunol.6:292,1976; the Hammerling et al, in the name of,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). The antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the SMMHC 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 a human ECM1 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).

Antibodies against SMMHC proteins can be used in immunohistochemical techniques to detect SMMHC proteins in a sample, particularly a plasma sample.

Affinity is a characteristic parameter of the relative state between antigen, antibody and antigen-antibody complex during a reversible reaction, which is more specialized and termed by the dissociation equilibrium constant KD. In the present invention, the EC50 (half maximal effect concentration for 50% of maximum effect, EC50) of the antibody secreted from hybridoma cell line 10B9E3-1 or hybridoma cell line 2D5C12-1 and SMMHC protein is equivalent to the KD value of the antibody and SMMHC protein. The smaller the EC50 value, the stronger the affinity, and the stronger the affinity determines the relative amount of each component of the reversible reaction at the end of the reaction.

The antibody secreted by the hybridoma cell strain 10B9E3-1 (the preservation number is CCTCC NO: C2021117) or the hybridoma cell strain 2D5C12-1 (the preservation number is CCTCC NO: C2021119) has good affinity.

In the specific embodiment of the invention, the EC50 of the antibody secreted by the hybridoma cell strain 10B9E3-1 (the preservation number is CCTCC NO: C2021117) and SMMHC protein is 0.50-2.00ng/mL; preferably 0.60-1.85ng/mL; the EC50 of the antibody secreted by the hybridoma cell strain 2D5C12-1 (the preservation number is CCTCC NO: C2021119) and SMMHC protein is 0.55-2.10ng/mL, preferably 0.70-1.80ng/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 become 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 adding a sample to be detected, and if the sample contains the target antigen, capturing the target antigen by the coated antibody. The antigen (antigen of interest) according to the present invention is a SMMHC protein.

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

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 detection of the label using the property of the detection antibody to bind specifically to the antigen of interest. In a preferred embodiment of the invention, the detection antibody binds with high affinity and high specificity to the SMMHC protein.

In a preferred embodiment of the invention, the detection antibody is generated by a hybridoma cell strain CCTCC NO: C2021119.

Detection system and detection kit

The invention provides a detection system for detecting SMMHC 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 that specifically binds to an SMMHC protein and is coupled to or detectably labeled;

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

In another preferred embodiment, when the detection system contains the SMMHC protein to be detected, a complex represented by formula I can be formed in the detection system:

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 that specifically binds to an SMMHC protein to be detected and is coupled to or carries a detectable label; wherein the binding between the B and SMMHC proteins is non-competitive with the binding between the A and SMMHC proteins;

c is SMMHC 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:

(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 located in the fifth container; and

(f) Optionally a sixth container and a wash liquor 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 particle is 0.2-4mg/mL, and the concentration range of the antibody is 0.5-25 mug/mL; the concentration of the antibody in the luminous combination is 0.2-1.8 mu g/mL.

The kit of the present invention further contains a calibrator. Usually refers to the antigen sample diluted by antigen diluent at a certain concentration into a series of concentrations in the mother night, and the dilution into 6 concentrations is usually: 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 0.755, the sensitivity and the specificity are respectively 80.5% and 95.0%, and the reference value (cutoff value) of the SMMHC is preliminarily obtained to be 1.20ng/mL. Wherein if the SMMHC concentration of the sample from the test subject is higher than 1.20ng/ml, the subject has a greater probability of aortic dissection than 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 SMMHC chemiluminescence detection kit of the present invention is used in combination with a chemiluminescence analyzer for detecting SMMHC in a serum sample.

Application of the invention

The kit is used for accurately detecting the content of SMMHC in human serum and detecting the variable quantity of the SMMHC in the human serum, and can be used for the 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 SMMHC monoclonal antibody has the advantages of high titer, good specificity and the like, and can be widely applied to the field of SMMHC detection, such as the field of preparation of detection reagents or detection equipment and the like;

(2) The hybridoma cell strain provided by the invention secretes monoclonal antibody SMMHC antibody with IgG type, and has extremely strong specificity and sensitivity in combination with SMMHC protein;

(3) The antibody secreted by the hybridoma cell strain can be applied to the 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 an SMMHC detection kit, has the advantages of high sensitivity, good specificity, short detection time and the like, and can well assist 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 followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

EXAMPLE 1 screening and preparation of hybridoma cell lines

1.1 animal immunization

BlAB/C mice (only) were immunized after conjugation of the prepared myostatin-11isoform SM1A protein to Immunoplus for a total of 5 immunizations (20 ug each using multiple immunizations) for 2 weeks as one immunization cycle.

1.2 cell fusion and selection

3 days before fusion, myostatin-11isofomm SM1A protein is coated on a plate, the tail serum titer of the mice is detected by an indirect Elisa method, and finally, high-immune-titer BLAB/C mice (1. After 3 days, the spleens of immunized mice were crushed and fused with SP2/0 mouse myeloma cell line by the action of PEG (Sigma, P7181).

1.3 screening of hybridoma-Positive cell lines

The fused cells were added to a 96-well plate previously plated with feeder cells, and after 7 to 10 days, supernatants of the fused cells were screened by indirect ELISA, and supernatants positive for the target protein myostatin-1isofom SM1A were selected. 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: 2D5C12, 7F11C4, 8C8E11, 10B9E3, 13D7E5, 15D5A3

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 which stably secretes the anti-myostatin-1isofom SM1A protein can be screened. The subclones obtained at this stage were: 2D5C12-1, 2D5C12-2, 7F11C4-1, 7F11C4-2, 8C8E11-1, 8C8E11-2, 10B9E3-1, 10B9E3-2, 13D7E5-1, 13D7E5-2, 15D5A3-1, 15D5A3-2.

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 SMMHC antigen was selected, diluted to 1. Mu.g/mL with the coating solution, and 100. Mu.L/well was added to a polystyrene 96 reaction well plate and left overnight at 4 ℃.

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

9) Color development: substrate 100. Mu.L/well was added, and the mixture was left at room temperature in the dark for 5 to 30min to show blue color.

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

1) The 12 subclones of the strain in the experiment are determined to have certain titer and are all effective cell strains.

2) Combining an ELISA indirect method to measure titer determination standard (OD sample absorbance/OD control sample absorbance is more than or equal to 2.1, the data is effective, and the dilution can be continued), selecting subcloned cells with higher OD ratio under the same dilution degree, namely the subcloned cells with higher titer, which are respectively: 2D5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1 and 15D5A3-1.

EXAMPLE 2 preparation, purification and screening of monoclonal antibodies

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.

Specific screening pairing results are shown in table 2:

TABLE 2 antibody Cross-pairing test results

Note: b is a detection antibody recommended for labeling purchased from commercial sources; p is an antibody purchased from the market.

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, wherein the following pairs have good reactivity: 2D5C12-1 (coating) -13D7E5-1 (marker); 7F11C4-2 (coated) -2D5C12-1 (labeled); 8C8E11-2 (coating) -15D5A3-1 (marker); 10B9E3-1 (coating) - -2D5C12-1 (marker); 13D7E5-1 (coating) -15D5A3-1 (marker); 15D5A3-1 (coating) -7F11C4-2 (label).

2) The purchased antibody P (coating) and antibody B (recommended for labeling) were cross-paired with some reactivity.

3) Cross-pairing of the purchased antibody B (recommended for labeling) and the 6 selected antibodies (2D 5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1, 15D5A 3-1) was non-reactive.

4) The purchased antibody P (used as a marker) and 6 screened antibodies (2D 5C12-1, 7F11C4-2, 8C8E11-2, 10B9E3-1, 13D7E5-1, 15D5A 3-1) were cross-paired, with 10B9E3-1 (coated) -P (marker) being the best reactivity.

To sum up 4 conclusions: preliminarily screening out the following pairs of 2D5C12-1 (coating) -13D7E5-1 (label); 7F11C4-2 (coated) -2D5C12-1 (labeled); 8C8E11-2 (coating) -15D5A3-1 (marker); 10B9E3-1 (coating) - -2D5C12-1 (marker); 13D7E5-1 (coating) -15D5A3-1 (label); 15D5A3-1 (coating) -7F11C4-2 (marker); p (coating) -B (label); 10B9E3-1 (coating) - - -P (labeling).

In view of the concentration proportion relationship of the antigen samples CAL1-CAL6 used in the pairing test, the optimal luminescence values of CAL2-CAL5 are sequentially in a 2-time increasing relationship, and the luminescence value of the CAL6 antigen sample is about 5 times of that of the CAL5 antigen sample. And simultaneously, the principle of priority of luminescence value is taken into consideration, namely 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, and therefore, 10B9E3-1 (coating) -2D5C12-1 (marking) is finally selected as a final paired antibody. Secretory cells corresponding to the 10B9E3-1 and 2D5C12-1 antibodies are the cell strains provided by the invention.

The antibody titer of 10B9E3-1 and 2D5C12-1 in the serum and the secreted antibody titer are respectively shown in figure 1 and figure 2 after the mice are immunized for four times, and the antibody titer in the serum is 1. The purified SMMHC recombinant antigen and the SDS-PAGE protein electrophoresis test report of the monoclonal antibody are respectively shown in fig. 3, fig. 4 and fig. 5, and the molecular weight of the SMMHC recombinant antigen can be obtained to be 90kDa, and the molecular weights of the heavy chain and the light chain of the anti-SMMHC monoclonal antibody are respectively 50kDa and 26kDa.

The experimental steps of the EC50 of the antibody and SMMHC protein secreted by the hybridoma cell strain 10B9E3-1 (with the preservation number of CCTCC NO: C2021117) and 2D5C12-1 (with the preservation number of CCTCC NO: C2021119) are as follows:

1. preparation of SMMHC coated ELISA plate

Dissolving SMMHC antigen with the 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/hole 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 closed 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-fold gradient from 100000ng/mL according to the layout, and adding 100 muL/hole into a microplate according to the layout;

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

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 a color developing solution corresponding to the enzyme-linked antibody for 20 minutes before use, balancing to room temperature, 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. placing the color developing solution at room temperature in a dark place for 10-30 minutes;

12. stopping reaction by adding 100 mul/hole stop solution with 8 rows of guns; (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

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. EC50 (half maximal effect concentration (50% of maximum effect, EC50) means a concentration that causes 50% of the maximal effect) is equivalent to the KD value. The smaller the EC50 value, the stronger the affinity, and the stronger the affinity determines the relative amount of each component of the reversible reaction at the end of the reaction.

The EC50 of the antibody secreted by the hybridoma cell strain 10B9E3-1 (the preservation number is CCTCC NO: C2021117) and the 2D5C12-1 (the preservation number is CCTCC NO: C2021119) and the antibody secreted by SMMHC protein are respectively 0.60-1.85ng/mL and 0.70-1.80ng/mL.

EXAMPLE 3 specific detection of anti-SMMHC monoclonal antibodies secreted by hybridoma cells 10B9E3-1 and 2D5C12 provided by the invention

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

3.1 coating of ELISA plates

With coating liquid (CBS: na) ₂ CO ₃ 0.8g，NaHCO ₃ 1.46g, procling 300 mL, distilled water, pH 9.0, 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 incubation was carried out in a thermostat at 37 ℃ for 2h.

3.3 Elisa plate

The microplate incubated at 37 ℃ for 2 hours 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 4.2 g, na2HPO4.12H2O 2.9g, naCl 8.0g, KCl 0.2g and Tween-20.5mL, and adding water to a constant volume of 1L. ) The plate washer cleans 5 times.

3.4 detection of anti-SMMHC antibodies by indirect ELISA.

After the ELISA plate is cleaned and dried, hybridoma cell strains 10B9E3-1 and 2D5C1 are respectively added into the ELISA plate with different coating antigens2 secretion of anti-SMMHC monoclonal antibody 100 muL/hole, adding physiological saline in a contrast test, incubating in a constant temperature cabinet at 37 ℃ for 2h, washing a plate washing machine for 5 times, adding a goat anti-mouse IgG secondary antibody marked by HRP (horse radish peroxidase) 100 muL/hole, incubating in a constant temperature cabinet at 37 ℃ for 1h, washing the plate washing machine for 5 times, adding a TMB substrate (purchased from Sigma) 100 muL/hole, incubating in a constant temperature cabinet at 37 ℃ for 10min, displaying blue, adding 2M concentrated sulfuric acid to stop reaction, changing color to yellow, and measuring OD (OD) by using a microplate reader _450nm And comparing and analyzing with a control test. The final result is: as can be seen from fig. 6 and 7, the anti-SMMHC monoclonal antibody in example 2 can detect only SMMHC protein antigen and the antibody detection titer is 1; as can be arbitrarily derived from fig. 8 and 9, the anti-SMMHC monoclonal antibody is non-reactive to MYO protein; from fig. 10, 11, anti-SMMHC monoclonal antibodies were non-reactive to CTNI protein; from fig. 12, 13, the anti-SMMHC monoclonal antibody was non-reactive to the CNN1 protein. From the above, it can be concluded that the anti-SMMHC monoclonal antibody provided by the invention has better specificity.

Example 4 preparation of kits of the invention

The detection kit for detecting SMMHC based on the chemiluminescence platform mainly comprises two parts, namely a magnetic particle solid phase conjugate and a luminescence conjugate. Wherein, the magnetic particle solid phase conjugate is prepared by coating magnetic particles with anti-SMMHC monoclonal antibody secreted by hybridoma cell 10B9E3-1 in example 2, the concentration range of the magnetic particles in the solid phase conjugate system is 0.2-4mg/mL, and the concentration range of the antibody is 0.5-25 mug/mL; the luminous conjugate is prepared by labeling acridinium ester with an anti-SMMHC monoclonal antibody secreted by the hybridoma cells 2D5C12-1 in example 2, and the antibody concentration in the luminous conjugate is 0.2-1.8 mu g/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 methods of using kits

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 immunoassay analyzer (Coosmei Smart 6500 or TeSMI i 100) for reagent loading detection;

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

3) 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;

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

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

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 detector, 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, wherein each step of washing is 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 reviewed and validated for determination.

5.3 sample testing:

from the above, if the quality control product is in 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 detector, wherein the sample adding amount is 50 mu L (a plasma sample and a serum sample);

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;

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

Example 6 kit Properties provided by the invention

6.1 lowest detection Limit (LOD)

Detecting with zero concentration calibrator or sample diluent as sample, repeatedly measuring 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, substituting 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, i.e. minimum detection limit

According to the test result, fitting a four-parameter Logistic curve to obtain a calibration curve:

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

A＝6.04309

B＝-0.57649

C＝35.77110

D＝2.58059

r^2＝0.99986

The lowest detection limit is: 0.13ng/mL.

6.2 accuracy

Adding the sample (A) to the sample (B) in a volume ratio of 1:9,

the recovery rate is 85-115%.

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 the solution B;

cs-concentration of liquid A.

The concentration values of the sample A, B and the mixed sample were measured as follows: 88.27ng/ml, 12.22ng/ml and 21.03ng/ml, the recovery rate of the test is 113.6%, which meets the requirement.

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

1. Clinical serum sample collection

41 aortic dissection patient samples and 180 healthy physical examination samples were collected from a certain Shanghai hospital and tested using the kit provided in example 5, and the results of SMMHC test of patients and healthy persons are shown in Table 3.

TABLE 3 serum test results for healthy and aortic dissection patients

In the invention, the reference value of SMMHC detected by the kit can be fitted by using the ROC curve method to obtain an ROC curve chart shown in figure 14 and the area under the ROC curve shown in table 4 for healthy people and aortic dissection patients.

Table 4 area under curve

Variable of test result SMMHC test

a. Under the nonparametric assumption

b. Zero hypothesis: real area =0.5

From the above table of the area of the ROC curve of 0.964 (95% CI, 0.940-0.989), p values of 0.000-0.05, it was concluded that the model simulations were statistically significant, and that the reference value for SMMHC was initially found to be 1.20ng/mL when the john index was 0.755, and the sensitivity and specificity were 80.5% and 95.0%, respectively. The reference value is combined, so that the detection kit provided by the invention has higher accuracy with clinical comparison. As can be seen from FIG. 15, the mean values of healthy persons and patients are (0.41. + -. 0.19) ng/mL and (7.55. + -. 2.88) ng/mL, respectively, and the serum level of SMMHC patients is 18 times that of healthy persons (p < 0.05), again indicating that the kit of the present invention has much higher specificity and sensitivity.

Example 8 clinical Cross-sample validation

10 myocardial injury clinical samples with CTNT concentration level of 0.014-0.06ng/ml are taken, the kit provided by the invention is used for detection, and the test results are shown in Table 5.

TABLE 5 myocardial injury sample test results

As can be seen from the table 5, the test results of 10 samples tested clinically by the kit provided by the invention for myocardial damage are all less than 1.20ng/ml and are all negative (the cutoff value of the kit provided by the invention is 1.20 ng/ml), so that the aortic dissection sample and the myocardial damage sample tested by the kit provided by the invention are not crossed.

Strain preservation

The hybridoma cell strain 10B9E3-1 for producing the anti-Smooth Muscle Myosin Heavy Chain (SMMHC) monoclonal antibody is preserved in China center for type culture Collection (CCTCC, china, wuhan) at 27/4.2021, and the preservation number of the hybridoma cell strain 10B9E3-1 is CCTCC NO: C2021117.

The hybridoma cell strain 2D5C12-1 for producing the anti-Smooth Muscle Myosin Heavy Chain (SMMHC) monoclonal antibody is preserved in China center for type culture Collection (CCTCC, china, wuhan) at 27.4.2021, and the preservation number of the hybridoma cell strain 2D5C12-1 is CCTCC NO: C2021119.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and 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 present invention as defined by the appended claims.

Sequence listing

<110> Shanghai perspective diagnosis science and technology Limited

SHANGHAI TELLGEN LIFE SCIENCE Co.,Ltd.

<120> hybridoma cell strains 10B9E3-1 and 2D5C12-1 and application of antibody secretion thereof

<130> P2021-1160

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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Met Ala Gln Lys Gly Gln Leu Ser Asp Asp Glu Lys Phe Leu Phe Val

1 5 10 15

Asp Lys Asn Phe Ile Asn Ser Pro Val Ala Gln Ala Asp Trp Ala Ala

20 25 30

Lys Arg Leu Val Trp Val Pro Ser Glu Lys Gln Gly Phe Glu Ala Ala

35 40 45

Ser Ile Lys Glu Glu Lys Gly Asp Glu Val Val Val Glu Leu Val Glu

50 55 60

Asn Gly Lys Lys Val Thr Val Gly Lys Asp Asp Ile Gln Lys Met Asn

65 70 75 80

Pro Pro Lys Phe Ser Lys Val Glu Asp Met Ala Glu Leu Thr Cys Leu

85 90 95

Asn Glu Ala Ser Val Leu His Asn Leu Arg Glu Arg Tyr Phe Ser Gly

100 105 110

Leu Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Val Val Asn Pro Tyr

115 120 125

Lys His Leu Pro Ile Tyr Ser Glu Lys Ile Val Asp Met Tyr Lys Gly

130 135 140

Lys Lys Arg His Glu Met Pro Pro His Ile Tyr Ala Ile Ala Asp Thr

145 150 155 160

Ala Tyr Arg Ser Met Leu Gln Asp Arg Glu Asp Gln Ser Ile Leu Cys

165 170 175

Thr Gly Glu Ser Gly Ala Gly Lys Thr Glu Asn Thr Lys Lys Val Ile

180 185 190

Gln Tyr Leu Ala Val Val Ala Ser Ser His Lys Gly Lys Lys Asp Thr

195 200 205

Ser Ile Thr Gly Glu Leu Glu Lys Gln Leu Leu Gln Ala Asn Pro Ile

210 215 220

Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Lys Asn Asp Asn Ser Ser

225 230 235 240

Arg Phe Gly Lys Phe Ile Arg Ile Asn Phe Asp Val Thr Gly Tyr Ile

245 250 255

Val Gly Ala Asn Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg Ala Ile

260 265 270

Arg Gln Ala Arg Asp Glu Arg Thr Phe His Ile Phe Tyr Tyr Met Ile

275 280 285

Ala Gly Ala Lys Glu Lys Met Arg Ser Asp Leu Leu Leu Glu Gly Phe

290 295 300

Asn Asn Tyr Thr Phe Leu Ser Asn Gly Phe Val Pro Ile Pro Ala Ala

305 310 315 320

Gln Asp Asp Glu Met Phe Gln Glu Thr Val Glu Ala Met Ala Ile Met

325 330 335

Gly Phe Ser Glu Glu Glu Gln Leu Ser Ile Leu Lys Val Val Ser Ser

340 345 350

Val Leu Gln Leu Gly Asn Ile Val Phe Lys Lys Glu Arg Asn Thr Asp

355 360 365

Gln Ala Ser Met Pro Asp Asn Thr Ala Ala Gln Lys Val Cys His Leu

370 375 380

Met Gly Ile Asn Val Thr Asp Phe Thr Arg Ser Ile Leu Thr Pro Arg

385 390 395 400

Ile Lys Val Gly Arg Asp Val Val Gln Lys Ala Gln Thr Lys Glu Gln

405 410 415

Ala Asp Phe Ala Val Glu Ala Leu Ala Lys Ala Thr Tyr Glu Arg Leu

420 425 430

Phe Arg Trp Ile Leu Thr Arg Val Asn Lys Ala Leu Asp Lys Thr His

435 440 445

Arg Gln Gly Ala Ser Phe Leu Gly Ile Leu Asp Ile Ala Gly Phe Glu

450 455 460

Ile Phe Glu Val Asn Ser Phe Glu Gln Leu Cys Ile Asn Tyr Thr Asn

465 470 475 480

Glu Lys Leu Gln Gln Leu Phe Asn His Thr Met Phe Ile Leu Glu Gln

485 490 495

Glu Glu Tyr Gln Arg Glu Gly Ile Glu Trp Asn Phe Ile Asp Phe Gly

500 505 510

Leu Asp Leu Gln Pro Cys Ile Glu Leu Ile Glu Arg Pro Asn Asn Pro

515 520 525

Pro Gly Val Leu Ala Leu Leu Asp Glu Glu Cys Trp Phe Pro Lys Ala

530 535 540

Thr Asp Lys Ser Phe Val Glu Lys Leu Cys Thr Glu Gln Gly Ser His

545 550 555 560

Pro Lys Phe Gln Lys Pro Lys Gln Leu Lys Asp Lys Thr Glu Phe Ser

565 570 575

Ile Ile His Tyr Ala Gly Lys Val Asp Tyr Asn Ala Ser Ala Trp Leu

580 585 590

Thr Lys Asn Met Asp Pro Leu Asn Asp Asn Val Thr Ser Leu Leu Asn

595 600 605

Ala Ser Ser Asp Lys Phe Val Ala Asp Leu Trp Lys Asp Val Asp Arg

610 615 620

Ile Val Gly Leu Asp Gln Met Ala Lys Met Thr Glu Ser Ser Leu Pro

625 630 635 640

Ser Ala Ser Lys Thr Lys Lys Gly Met Phe Arg Thr Val Gly Gln Leu

645 650 655

Tyr Lys Glu Gln Leu Gly Lys Leu Met Thr Thr Leu Arg Asn Thr Thr

660 665 670

Pro Asn Phe Val Arg Cys Ile Ile Pro Asn His Glu Lys Arg Ser Gly

675 680 685

Lys Leu Asp Ala Phe Leu Val Leu Glu Gln Leu Arg Cys Asn Gly Val

690 695 700

Leu Glu Gly Ile Arg Ile Cys Arg Gln Gly Phe Pro Asn Arg Ile Val

705 710 715 720

Phe Gln Glu Phe Arg Gln Arg Tyr Glu Ile Leu Ala Ala Asn Ala Ile

725 730 735

Pro Lys Gly Phe Met Asp Gly Lys Gln Ala Cys Ile Leu Met Ile Lys

740 745 750

Ala Leu Glu Leu Asp Pro Asn Leu Tyr Arg Ile Gly Gln Ser Lys Ile

755 760 765

Phe Phe Arg Thr Gly Val Leu Ala His Leu Glu Glu Glu Arg Asp Leu

770 775 780

Lys Ile Thr Asp Val Ile His His His His His His His His

785 790 795

Claims (10)

1. An anti-Smooth Muscle Myosin Heavy Chain (SMMHC) monoclonal antibody, which is characterized in that the antibody can be specifically combined with SMMHC protein, and is produced by a hybridoma cell strain 10B9E3-1 with the preservation number of CCTCC NO: C2021117; or the antibody is generated by a hybridoma cell strain 2D5C12-1 with the preservation number of CCTCC NO: C2021119.

2. The antibody of claim 1, wherein the EC50 of the antibody secreted by the hybridoma cell line 10B9E3-1 (CCTCC NO: C2021117) and SMMHC protein is 0.50-2.00ng/mL, preferably 0.60-1.85ng/mL; and/or

The EC50 of the antibody secreted by the hybridoma cell strain 2D5C12-1 (the preservation number is CCTCC NO: C2021119) and SMMHC protein is 0.55-2.10ng/mL, preferably 0.70-1.80ng/mL.

3. A hybridoma cell strain is characterized in that the preservation number is CCTCC NO of C2021117; or the preservation number is CCTCC NO of C2021119; the hybridoma cell line is capable of producing the anti-SMMHC monoclonal antibody of claim 1.

4. A recombinant protein, said recombinant protein having:

(i) A monoclonal antibody of claim 1;

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

5. An assay system for the detection of SMMHC proteins, wherein the assay 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

(iii) A second binding protein B that specifically binds to an SMMHC protein and is coupled to or detectably labeled;

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

6. The test system of claim 5, wherein when the test system comprises an SMMHC protein to be tested, a complex according to formula I is formed in the test system:

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 the monoclonal antibody of claim 1 or the recombinant protein of claim 4, coated on a solid support surface;

b is a second binding protein that specifically binds to an SMMHC protein to be detected and is coupled to or carries a detectable label; wherein the binding between the B and SMMHC proteins is non-competitive with the binding between the A and SMMHC proteins;

c is SMMHC 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. Use of the monoclonal antibody according to claim 1, the hybridoma cell line according to claim 3 or the recombinant protein according to claim 4 for the preparation of a reagent or a kit for the detection of SMMHC proteins.

9. A method for detecting the presence of an SMMHC protein in a sample, comprising:

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

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

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

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

(c) Comparing the expression level of SMMHC determined in said (b) with a control reference value; wherein the control is a control of the plurality of control cells,

if the expression level of SMMHC 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.

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