CN115651914A - Hybridoma cell, monoclonal antibody and P4HB protein detection reagent - Google Patents
Hybridoma cell, monoclonal antibody and P4HB protein detection reagent Download PDFInfo
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- CN115651914A CN115651914A CN202210999676.0A CN202210999676A CN115651914A CN 115651914 A CN115651914 A CN 115651914A CN 202210999676 A CN202210999676 A CN 202210999676A CN 115651914 A CN115651914 A CN 115651914A
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Abstract
The invention discloses a hybridoma cell, wherein the preservation number of the hybridoma cell is CCTCCNO: C2022108. The hybridoma cell can secrete the monoclonal antibody specifically bound with the P4HB protein, and the monoclonal antibody has high titer, high purity, high affinity with the P4HB protein and good specificity, can effectively perform qualitative and/or quantitative detection on the P4HB protein, further realizes the diagnosis or prognosis of cancer cachexia by performing qualitative and/or quantitative detection on the P4HB protein, simplifies the diagnosis and detection process of the cancer cachexia, and reduces the evaluation cost of the cancer cachexia.
Description
Technical Field
The application relates to the technical field of immunodetection, in particular to a hybridoma cell, a monoclonal antibody and a P4HB protein detection reagent.
Background
Cachexia (cachexia), also known as cancer cachexia or cancer anorexia cachexia, is a wasting syndrome. It is a condition of loss of muscle, fat content due to chronic diseases such as cancer, and not taking sufficient nutrition (malnutrition). Cancer-related cachexia usually does not occur early in the development of cancer, but generally occurs late in the cancer and when metastases develop in the tumor. Cancer cachexia affects approximately 50% -80% of cancer patients, and approximately 20% of cancer patients die of cachexia. Cachexia reduces the patient's weight bearing capacity and therapeutic effect on chemotherapy, radiation therapy and surgery, and also directly affects the patient's quality of life and prognosis.
Clinically, the weight is reduced by 5 percent or the body mass index BMI is less than 20kg/m 2 Or the weight of the patient with the skeletal muscle mass reduction is reduced by 2 percent as the diagnosis standard of the cachexia, so the existing diagnosis and monitoring process of the cachexia is complex and the evaluation cost is high.
Studies have shown that P4HB mediates skeletal muscle atrophy leading to cachexia, and that inhibition of P4HB can block cachexia from worsening and increase survival in cancer cachexia mice. Epigenomic association analysis has reported that P4HB gene is strongly related to human Body Mass Index (BMI) (1.7X 10) -16 ). Relevant functional studies revealed that the P4HB protein can accelerate glycosylation end product-induced smooth muscle cell apoptosis. Thus, P4HB may be used as a diagnostic or prognostic marker for cancer cachexia.
However, there is currently a lack of effective P4HB protein detection reagents for the diagnosis or prognosis of cancer cachexia.
Disclosure of Invention
In order to solve the above problems and provide an effective P4HB protein detection reagent, a first object of the present application is to provide a hybridoma cell having a preservation number of CCTCC NO: C2022108 which is capable of secreting a monoclonal antibody specifically binding to P4HB protein for detection of P4HB protein and further for diagnosis or prognosis of cancer cachexia.
A second object of the present application is to provide a monoclonal antibody secreted by the above hybridoma cell.
In one embodiment, the monoclonal antibody is an IgG antibody.
The third purpose of the application is to provide an application of the monoclonal antibody in preparing a P4HB protein detection reagent.
In one embodiment, the P4HB protein detection reagent is used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay, immunofluorescence, immunocolloidal gold, immunochemiluminometry, immunoturbidimetry, immunoblotting and dot blotting.
The fourth purpose of the application is to provide a P4HB protein detection reagent, which comprises a monoclonal antibody secreted by a hybridoma cell with the preservation number of CCTCC NO: C2022108.
In one embodiment, the monoclonal antibody is used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunocolloidal gold, immunochemical luminescence, immunoturbidimetry, immunoblotting and dot blotting.
In one embodiment, the kit further comprises a monoclonal antibody to perform at least one of the detection methods described above.
In one embodiment, the detection reagent further comprises a labeled antibody for detecting the monoclonal antibody.
In one embodiment, the labeled antibody is an anti-IgG antibody.
In one embodiment, the labeled antibody is linked to a label selected from at least one of a chromophore, a digoxigenin-labeled probe, an electron dense substance, colloidal gold, and an enzyme that produces a detectable signal.
In one embodiment, the label comprises horseradish peroxidase.
A fifth object of the present application is to provide a method for detecting a P4HB protein for non-disease diagnosis purposes, comprising: the use of the reagent of claim for the detection of P4HB protein.
The sixth purpose of the present application is to provide the use of the reagent in the preparation of a kit for detecting cachexia.
The seventh purpose of the present application is to provide a kit for detecting cachexia, which comprises the above reagent.
In one embodiment, the kit further comprises at least one of an antibody positive control reagent, a negative control reagent, a buffer for dilution, a deparaffinization reagent, a hydration reagent, an antigen retrieval reagent, a blocking reagent, a color reagent, a counterstain reagent, and a mounting reagent.
An eighth object of the present invention is to provide a method for preparing the hybridoma, comprising: taking human P4HB protein as immunogen to immunize BALB/c mice, taking immune mouse spleen cells to mix with SP2/0 myeloma cells and mouse feeder cells for cell fusion, and then screening out positive fusion cells capable of secreting P4HB antigen monoclonal antibodies, namely hybridoma cells.
The hybridoma cell can secrete the monoclonal antibody specifically bound with the P4HB protein, the monoclonal antibody is high in titer and purity, high in affinity and good in specificity with the P4HB protein, qualitative and/or quantitative detection can be performed on the P4HB protein specifically, diagnosis and detection processes of cancer cachexia are simplified after diagnosis or prognosis of the cancer cachexia is realized through qualitative and/or quantitative detection on the P4HB protein, and evaluation cost of the cancer cachexia is reduced.
Drawings
In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram showing the results of an experiment for screening anti-human P4HB protein-specific hybridoma cells by ELISA in example 1 of the present application;
FIG. 2 is a schematic diagram showing the results of the experiment for detecting the titer of monoclonal antibody 3B1D9 by ELISA in example 1 of the present application;
FIG. 3 is a graph showing the result of subtype identification of the monoclonal antibody 3B1D9 in example 3 of the present application;
FIG. 4 is a graph showing the purity test results of the monoclonal antibody 3B1D9 in example 3 of the present application;
FIG. 5 is a diagram showing the results of specific detection of the monoclonal antibody 3B1D9 in example 3 of the present application;
FIG. 6 is a sensor graph showing the binding between a monoclonal antibody and a human P4HB protein in example 3 of the present application;
FIG. 7 is a graph of a Steady State Affinity fit between a monoclonal antibody and human P4HB proteins in example 3 of the present application;
FIG. 8 is a graph showing the results of the test kit of example 4 of the present application for detecting P4HB protein in tumor tissue of 5 patients with esophageal cancer among patients with cachexia;
FIG. 9 is a graph showing the results of the test kit of example 4 of the present application for detecting P4HB protein in tumor tissues of 3 pancreatic cancer patients among cachexia patients;
FIG. 10 is a graph showing the results of the test kit of example 4 of the present application for detecting P4HB protein in tumor tissues of 1 gastric cancer patient out of cachexia patients;
FIG. 11 is a graph showing the result of detecting P4HB protein in a healthy human pancreatic cancer tissue by the kit of example 4 of the present application;
FIG. 12 is a graph showing the results of the test kit of example 4 of the present application for detecting P4HB protein in healthy human colon cancer tissue;
FIG. 13 is a schematic diagram showing the differential analysis of the expression profiles of P4HB proteins in a cachectic patient and a healthy human in example 4 of the present application.
Detailed Description
Reference will now be made in detail to embodiments of the present application, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present application. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the scope or spirit of the application. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present application cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present application.
As described above, the existing cachexia diagnosis and monitoring process is complicated, the evaluation cost is high, and an effective marker detection reagent is not available for the diagnosis and monitoring of cachexia.
In order to at least partially solve at least one of the above technical problems, a first aspect of the present application provides a hybridoma cell, deposited at the chinese type culture collection (CCTCC), also known as the university of wuhan collection, at 2022, day 5 and 18 and identified as viable by the collection at 2022, day 5 and 22, address: eight paths 299 in Wuhan city, hubei province, the preservation number of the hybridoma cell is CCTCC NO: C2022108, and the classification and the naming are as follows: hybridoma cell line 3B1D9.
Specifically, the hybridoma is a B-cell hybridoma obtained by fusing a sensitized B-cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability, based on a cell fusion technique. The hybridoma cell with the preservation number of CCTCC NO: C2022108 can secrete a novel monoclonal antibody which is specifically combined with P4HB and is marked as 3B1D9.
Accordingly, a second aspect of the present application provides a method for preparing the above hybridoma cell, comprising: taking human P4HB protein as immunogen to immunize BALB/c mice, taking splenocytes of the immunized mice and SP2/0 myeloma cells to carry out cell fusion in the presence of mouse feeder cells, and then screening out positive fusion cells which can secrete P4HB antigen monoclonal antibodies, namely hybridoma cells.
P4HB is located on chromosome 17, the protein of which contains 508 amino acids, and the Genbank accession number of the P4HB Gene on NCBI is Gene ID:5034, and the function of which is mainly to catalyze the formation, isomerization and reduction of disulfide bonds of a substrate protein.
An "antibody", also known as an immunoglobulin, is a large Y-shaped protein secreted by B lymphocytes, an immunoglobulin molecule capable of specifically binding a target antigen, such as a protein, sugar, polynucleotide, lipid, polypeptide, small molecule compound, or the like.
By culturing a population of adult cells with a single hybridoma cell, a highly homogeneous, specific antibody directed against only one epitope, i.e., a monoclonal antibody, produced by a single B cell clone can be prepared. In the preparation method of the hybridoma, the target antigen is P4HB, and the prepared monoclonal antibody is a specific antibody only aiming at one P4HB epitope. Therefore, the third aspect of the application provides a monoclonal antibody, wherein the monoclonal antibody is secreted by the hybridoma with the preservation number of CCTCC NO: C2022108 and is marked as 3B1D9.
The monoclonal antibody was identified as an IgG antibody. The application discovers for the first time that the monoclonal antibody specifically recognizing the P4HB protein can realize the diagnosis or prognosis of cancer cachexia, simplify the diagnosis and detection process of the cancer cachexia and reduce the evaluation cost of the cancer cachexia.
The monoclonal antibody 3B1D9 secreted by the hybridoma with the preservation number of CCTCC NO: C2022108 has high titer, high purity and high affinity with P4HB protein, can effectively perform qualitative and/or quantitative detection on the P4HB protein, can be applied to the preparation of products for detecting the P4HB protein, and can further realize the diagnosis or prognosis of cancer cachexia by performing qualitative and/or quantitative detection on the P4HB protein, simplify the diagnosis and detection process of the cancer cachexia and reduce the evaluation cost of the cancer cachexia.
Accordingly, the fourth aspect of the present application provides a use of a monoclonal antibody in the preparation of a P4HB protein detection reagent.
It can be understood that the present application provides a novel monoclonal antibody 3B1D9, which has high potency, high purity, high affinity with P4HB protein, good specificity, and binds to only one P4HB epitope, thus effectively realizing P4HB protein detection.
In some embodiments, the above P4HB protein detection reagents are used to perform at least one of the following detection methods to achieve detection of the expression level of P4HB protein, the detection methods comprising: enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunocolloidal gold, immunochemiluminescence, immunoturbidimetry, immunoblotting and dot blotting.
Among them, enzyme-linked immunoassay is the most widely used immunoassay method at present. The method combines the secondary antibody with enzyme, the specificity of antigen-antibody reaction and the action of enzyme catalysis substrate, and judges the test result according to the color development change after the enzyme acts on the substrate, and the sensitivity can reach ng level. Common enzymes for labeling are horseradish peroxidase (HRP), alkaline Phosphatase (AP), and the like. The enzyme linked immunosorbent assay method does not need special instruments, and is simple in detection, so that the enzyme linked immunosorbent assay method is widely applied to disease detection.
Commonly used enzyme-linked immunoassays are the indirect method, the sandwich method and the BAS-ELISA. Specifically, the indirect method is to coat the protein to be detected in a pore plate, then add primary antibody, enzyme-labeled secondary antibody and substrate in sequence for color development, and quantitatively detect the antigen through an instrument (such as an enzyme-labeling instrument). The sandwich method utilizes two primary antibodies to capture and fix a target antigen, thereby greatly improving the specificity of the reaction while ensuring the sensitivity.
The immunofluorescence method is a method for detecting the antigen in the specimen by using fluorescein, such as fluorescein isothiocyanate, rhodamine and the like to mark the antibody. Immunofluorescence techniques also encompass two basic types, namely fluorescent antibody staining and fluorescent immunoassays.
The fluorescent antibody staining is to dip and stain cells or tissue sections possibly containing antigens by using the fluorescent antibody, if corresponding antigens exist, the antigens are combined with the fluorescent antibody to prevent fluorescein from being eluted, and objects which can emit light can be seen under a fluorescent microscope, so that the aim of positioning detection is fulfilled. The direct method and indirect method can be divided according to the difference of fluorescent antibody, the former uses the first antibody which is marked by fluorescence to directly detect the antigen on the sample slice, such as virus and some protein components; the latter is followed by treatment of the sample piece with an unlabeled corresponding antibody (primary antibody) and then with a fluorescently labeled anti-globulin antibody (secondary antibody), whereby various antigens and antibodies can be detected. Compared with the direct method, the indirect method can adapt to the detection of a plurality of antigen-antibody systems only by labeling one second antibody, and has higher sensitivity.
Fluorescence immunoassays, like enzyme immunoassays, can be classified into homogeneous and heterogeneous methods. Homogeneous methods often use certain properties of fluorescence, such as excitation, absorption, quenching, etc., to design assays without the need for bound and free label separation. The double labeling method is one type of homogeneous phase fluorescence immunoassay, the detection reagent is FITC labeled antigen and rhodamine labeled antibody, when the two labeled antigen and antibody are specifically combined, the two fluorescein approaches, and the fluorescence of FITC is obviously weakened because the emission spectrum of FITC can be absorbed by rhodamine. During the test, the sample possibly containing the antigen reacts with the two markers together, and the sample can compete with the FITC-labeled antigen to be combined with the rhodamine-labeled antibody, so that the absorption of the rhodamine on the FITC emission spectrum is reduced. The amount of antigen in the sample can be deduced by FITC fluorescence measurement, which is proportional to the fluorescence intensity.
Heterogeneous methods are limited to laboratory conditions, nonspecific fluorescence interference of reagents and containers or carriers, etc., and are not as widely used as ELISA. The time-resolved fluoroimmunoassay utilizes the fact that the chelate of rare earth metals (europium, terbium, etc.) has a very long fluorescence lifetime, labels the antibody and prolongs the measurement time so as to fade short-lived non-specific fluorescence, thereby measuring the fluorescence of the uniform rare earth chelate with a long lifetime. The difference between the excitation light absorption peak (340 nm) and the fluorescence emission peak (613 nm) of the rare earth chelate is obvious, and the interference of non-specific fluorescence can be eliminated. Can be used for measuring the level of trace serum components such as IgE and the like, hormones and certain medicines.
Immunohistochemistry is also called immunocytochemistry, and refers to a technology for qualitatively, positionally and quantitatively determining corresponding antigens by antigen-antibody reaction and histochemical color reaction of specific antibodies with color developing agent marks in situ in tissue cells. Immunohistochemistry skillfully combines the specificity of immune reaction and the visibility of histochemistry, and detects various antigen substances such as proteins, polypeptides, enzymes, hormones, pathogens, receptors and the like at cellular and subcellular levels by means of the development and amplification of microscopes such as fluorescence microscopes and electron microscopes.
The immunochemiluminescence method is that chemical luminescent agent is used to mark antibody directly, chemical luminescent agent marker is used to react with corresponding antigen and magnetic granular anti-antibody in the sample to be detected, the combined state (precipitation part) and free state chemical luminescent agent marker are separated by magnetic field, then luminescence reaction is carried out by adding luminescence promoter, and quantitative or qualitative detection is carried out by detecting luminescence intensity.
Immunonephelometry (immunonephelometry) is a method in which a sample of a certain volume is added at a certain antibody concentration, and the turbidity of the reaction liquid is measured by a light scattering turbidimeter (nephelometry) after a certain period of time to calculate the antigen content in the sample. When the concentration of the antibody is high, a small amount of soluble antigen is added, so that small immune complexes which cannot be seen by naked eyes can be formed, light beams passing through liquid can be scattered, the formed immune complexes are increased along with the increase of the added antigen, and the light scattering phenomenon is correspondingly enhanced. The immunoturbidimetry is sensitive, rapid, simple and convenient, and can be used for measuring the concentration of protein.
The immunoblotting method is also called Western blotting method, and combines gel electrophoresis with solid phase immunization, and firstly transfers the protein to be distinguished to the solid phase carrier such as NC membrane by means of protein electrophoresis technology, and then utilizes the techniques of enzyme immunization and radioimmunoassay to make determination. The method can separate proteins with different molecular sizes and determine the molecular mass of the proteins.
Dot blotting is a method of blotting a dot sample onto a specific membrane for detection. For example, in the dot immuno-blotting assay (DIBA), a nitrocellulose membrane (NC membrane) or a cellulose acetate membrane is used as a solid support, colonies or plaques on a solid culture medium are blotted, bacteria are lysed in situ on the membrane or the phage are lysed, and then the membrane is hybridized with a specific labeled probe, so that the required clone is directly screened from a transformed DNA library or a phage library; the proteins with different dilutions are adsorbed on the membrane for color reaction, so that semi-quantification of trace proteins can be performed. The method has the advantages of low consumption, high speed, low cost, and convenience.
Therefore, the fifth aspect of the application provides a P4HB protein detection reagent, which comprises a monoclonal antibody secreted by a hybridoma cell with the preservation number of CCTCC NO: C2022108, so as to qualitatively and/or quantitatively detect the P4HB protein.
Specifically, the P4HB protein can be detected using any of the detection methods described above using a monoclonal antibody, and therefore, the kit further comprises other detection reagents other than the monoclonal antibody for performing at least one of the detection methods described above. Specifically, the detection reagent further comprises a labeled antibody for detecting the monoclonal antibody.
In some embodiments, the labeled antibody is linked to a label that is labeled on the antibody that binds to monoclonal antibody 3B1D9, reflecting the amount of monoclonal antibody that binds to the P4HB antigen by detecting the signal from the label or the signal generated upon reaction with the substrate, and thus reflecting the amount of P4HB antigen. The label is selected from at least one of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, and an enzyme that generates a detectable signal, depending on the detection method. In some embodiments, the label comprises horseradish peroxidase to allow detection of P4HB antigen by DAB chromogenic reflection. Of course, in other embodiments, the label is not limited to the above, but may be other labels that can be used for detection.
Correspondingly, according to the kit, the application also provides a P4HB protein detection method, which comprises the step of detecting the P4HB protein by using the P4HB protein detection reagent so as to realize the qualitative and/or quantitative detection of the P4HB protein.
The P4HB is a potential key target point in the process of inducing the tumor cachexia, the P4HB protein detection reagent is used for detecting the P4HB protein of the cachexia tumor tissue, the monoclonal antibody can be specifically combined with the P4HB in the tumor tissue of the cachexia patient, and the false positive condition is less existed. The application proves that the differential expression of the P4HB protein in a cachexia patient in a tumor tissue and a normal tissue is proved by utilizing the monoclonal antibody through the specific binding of the monoclonal antibody to the P4HB protein for the first time, and the diagnosis of the cachexia by taking the P4HB as a marker is realized. In some embodiments, the cachexia detection kit described above is used to perform immunohistochemistry to detect the expression level of P4HB protein in cancer tissues, thereby verifying differential expression of P4HB protein in tumor tissues and normal tissues in cachectic patients.
In some embodiments, the kit further comprises at least one of an antibody positive control reagent, a negative control reagent, a buffer for dilution, a dewaxing reagent, a hydration reagent, an antigen retrieval reagent, a blocking reagent, a chromogenic reagent, a counterstain reagent, and a mounting reagent. The positive standard reagent can be used as a positive control in qualitative detection and can also be used for preparing a standard curve in quantitative analysis. Optionally, the positive standard reagent is human P4HB protein, and is diluted by using a buffer solution gradient for use. The positive standard reagent can be used for qualitative or quantitative analysis of the P4HB protein through the color reaction of an indirect method. The dilution buffer is used for diluting each component, and specifically, it may be a Tris buffer solution having a pH of 7.2, and further, the concentration of the Tris buffer solution is 0.05M. The negative control reagent includes water.
In some embodiments, the dewaxing agent includes xylene for at least one dewaxing treatment of the tissue section to be tested. Further, the hydration reagent comprises at least one of xylene, gradient ethanol, PBS buffer and water, and is used for enabling the paraffin section sample to enter the water. Further, the antigen retrieval reagent comprises sodium citrate buffer solution, and is used for performing antigen retrieval on the paraffin section sample. Further, the blocking reagent comprises at least one of hydrogen peroxide, PBS buffer solution and BSA, and is used for blocking endogenous catalase and endogenous antigen to reduce non-specific staining. Further, the color reagent comprises DAB color solution for color reaction with the marker of the labeled antibody. Further, the counterstaining reagent includes a hematoxylin counterstaining reagent. Further, the mounting reagent includes a resin.
Further, the tissue to be tested can be semi-quantitatively analyzed on the basis of the result of qualitative analysis of P4HB protein in the cells of the cancer tissue, thereby achieving detection of cachexia (see the description of the method steps for detecting P4HB protein of the above-mentioned kit below for details).
Therefore, the present application also provides a method for detecting cachexia, comprising:
preparing a tissue sample to be detected, and performing antigen heat restoration on the tissue sample to be detected;
sequentially dripping a primary antibody and a secondary antibody on the tissue sample to be detected subjected to antigen heat repair, and respectively incubating and washing, wherein the primary antibody is the monoclonal antibody, and the secondary antibody is a labeled antibody;
and adding a color reagent into the tissue sample to be detected for color reaction, and analyzing the detection result of the tissue sample to be detected after color development.
Specifically, the staining is stopped after the color development, and the detection result of the tissue to be detected is analyzed by a microscope through counterstaining, dehydration, tissue transparency and mounting. Further, determining cells in a tumor tissue region which are positively stained due to the combination of the P4HB protein and the monoclonal antibody 3B1D9 according to a color development result of the tissue to be detected, analyzing the number of the positive cells in the tumor region of the tissue to be detected, further calculating a tumor proportion score according to the number of the positive cells and the total number of the cells in the tumor region of the tissue to be detected, and judging whether the tissue to be detected is a cancer cachexia tissue according to the combination of the tumor proportion score and the staining depth, wherein the cancer cachexia tissue is the tumor tissue of a cancer cachexia patient.
In some embodiments, the count can be observed through a microscope to calculate a tumor proportion score for use in differentiating cancer cachexia tissue from para-cancerous tissue or healthy human tissue.
Embodiments of the present application will be described in detail below with reference to examples, but the present application is not limited to these examples. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of anti-human P4HB hybridoma cell lines
The embodiment provides a hybridoma cell strain 3B1D9 capable of secreting a P4HB monoclonal antibody, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2022108.
1. Animal immunization
Human P4HB protein (purchased from Beijing Yiqiao Shen science, inc.) was emulsified with Freund's complete adjuvant and BALB/c mice (50 ug/mouse) were injected subcutaneously. Four weeks later, 3 consecutive boosts (25 ug/mouse) were made, with two weeks of each interval, and the human P4HB protein was emulsified with Freund's incomplete adjuvant. 3 days after the last booster immunization, fusion experiments of mouse splenocytes and mouse myeloma cells were performed.
2. Cell fusion
(1) Preparation of spleen cells
The immune mice are picked up by eyeballs and blood is taken, the mice are killed by cervical vertebra breakage and then are placed in 75% alcohol for soaking for 10 minutes, the spleens of the mice are taken out from a sterile operating platform, the spleens are squeezed by an inner core of a syringe, the splenocytes are placed in a DMEM medium, and the cells are resuspended to prepare single cell suspension.
(2) Preparation of feeder cells
Taking one female BALB/c mouse of 4-6 weeks old, picking eyeball to obtain negative serum, and soaking in 75% alcohol for 10 minutes after cervical vertebra is cut off; the abdominal skin was aseptically uncovered, the peritoneum was exposed, about 10mL of DMEM medium containing 20% fetal bovine serum was injected into the abdominal cavity of the mouse using a syringe, after several puffs, the medium containing macrophages was aspirated and injected into 20% DEME medium, which was then plated at 100. Mu.L/well in 96-well cell culture plates for use.
(3) Cell fusion
Selecting a mouse myeloma cell line SP2/0 in a logarithmic growth phase, adding about 108 spleen cells and 2 x 107 SP2/0 cell lines into a fusion tube, uniformly mixing, centrifuging at 1000rpm for 5 minutes, then removing supernatant, and placing the fusion tube on a palm to gently rub back and forth to loosen precipitates. Adding 1mL of preheated PEG within 60 seconds, adding 4mL of the culture medium in batches, adding 1mL of the culture medium for 1min, adding 2mL of the culture medium for 30s, adding 3mL of the culture medium for 30s, adding 4mL of the culture medium for 15s (the centrifugal tube is rotated by the left hand at a constant speed halfway), adding 45-50 mL of the DMEM culture medium, stopping, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 2 HAT culture medium, mixing completely, adding 100 mu L of the mixture into the 96-well cell culture plate, setting the temperature at 37 ℃ and 5 CO 2 Cultured in a cell culture box. After one week, the supernatant was collected and examined.
3. Screening of anti-human P4HB protein specific hybridoma cell strain by ELISA method
(1) Preparation of a detection plate: diluting the human P4HB protein (purchased from Beijing Yiqiao Shenzhou science, inc.) to 2ug/mL by using coating diluent, coating a 96-hole ELISA plate with 100 mu L/hole, coating overnight at 2-8 ℃, and discarding liquid in the hole; add 200. Mu.L of blocking solution to each well, block for 1h at 37 ℃, wash, and dry for use.
(2) Screening of positive clones: adding 100 μ L/well of cell culture supernatant to be tested into the above detection plate, and allowing to act at 37 deg.C
Washing and drying after 1h, adding 100 mu L/hole of HRP-labeled goat anti-mouse IgG, acting at 37 ℃ for 1h, washing and drying, adding 100 mu L/hole of developing solution, developing for 15min in a dark place at 37 ℃, adding 50 mu L of stop solution into each hole to stop the reaction, and reading the value with OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1.
(3) Selecting positive clone wells, performing cell cloning screening, diluting the positive clones on 96-well plate to 1 cell/well as shown in FIG. 1, 37 deg.C, 5% CO 2 Culturing in a cell culture box, then screening positive clones by an ELISA method again until the positive rate of the monoclonal cell strain is 100 percent, determining the monoclonal cell strain as a stable cell strain, and performing strain determination on the cell strain. From these, a monoclonal cell line 3B1D9 was selected.
4.ELISA method for detecting titer of monoclonal cell strain 3B1D9
(1) Preparation of a detection plate: diluting the human P4HB protein (purchased from Beijing Yiqiao Shenzhou science, inc.) to 1ug/mL by using coating diluent, coating a 96-hole ELISA plate with 100 mu L/hole, coating overnight at 2-8 ℃, and discarding liquid in the hole; add 200. Mu.L of blocking solution to each well, block for 1h at 37 ℃, wash, and dry for use.
(2) The supernatant of the monoclonal cell strain 3B1D9 was subjected to gradient dilution from A to G wells at once in a range of 2000-fold, 4000-fold, 8000-fold, 16000-fold, 32000-fold, 64000-fold, 128000-fold, 256000-fold, and 100. Mu.L/well in the above assay plate, and the dilution was repeated five times, and the dilution was applied at 37 ℃ for 1 hour, washed and patted dry, and 100. Mu.L/well of HRP-labeled goat anti-mouse IgG was added, and the dilution was applied at 37 ℃ for 1 hour, washed and patted dry, 100. Mu.L/well of a developing solution was added, and the reaction was stopped by adding 50. Mu.L of a stopping solution to each well, and the reaction was observed (see FIG. 2).
According to the detection results in FIG. 2, the antibody was diluted 256000 times, i.e., the antigen could be detected by G-well ELISA, indicating that the monoclonal cell line 3B1D has higher titer.
EXAMPLE 2 preparation and purification of monoclonal antibodies
(1) Preparation of monoclonal antibodies
Taking healthy BALB/c female mice, injecting 0.5mL of liquid paraffin into each abdominal cavity, and keeping for 1-2 weeks. Adjusting the positive clone hybridoma cells to 106/mL, injecting 1mL of positive clone hybridoma cells into the abdominal cavity of each pretreated mouse, collecting ascites after 7-9 days, centrifuging at 3000rpm for 10min, discarding a lipid layer and a cell layer, collecting an intermediate clarification layer, subpackaging, and storing at-20 ℃.
(2) Purification of monoclonal antibodies
Adding equal volume of barbital buffer saline into the ascites, adding appropriate amount of silicon dioxide powder, centrifuging at room temperature of 30min,4 deg.C, 2000g for 20min to obtain clarified ascites;
adding two portions of 0.06M acetic acid buffer solution of PH5.0 into the clarified ascites, adjusting the pH to 4.8, then dropwise and slowly adding octanoic acid within 20-30 min, standing for 2h at 4 ℃, centrifuging for 30min at 10000-15000 rpm, discarding the precipitate, filtering at 0.45 mu m, adding 1/10 volume of PBS, and adjusting the pH to 7.4. Ammonium sulfate of 0.2-0.5 g/mL is added within 30min at 4 ℃ in ice bath, and the mixture is kept stand for 1h. Centrifuged at 10000-15000 rpm for 30min, the supernatant was discarded and dissolved in PBS containing 137mM NaCl,2.6mM KCl and 0.2mM EDTA at pH7.4, dialyzed overnight and stored at-20 ℃.
EXAMPLE 3 identification of monoclonal antibodies
1. Antibody subclass and subtype identification
The subclass and subtype of the monoclonal antibody are identified by using a monoclonal antibody subtype identification ELISA kit of the same Biotechnology (Shanghai) Co., ltd, and simultaneously using a negative control and a positive control in the ELISA kit, and detecting OD values of the monoclonal antibody, the negative control and the positive control, wherein the detection results are specifically shown in FIG. 3 and Table 1. The subclass and subtype analysis results of the monoclonal antibody indicate that the monoclonal antibody is of the IgG subclass and comprises light chain KAPA.
TABLE 1 antibody subclass OD values
| OD value | Negative control | Positive control | 3B1D9 |
| IgG1 | 0.051 | 4.711 | 2.697 |
| IgG2a | 0.056 | 4.580 | 0.122 |
| IgG2b | 0.055 | 4.885 | 0.066 |
| IgG3 | 0.044 | 4.642 | 0.045 |
| IgM | 0.048 | 4.586 | 0.046 |
| IgA | 0.044 | 4.572 | 0.044 |
| kappa | 0.050 | 4.656 | 3.447 |
| Lambda | 0.044 | 4.495 | 0.044 |
2. Antibody purity identification
Polyacrylamide gel electrophoresis: preparing 10% separation gel and 5% concentrated gel, loading standard protein and antibody, respectively, and performing electrophoresis at constant pressure for 1 hr.
According to the detection results in fig. 4, only the light chain band and the heavy chain band of the antibody exist in the bands, no other obvious hybrid band exists, and the purity of the antibody in the application is more than 95%.
3. Identification of the specificity of antibodies
(1) Polyacrylamide gel electrophoresis: preparing 10% separation gel and 5% concentrated gel, repeatedly loading two human P4HB proteins, and performing constant voltage electrophoresis for 1h.
(2) Film transfer: the membrane was rotated at constant pressure for 1h, and the proteins on the polyacrylamide gel were transferred to a nitrocellulose membrane.
(3) Soaking the nitrocellulose membrane in TBST, washing for 5min, sealing with TBST containing 3% skimmed milk for 1h, and washing in TBST buffer on a shaker for 5min.
(4) A first antibody: the 3B1D9 antibody was mixed well with 3% BSA in TBST buffer 1 at 10000 ratio, incubated overnight at 4 ℃ with nitrocellulose membrane, and washed 5 times for 5min each.
(5) Secondary antibody: the HRP-labeled goat anti-mouse IgG was mixed with TBST buffer 1 containing 3% skim milk at a ratio of 10000, the nitrocellulose membrane was incubated at room temperature for 1h, and TBST was washed 5 times for 5min each time.
(6) Color development: sucking up residual liquid on the nitrocellulose membrane, adding 2mLTCL color development liquid, uniformly wetting the surface of the nitrocellulose membrane, reacting at room temperature in a dark place for 1min, and then photographing in a gel imaging system (as shown in figure 5).
The detection result shows that the antibody has better specificity to the two repeatedly loaded P4HB proteins, and can specifically detect the human P4HB protein.
Detection of affinity of antibody and human P4HB protein by SPR method
The 3B1D9 antibody is coupled and fixed on a CM5 chip through amino groups, the fixing level reaches 18000RU, human P4HB protein is used as an analyte, an experiment is carried out, and the affinity between the antibody and the protein is detected. FIG. 6 is a sensorgram showing the binding between the 3B1D9 antibody and human P4HB proteins, and FIG. 7 is a graph showing the Steady State Affinity fit between the 3B1D9 antibody and human P4HB proteins.
From the experimental results shown in FIGS. 6 and 7, the affinity constant of the 3B1D9 antibody to the human P4HB protein was 2.049E-7M.
Example 4 application of cancer cachexia detection
This example utilizes immunohistochemistry to detect cancer cachexia and the P4HB content of healthy human specimens.
1. Kit Components
The immunohistochemical detection kit comprises a P4HB antibody diluent specifically binding with a target protein, wherein the P4HB antibody diluent is diluted by using a 0.05M Tris buffer solution, and the specific components comprise: 5ug/ml monoclonal antibody 3B1D9, 0.01M EDTA, 0.05% PC300, 0.03% Evans blue, 0.05% Triton X-100 and 0.3% BSA. Wherein, PC300 is preservative, evans blue is nuclear staining dye, triton X-100 is detergent, which is used for cell perforation, is beneficial to the combination of monoclonal antibody and target protein P4HB, BSA is bovine serum albumin, which is used for preventing nonspecific staining.
2. Detection method
(1) After dewaxing at 60 ℃ for 20min conventionally, the paraffin sections were immersed in xylene I and xylene II for 10min each. Then gradually reducing the alcohol concentration into water, and washing for 3-5 min each time.
(1) Washing for 1 time and 3-5 min without water;
(2) washing with 90% alcohol for 1 time and 3-5 min;
(3) washing with 85% alcohol for 1 time, 3-5 min;
(4) washing with 75% alcohol for 1 time and 3-5 min;
(5) washing with PBS for 5min for 3 times;
(2) A heat-repairing antigen: placing the slices in 0.01mol/L pH6.0 sodium citrate buffer solution, heating to boil in microwave oven, and powering offRepeating for 1-2 times at intervals of 5-10min, and naturally cooling at room temperature. 0.01mol/L PBS pH7.4 washing 3 times, each time for 5min, and then with 3% H 2 O 2 Incubating for 5-10min at room temperature to eliminate the activity of endogenous peroxidase, and washing for 5min for 2-3 times with PBS.
(3) Dropping primary antibody: the primary antibody was added dropwise to the cut pieces, incubated at 37 ℃ for about 1 hour, and washed 3 times with PBS (pH7.4) for 5min each.
(4) Secondary antibody was added, incubated at 37 ℃ for 0.5 hour, removed and washed 3 times with PBS for 5min each.
(5) DAB color development: taking 1ml of DAB color development liquid I, then adding 50ul of color development liquid II to prepare DAB working solution, dropwise adding the DAB working solution on the slices, developing at room temperature, controlling the reaction time under a mirror, and reacting for about 5-10 min. The distilled water was sufficiently washed to terminate the reaction.
(6) Observing color development, washing with tap water, counterstaining with hematoxylin for 1-2min, washing with tap water for 10min, dehydrating with gradient alcohol, clearing with xylene, sealing with neutral gum, drying, analyzing, and taking picture.
3. Interpretation of the results
Under the optical microscope, the observed blue area is cell nucleus, and the brown yellow area is positive coloration. Positive staining tissue samples were evaluated for P4HB expression by staining intensity, and staining site. The color density is a ratio of a region area of the colored portion to a region area of the entire visual field. In this example, the stained site should be stained by the cell membrane or cytoplasm, and the evaluation criteria are shown in Table 2.
TABLE 2
| Evaluation of | Description of evaluation of coloring depth | Description of evaluation of coloring Density |
| - | No or slight coloration | The positive cells are less than 14% |
| + | Slight coloration | The positive cells account for 15 to 39 percent |
| ++ | Moderate coloration | The positive cells account for 40 to 69 percent |
| +++ | Moderate to severe coloration | Over 70% of positive cells |
4. Comparison of expression levels of P4HB in cachectic patient and Normal patient tissues
In this example, by purchasing a tissue chip of a core super-organism, according to the follow-up information of patients provided by the company, tumor tissue samples (including 5 esophageal cancers, 3 pancreatic cancers and 1 gastric cancer) of 9 cancer cachexia patients and tissue samples (3 pancreatic cancers and 2 colon tissues) of 5 healthy people are found, and these tissue samples are detected by using the kit. Among them, the diagnosis of cachexia patients is agreed with 8 national experts on the standard of cachexia stage by Lancet Oncol.
The detection results of the kit of this example are shown in FIGS. 8 to 12, where FIG. 8 shows the expression of P4HB in tumor tissues of 5 esophageal cancer patients of cachexia, FIG. 9 shows the expression of P4HB in tumor tissues of 3 pancreatic cancer patients of cachexia, FIG. 10 shows the expression of P4HB in tumor tissues of 1 gastric cancer patient of cachexia, FIG. 11 shows the expression of P4HB in pancreatic tissues of healthy persons, and FIG. 12 shows the expression of P4HB in colon tissues of healthy persons. According to the detection result of the kit, compared with healthy people, the staining range of the tumor tissue of the cachexia patient is remarkably increased, and the staining color is remarkably deepened. In addition, the positive rate of healthy people in the detection sample is 20%, the positive rate of cachexia patients is 100%, the specificity of detection is 80%, and the sensitivity is 100%.
The expression profile of 9 cachectic patients and 5 healthy people was statistically analyzed. Specifically, the SPSS software is used for performing significance analysis on P4HB expression conditions of cachexia patients and normal people in a T detection mode, the analysis result is shown in figure 13, and the P value is less than 0.001, so that the kit has a high significance for the difference of the detection results of the cachexia patients and the healthy people.
All possible combinations of the technical features in the above embodiments may not be described for the sake of brevity, but should be construed as being within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (15)
1. The hybridoma cell is characterized in that the preservation number is CCTCC NO: C2022108.
2. A monoclonal antibody secreted by the hybridoma cell of claim 1.
3. The monoclonal antibody of claim 2, wherein the monoclonal antibody is an IgG antibody.
4. Use of the monoclonal antibody of claim 2 or 3 in the preparation of a reagent for detecting a P4HB protein.
5. The use of claim 4, wherein the P4HB protein detection reagents are used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay, immunofluorescence, immunocolloidal gold, immunochemiluminescence, immunoturbidimetry, immunoblotting and dot blotting.
6. The P4HB protein detection reagent is characterized by comprising a monoclonal antibody secreted by a hybridoma cell with the preservation number of CCTCC NO: C2022108.
7. The reagent of claim 6, wherein the kit is used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunocolloidal gold, immunochemiluminescence, immunoturbidimetry, immunoblotting and dot blotting.
8. The reagent of claim 7, wherein the kit further comprises a detection reagent other than the monoclonal antibody for performing at least one of the detection methods.
9. The reagent of claim 8, wherein the detection reagent further comprises a labeled antibody for detecting the monoclonal antibody.
10. The reagent according to claim 9, wherein the labeled antibody satisfies at least one of the following characteristics:
(1) The labeled antibody is an anti-IgG antibody;
(2) The labeled antibody is linked with a label, optionally, the label is selected from at least one of a chromophore, a digoxin labeled probe, an electron dense substance, colloidal gold, and an enzyme that generates a detectable signal, further optionally, the label comprises horseradish peroxidase.
11. A method for detecting a P4HB protein for non-disease diagnosis purposes, which is characterized by comprising the following steps: detecting a P4HB protein using the reagent of any one of claims 6 to 10.
12. Use of a reagent according to any one of claims 6 to 10 in the preparation of a kit for the detection of cachexia.
13. A kit for detecting cachexia, comprising the reagent according to claims 6 to 10.
14. The kit of claim 13, further comprising at least one of an antibody positive control reagent, a negative control reagent, a dilution buffer, a dewaxing reagent, a hydration reagent, an antigen retrieval reagent, a blocking reagent, a chromogenic reagent, a counterstaining reagent, and a mounting reagent.
15. A method of preparing the hybridoma cell of claim 1, comprising: immunizing BALB/c mouse with human P4HB protein as immunogen, taking immune mouse spleen cell, mixing with SP2/0 myeloma cell and mouse feeder cell to make cell fusion, then screening out positive fusion cell capable of secreting P4HB antigen monoclonal antibody, namely said hybridoma cell.
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| Title |
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| JESSICA L. O. CAMPOS等: "Protein Disulfide Isomerase Modulates the Activation of Thyroid Hormone Receptors" * |
| M CHEN.等: "Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction" * |
| RUIJIAN ZHANG等: "Inhibition of autophagy using 3-methyladenineincreases cisplatin-induced apoptosis by increasing endoplasmic reticulumstress in U251 human glioma cells" * |
| 高晓寒: "食管鳞癌来源的细胞外囊泡中的蛋白P4HB调控恶病质骨骼肌萎缩的作用及机制研究" * |
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