CN108384761B - Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof - Google Patents
Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention relates to a hybridoma cell strain and a monoclonal antibody secreted by the hybridoma cell strain, wherein the antibody can be specifically combined with human IgM, can be used for in vitro detection of the human IgM, and is particularly suitable for early diagnosis of hepatitis B viruses. The invention also relates to a kit comprising the hybridoma cell strain or the monoclonal antibody.
Description
Technical Field
The invention relates to a monoclonal antibody, in particular to an anti-human IgM monoclonal antibody.
Background
Immunodiagnostic reagents are one of the main types of in vitro diagnostic reagents. The reagent utilizes the specific reaction of combining antigen and antibody to perform qualitative or quantitative diagnosis, and is developed most rapidly in all diagnostic reagent products at present, whether the technology or the market.
After the pathogenic microorganisms infect a human body, IgM antibody is firstly generated in the process of stimulating and inducing humoral immune response, the early diagnosis of infectious diseases is the premise of carrying out early and effective treatment, and particularly, the early diagnosis is important for the infectious diseases with acute onset and serious illness, and the antibody which is firstly generated by an organism after infection is the IgM antibody, so that the detection of the specific IgM antibody in the blood of a patient at the early stage of pathogenesis has important significance in clinical early diagnosis.
In recent years, there have been some studies on anti-human IgM monoclonal antibodies, and for example, WO 2010026758 a1 discloses an anti-human IgM monoclonal antibody, which aims to solve the problem of low polyclonal antibody specificity and focuses on the property of monoclonal antibodies to induce agglutination between human IgM and to verify the effect of monoclonal antibodies to inhibit non-specific reactions, but does not pay attention to the systematic evaluation (e.g., antibody titer, cross-reactivity, stability, detection sensitivity and specificity, etc.) of the anti-human IgM monoclonal antibodies when used in vitro diagnostic reagents, and thus it is unclear whether the antibodies are suitable for the preparation of in vitro diagnostic reagents.
For example, the mansion organism anti-human IgM monoclonal antibody is a common commercial anti-human IgM monoclonal antibody, but it is not good enough in stability to be used as an immunodiagnostic antibody.
In fact, screening monoclonal antibodies for preparing in vitro diagnostic reagents is a complex process, and first a good antigen is obtained to prepare enough antibodies, and then the antibodies are systematically evaluated to obtain candidate antibodies with clinical relevance, and then the candidate antibodies are developed into detection reagents. The antibody titer, the cross reactivity, the stability, the clinical effect and the like are important evaluation factors, for example, the antibody titer reflects the lowest titer of the antigen reaction under a certain concentration, and the lower the titer is, the higher the titer is; antibody cross-reactivity may affect the specificity of the antibody; the stability of the antibody directly affects the reliability of the final result, and the antibody with poor stability has higher requirements on storage conditions and operation conditions, so that the practicability of the antibody as a diagnostic reagent and the reliability of the result are reduced, and the increase of the cost is inevitably caused; clinical trials are used to demonstrate the efficacy of antibodies in diagnosing a particular disease.
Therefore, for in vitro diagnostic applications, there is a great need in the art for monoclonal antibodies that provide better results for systemic evaluation, and are particularly useful for early diagnosis of pathogenic infections.
Disclosure of Invention
The invention aims to provide an anti-human IgM monoclonal antibody, a hybridoma cell strain secreting the anti-human IgM monoclonal antibody, a kit containing the monoclonal antibody or the hybridoma cell strain and application of the monoclonal antibody or the hybridoma cell strain in detection of human IgM. Through systematic evaluation, the antibody has better performance in all aspects, so that the antibody is suitable to be used as an immunodiagnostic reagent for preparing an in vitro diagnosis kit, and is particularly suitable to be used for preparing an early diagnosis kit for detecting hepatitis B virus.
Therefore, the inventor carries out a great deal of research, immunizes a mouse by using human IgM, clones at least 4 times by a limiting dilution method after cell fusion until the monoclonal antibody is reached, screens 1 novel Hybridoma cell strain (Hybridoma) IgM-10 capable of stably secreting the antibody from the obtained clones, and stores the Hybridoma cell strain in a China center for type culture collection, Wuchang Lojiashan university in Wuhan city, Hubei province in 2018 and 8 days in 8 months, wherein the preservation number is CCTCC NO: C201852, thereby completing the invention.
In a first aspect, the invention provides a hybridoma cell strain which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C201852.
In a second aspect, the invention provides a monoclonal antibody capable of specifically binding to human IgM.
In one embodiment, the monoclonal antibody does not bind to human IgG, murine IgG, rabbit IgG, or bovine IgG.
In another embodiment, the monoclonal antibody has a median potency of 296600 and superior stability and reliability under repeated freeze-thaw, long-term storage, heat accelerated harsh conditions.
In a preferred embodiment, the monoclonal antibody is an antibody secreted by the hybridoma cell line of the invention.
In a third aspect, the invention provides a kit, which comprises the hybridoma cell strain or the monoclonal antibody of the invention.
In a specific embodiment, the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.
In a preferred embodiment, the kit is a chemiluminescent kit.
In another embodiment, the kit is a microfluidic chip.
In a fourth aspect, the invention provides the use of the hybridoma cell strain or monoclonal antibody of the invention in the preparation of a kit.
In one embodiment, the kit is based on immunoassay, preferably, the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.
In a preferred embodiment, the kit is a chemiluminescent kit.
In another embodiment, the kit is a microfluidic chip, preferably, the microfluidic chip is based on immunoassay.
In one embodiment, the kit is for detecting human IgM.
In a preferred embodiment, the human IgM is a hepatitis b virus core IgM antibody.
In a specific embodiment, the pathogen is hepatitis b virus.
In addition, the invention also provides the application of the hybridoma cell strain or the monoclonal antibody in preparing a kit for diagnosing the hepatitis B virus, preferably a kit for early diagnosis of the hepatitis B virus.
The monoclonal antibody has the beneficial effects that firstly, the antibody titer is at least one order of magnitude higher than that of a commercially available human IgM antibody usually used in-vitro diagnosis, and the monoclonal antibody has a better immune effect; secondly, the antibody has no cross reaction with human IgG, mouse IgG, rabbit IgG and bovine IgG, and has good specific binding capacity; thirdly, it has superior long-term and thermal stability to commercially available human IgM antibodies, that is, it has an extended life and can accept relatively loose storage and handling conditions, thus greatly saving costs; finally, clinical experiments show that the monoclonal antibody can be used for a chemiluminescence kit for detecting hepatitis B virus core antibody IgM, the detection sensitivity can reach 100%, and the detection specificity can reach 100%.
That is, the monoclonal antibody of the invention shows good performance in various aspects such as antibody titer, cross reactivity, stability and detection effect, so that the invention provides an anti-human IgM monoclonal antibody which can be used for in vitro diagnosis through systematic evaluation and has outstanding comprehensive capacity.
Drawings
FIG. 1 shows a SDS-PAGE electrophoresis of the antibody IgM-Ab10 of the present invention;
FIG. 2 shows the results of the measurement of the antibody titer of the antibody IgM-Ab10 of the present invention with two commercial anti-human IgM antibodies, wherein the abscissa is Log (dilution factor) and the ordinate is OD450。
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Antibodies
As used herein, the term "antibody" refers to immunoglobulin molecules, including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, CDR-grafted antibodies, and antibody constructs, such as single chain fv (scfv) or antibody fusion proteins; furthermore, it relates to antibodies produced/synthesized recombinantly or synthetically.
In a preferred embodiment, the antibody is an antibody produced from a hybridoma cell line with the preservation number of CCTCC NO: C201852.
An "antibody fragment" typically includes the antigen-binding region, light and/or heavy chain variable regions, at least a portion of one or more (e.g., six) CDRs, of the parent antibody that retain at least some of the binding specificity of the parent antibody. In particular, the parent antibody herein refers to an antibody produced from a hybridoma cell line with the preservation number of CCTCC NO: C201852. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a dimeric molecule; a linear antibody; single chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, when the activity is expressed on a molar basis, the fragments retain at least 50% of the binding activity to human IgM. Preferably, the fragment retains at least 60%, 70%, 80%, 90%, 95%, or 100% of the binding activity to human IgM as compared to the parent antibody.
Preferably, an antibody fragment refers to the antigen binding region, the light and heavy chain variable regions or the six CDRs of an antibody.
"antibody derivatives" refer to conservative amino acid substitutions (referred to as "conservative variants") that may include antibodies whose biological activity is not substantially altered as compared to the parent antibody.
The invention provides monoclonal forms of the antibodies.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. Monoclonal antibodies are advantageous because they can be obtained by culture of hybridoma cell lines and are substantially free of contamination by other immunoglobulins.
Reagent kit
The detection kit of the present invention may take various forms, for example, a strip, a cartridge containing various reagents required for the test, a microfluidic chip, etc., and the kit may be manufactured according to standard procedures known to those skilled in the art.
Kits of the invention may include containers, chips, instructions for use, buffers, immunological aids, and/or other materials, structures, and/or reagents as desired for performing the diagnosis/assay.
The examples are illustrated using chemiluminescence assays, but it should not be understood that the kits of the present invention are limited to chemiluminescence assays.
The kit of the present invention includes an antibody produced from a hybridoma cell line having a preservation number of CCTCC NO: C201852, which may be present in a manner conventional in the art, for example, in a dissolved or dried form in a container, coated on a solid phase carrier (e.g., a film, a plate, a bead, a particle (e.g., a magnetic particle), etc.), and present in a dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.
Due to objective factors such as transportation and use places, the kit is often suitable for field detection in various complex environments, so that the stability of raw materials is one of important factors for restricting the kit result. As shown in example 9 below, the antibody IgM-Ab10 of the invention possesses better stability as a starting material for chemiluminescent kits than conventional IgM antibodies under extreme conditions, thereby enhancing the reliability of the results of the kit and reducing costs in phase change.
The antibody of the present invention may be coated at a concentration of 1 to 100. mu.g/mg, preferably 10 to 70. mu.g/mg, and more preferably 15 to 50. mu.g/mg.
Other materials required for diagnosis/detection in the kit of the present invention include, but are not limited to, anti-human IgM antibodies other than the antibodies of the present invention, antigens bound to human IgM antibodies, and/or human IgM. The other materials mentioned above may be present in a manner conventional in the art, for example, in dissolved or dried form in a container, coated on a solid support (e.g., a membrane, a plate, beads, particles (e.g., magnetic particles), etc.), in dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.
Other structures required for performing a diagnosis/test in the kit of the invention include, but are not limited to, structures for sampling, structures for performing controls, and/or structures for observing the course or result of the test.
Other reagents required for performing the diagnosis/detection in the kit of the present invention include, but are not limited to, detergents, visualization reagents and/or terminating reagents.
In one embodiment, the antibody in the kit of the invention is detectably labeled. Any label and labeling method known to those skilled in the art may be used. For example, the labels that may be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds, but the present invention is not limited thereto.
Commonly used labels may include enzymes (e.g., horseradish peroxidase, β -galactosidase, alkaline phosphatase, etc.), radioisotopes (e.g., horseradish peroxidase, β -galactosidase, etc.), and the like32P or125I) Etc., biotin, digoxin, colloidal metals (e.g., colloidal gold, etc.), fluorescent dyes (e.g., fluorescein, rhodamine, texas red, etc.), chemiluminescent compounds, or bioluminescent compounds (e.g., dioxetane, luminol, acridinium, etc.). Any labeling step well known in the art may be used, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, and the like.
In some embodiments, one or more of the other materials required for diagnosis/detection may also be detectably labeled.
In a preferred embodiment, the kit of the present invention is a kit for early diagnosis of a pathogen.
Use of
The anti-human IgM antibody or hybridoma cell strain of the present invention can be used for any purpose related to the specific reaction of human IgM. Preferably, the antibody or hybridoma cell strain can be used for detecting human IgM.
The antibody or hybridoma cell strain can be used for detecting biological samples from human beings.
As used herein, "biological sample" refers to semen, lymph, serum, plasma, urine, synovial fluid, or spinal fluid. In a preferred embodiment, the biological sample is blood, serum or plasma.
Preferably, a biological sample from a human at an early stage of onset is used.
The presence of human IgM can be detected quantitatively or qualitatively using immunoassay methods which typically involve incubating or sequentially contacting a biological sample with the antibodies of the invention and/or other materials required for detection and detecting the bound antibodies by a variety of techniques well known in the art.
Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, radioisotopic or non-radioisotopic methods, and the like. These methods include, inter alia, Western blotting, overlay assays, RIA (radioimmunoassay) and IRMA (immunoradioimmunoassay), GIA (colloidal gold immunoassay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescent immunoassay), and CLIA (chemiluminescent immunoassay).
The anti-human IgM monoclonal antibody can be combined with human IgM produced by various pathogenic infections, but when preparing in vitro diagnostic reagents, the diagnostic effect is inevitably different due to different detection systems or different properties of antibodies. As shown in example 8 below, the antibody of the present invention is superior in the effect on the detection of hepatitis B virus core IgM antibody caused by Hepatitis B Virus (HBV) to currently available detection kits.
Therefore, in a preferred embodiment, the antibody or hybridoma cell line of the invention is particularly suitable for detecting the core IgM antibody produced by hepatitis B virus infection, thereby diagnosing whether an individual is infected with hepatitis B virus.
The core IgM antibody generated by hepatitis B virus infection is also called hepatitis B core antibody IgM type, is an early sign of hepatitis B infection, and is one of indexes affected by hepatitis B virus.
The antibodies or hybridomas of the present invention can be used to detect whether a subject is infected with hepatitis b virus or whether a subject has hepatitis b. Wherein the hepatitis B is acute or chronic.
The embodiments of the present invention will be described in detail with reference to examples, which do not indicate specific conditions, and are performed according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used are not indicated to the manufacturer, but are conventional products which are commercially available.
EXAMPLE 1 immunization of mice
Diluting human IgM (Sichuan Mike biological new material technology Co., Ltd., batch No. 150127) extracted from blood source to 1.0mg/ml with normal saline, mixing with quick immunization adjuvant quick antibody 5w (Beijing Boolong immunization technology Co., Ltd.) in equal volume, mixing well, injecting four limbs intramuscular with 50 μ l/one dose to BALB/c mouse (Chengdu Daokou laboratory animal center, 6 weeks old female, 2), enhancing immunity 21 days after the first immunization, collecting tail blood 35 days later, separating serum, measuring titer by indirect ELISA method, and the titer of all mice serum is more than 1: 106Can be used for fusion. 3 days before fusion, the human IgM was diluted to 1.0mg/ml with physiological saline, and then mixed with the same volume of physiological saline in the tail vein for additional immunization at a dose of 50. mu.l/mouse.
EXAMPLE 2 preparation of hybridoma cell lines
2-1 preparation of feeder cells
Normal 10-week-old BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c is used for taking eye blood and pulling neck to kill, 0.1% benzalkonium bromide is soaked for 1 minute, 75% alcohol is added for soaking for 1 minute, the abdominal skin is cut off by scissors under the aseptic operation in an ultra-clean bench, the peritoneum is exposed, 4.5ml of RPMI1640 basic culture solution is injected into the abdominal cavity by a syringe, then the eye blood is taken out by a dropper, 5.5ml of RPMI1640 basic culture solution is added to the abdominal cavity to carry out reverse reactionWashing again, recovering washing liquid, centrifuging at 1000rpm for 5min to obtain precipitate, re-suspending with RPMI1640 culture solution containing 20% newborn calf serum, and adjusting cell concentration to 3.2 × 105Adding into 96-well plate at 100 μ l/well, 37 deg.C and 5% CO2And (5) culturing.
2-2 preparation of immune splenocytes
Three days after additional immunization of the mice in example 1, the spleens were removed under aseptic conditions, placed in a dish, washed once with RPMI1640 basic medium, minced, ground, filtered to obtain dispersed splenocytes, centrifuged at 1000rpm for 5 minutes to leave a pellet, resuspended in RPMI1640 basic medium, and counted by 3% acetic acid dilution.
Preparation of 2-3 myeloma cells
Mouse myeloma cell Sp2/0 (preserved by Sichuan Mike biological new material technology Co., Ltd.) is screened by 8-azaguanine, cultured to logarithmic phase, and taken 5 bottles (75 cm)2) Making into cell suspension, centrifuging at 1000rpm for 5min to obtain precipitate, resuspending with RPMI1640 basic culture medium, counting, and mixing at 1.1 × 105The cells are cultured in flasks at an individual/ml cell concentration (15-30 ml of complete 1640 medium is generally replaced every 1-2 days).
2-4 cell fusion and HAT selective culture hybridoma
Myeloma cells and immune spleen cells were mixed at a ratio of 1:9, washed 1 time with RPMI1640 basic medium in a 50ml conical centrifuge tube, and centrifuged at 1000rpm for 5 minutes to leave a precipitate. The cells were mixed well and fused slowly with 0.5ml of 50% PEG4000, and fused for 1 minute, and then fused with 50ml of RPMI1640 basic medium to terminate the cell fusion. After centrifugation at 700rpm for 5 minutes, the cells were resuspended in 1640 medium containing 1% HAT and 20% newborn calf serum, and 9 sets of 96-well cell culture plates were dropped on average. 37 ℃ and 5% CO2The next day of culture, supplemented with 1640 medium containing 1% HAT and 20% newborn bovine serum to well-full. The medium was changed half after 5 days and again half after 7 days.
Screening of 2-5 Positive cell lines
Diluting human IgM (Sichuan Mekken BioNew Material technology Co., Ltd., lot No. 150127) with 0.06M pH9.6 carbonic acid buffer solution to 5. mu.g/ml in 96-well ELISA plateCoating 100 mul in each hole for detecting cell culture supernatant, placing in a refrigerator for overnight at 2-8 ℃, discarding liquid in the holes the next day, washing the plate with ELISA washing liquid three times, patting dry, using PBS containing 10% calf serum and having a pH of 0.01MpH7.2, sealing at 37 ℃ for 2 hours, patting dry, vacuum packaging for later use, on the ninth day after spleen cell fusion, taking 100 mul of cell supernatant into the above 96-hole detection plate, incubating at 37 ℃ for 40 minutes, washing five times with the ELISA washing machine, adding 100 mul of horse radish peroxidase-labeled goat anti-mouse IgG (produced by Sichuan Meyer biological New Material Co., Ltd.), incubating at 37 ℃ for 30 minutes, adding 100 mul of buffer solution containing 0.1% (M/V) o-phenylenediamine and 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer solution, incubating at 37 ℃ for 10 minutes, adding 50 mul 2M sulfuric acid solution into each hole, stopping reaction, measuring 450 mul of absorption value, adjusting the absorbance of mouse serum to a value, adding a proper amount of 0.1% (V/V) of 0.1% (V) of the buffer solution, adding the buffer solution into a vial, continuously diluting the buffer solution, adding the buffer solution of horse radish peroxidase-96-ml, performing continuous-96-mesh cloning, performing cell cloning, performing continuous-mesh cloning, performing cell cloning, performing the steps, and collecting the steps, and performing the steps of cloning6Counting the cells per ml (if the number of cells is not within the range, re-centrifuging the cells according to the total cell count, adding a proper amount of the frozen stock solution again, and finally making the cell concentration be 4-8 × 106One/ml) and then subpackaged in sterile freezing tubes, and 0.5ml of cell sap is added into each freezing tube. Cell fusion to obtain 11 strains of hybrid capable of stably secreting IgM monoclonal antibodyHybridoma cell lines, see table 1 below. The antibody secreted by the hybridoma cell 1F10-A12-C7-D7-G9 has the best effect when being used for detecting the core antibody IgM antibody in the early stage of hepatitis B virus infection, the hybridoma cell is marked as IgM-10 and is preserved in the China center for type culture collection on 3 to 8 days in 2018, and the preservation number is CCTCCNO: C201852.
TABLE 1 hybridoma cell lines stably secreting anti-human IgM monoclonal antibody
| Hybridoma cell | Hybridoma cell | Hybridoma cell |
| 9A6-F8-H6-H3-H1 | 6B11-C11-G8-E7-E1 | 8G9-C12-C10-D11 |
| 8A7-C9-E2-C1-D9 | 7G5-E1-B7-H7-D12 | 8A5-D9-D12-D8-C11 |
| 5C12-D1-G9-E12-E12 | 4F3-G10-G1-F1-G1 | 3A12-D2-G8-G4-C1 |
| 2G12-B7-F10-C11-C2 | 1F10-A12-C7-D7-G9 |
EXAMPLE 3 preparation of monoclonal antibodies
Selecting healthy BALB/c mice of 6-8 weeks, injecting 0.5ml of liquid paraffin (Tianjin Kemiou) into the abdominal cavity of each mouse, and injecting 1.1 × 10 into the abdominal cavity of each mouse 7 days later6And (3) hybridoma cells. Ascites can be generated 7-10 days after the cells are inoculated, the health condition and ascites symptoms of the animals are closely observed, the mice are sacrificed when the ascites is as much as possible and before the mice are dying, the ascites is sucked into a test tube by a dropper, and 1-5 ml of ascites can be obtained by one mouse. The collected ascites is centrifuged to take the supernatant, and a small sample is stored in a refrigerator at the temperature of minus 20 ℃. The ascites fluid was separately precipitated by saturation with ammonium sulfate and purified by protein A affinity column chromatography, and the purity of the antibody was more than 90% by SDS-PAGE (designated as IgM-Ab10), and the results of electrophoresis are shown in FIG. 1.
Example 4 hybridoma cell culture supernatant titer assay
Human IgM (Sichuan Mekken BioNew Material technology Co., Ltd., batch No. 150127) was diluted to 5. mu.g/ml with 0.06M carbonic acid buffer solution having a pH of 9.6, and each well of a 96-well microplate was coated with 100. mu.l. Placing the mixture in a refrigerator for overnight at 2-8 ℃, discarding liquid in the holes the next day, washing the mixture by an ELISA plate washing machine for three times, patting the mixture to be dry, sealing the mixture for 2 hours at 37 ℃ by using 0.01M PBS (phosphate buffer solution) containing 10% calf serum and pH7.2, 150 mu l/hole, and patting the mixture to be dry, and detecting culture supernatant, ascites and antibody titer of the hybridoma cells. Hybridoma cell culture supernatant titer detection the first well is original-fold supernatant culture solution, the second well to the fourth well are diluted by 10-fold with 0.01M PBS buffer solution with pH7.2, the fifth well to the tenth well are diluted by 2-fold, the eleventh well is diluted by 100-fold of mouse serum during fusion and is used as positive control, the twelfth well is used as negative control with RPMI1640 complete culture solution, and the sample volume of each well is 100 mul. Incubating for 40 minutes at 37 ℃, washing the plate for five times by an ELISA washing machine, adding 100 mu L of goat anti-mouse IgG (produced by Sichuan Mekkera New biological Material technology Co., Ltd.) diluted by 12000 times, incubating for 30 minutes at 37 ℃, adding 100 mu L of o-phenylenediamine containing 0.1% (M/V), 0.1% (V/V) hydrogen peroxide and pH5.0 citric acid phosphate buffer solution into each hole, incubating for 10 minutes at 37 ℃, adding 50 mu L of 2M sulfuric acid solution into each hole to terminate the reaction, and measuring the absorption value of 450 nm. The negative control OD value is less than 0.2, and the positive control OD value is greater than 1.8, and the detection system hasThe effect is that when the OD value is more than or equal to 2 × negative control OD value, the positive control OD value is positive, otherwise, the negative control OD value is negative, the dilution ratio corresponding to the positive hole with the lowest detection value is the titer of the hybridoma cell culture supernatant, and the titer of the antibody of the hybridoma cell culture supernatant is more than 1:6.4 × 103See table 2.
TABLE 2 culture supernatant titer results
| Enzyme-labeled |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| |
1 | 10 | 100 | 1000 | 2000 | 4000 | 8000 | 16000 | 32000 | 64000 | Positive control | Negative control |
| IgM-10 supernatant | 2.21 | 2.17 | 1.78 | 0.72 | 0.37 | 0.26 | 0.22 | 0.20 | 0.17 | 0.11 | 2.68 | 0.05 |
Example 5 ascites titer test
The ELISA detection method is different from that of example 4 in the dilution method, specifically, the first well is original ascites, the second well is diluted 10 times to the seventh well by PBS buffer solution with 0.01MpH7.2 step by step, the eighth well is diluted 2 times to the tenth well, the dilution ratio corresponding to the positive well with the lowest detection value is the ascites titer, the table 3 is the ascites titer, and the ascites titer prepared by the hybridoma IgM-10 is more than 1:8 × 106。
TABLE 3 ascites titre
| Enzyme-labeled |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| |
1 | 10 | 100 | 1000 | 10000 | 100000 | 1000000 | 2000000 | 4000000 | 8000000 | Positive control | Negative control |
| IgM-10 ascites | 2.61 | 2.75 | 2.78 | 1.72 | 1.37 | 1.26 | 0.52 | 0.21 | 0.19 | 0.16 | 2.67 | 0.05 |
Example 6 antibody titer detection
The purified antibody IgM-Ab10 prepared in example 3 was first diluted to 1mg/ml in 0.01M PBS buffer pH7.2, and then diluted 100-fold as the initial first well, and was diluted 5-fold from the second well to the tenth well, the eleventh well was a positive control in which the mouse serum was diluted 100-fold during fusion, and the twelfth well was a negative control in which PBS was used, and the sample volume of each well was 100. mu.l. Incubating for 50 minutes at 37 ℃, washing the plate for five times by an ELISA washing machine, adding 100 mu L of goat anti-mouse IgG (produced by Sichuan Mekken Biotech Co., Ltd.) diluted by 8000 times, incubating for 1h at 37 ℃, adding 100 mu L of o-phenylenediamine containing 0.1% (M/V), 0.1% (V/V) hydrogen peroxide and pH5.0 citric acid phosphate buffer solution into each hole, incubating for 15 minutes at 37 ℃, adding 50 mu L of 2M sulfuric acid solution into each hole to terminate the reaction, detecting the absorption value at 450nm (multifunctional reader, manufacturer Thermo, model Varioskan Flas), repeating for 3 times, taking the average OD valueThe determination standard of the antibody titer is characterized in that LOG (dilution) is used as an abscissa, an antibody OD average value is used as an ordinate to make a curve, the curve equation is y ^ min + (max-min)/(1+10 ((logEC50-x) × Hillslope)), the curve is fitted through sigmaplot data processing software, and the median titer is 10logEC50. The results show that the median titer of the present antibody IgM-Ab10 was 296600. The purity of commercial anti-human IgM antibody B (purchased from Xiamen Bosheng Biotechnology Co., Ltd.) and C (bougai Takei Biotechnology Co., Ltd., Loyang) were both greater than 90%, the concentrations were uniformly adjusted to 1mg/ml, B, C titers were detected by the same method as above, and by fitting, the median titer of the antibody B was 13600, the median titer of the antibody C was 8793, and the median titer of B, C was lower than that of the antibody secreted from the hybridoma of the present invention. The results of the titer determination are shown in table 4 and figure 2.
TABLE 4 antibody titer test
Example 7 Cross-reaction assay
Human IgG (Sichuan Michael Bionew materials technology Co., Ltd., lot No. 140120), mouse IgG (Sichuan Michael Bionew materials technology Co., Ltd., lot No. 140221), rabbit IgG (Sichuan Michael Bionew materials technology Co., Ltd., lot No. 140225) and bovine IgG (Sichuan Michael Bionew materials technology Co., Ltd., lot No. 140311) were diluted to 2.5. mu.g/ml with 0.06M of pH9.6 carbonate buffer solution, and each well of a 96-well plate was coated with 100. mu.l. Placing the plate in a refrigerator for 2-8 ℃ overnight, discarding the liquid in the holes the next day, washing the plate in an ELISA plate washing machine for three times, patting the plate dry, using 0.5% casein containing 10mM Tris-HCl (7.4) as a sealing solution, sealing the plate in 150 mu l/hole at 37 ℃ for 2 hours, discarding the liquid, and patting the plate dry for later use. The anti-human IgM monoclonal antibody IgM-Ab10 of the present invention was labeled with HRP and diluted to the same concentration (0.5mg/ml), and then diluted 500 times, 1000 times, 2000 times, 4000 times, 8000 times, and added to the above-mentioned coated ELISA plate wells, 50. mu.l of each well reacted with four IgG respectively, incubated at 37 ℃ for 30 minutes, then washed five times by ELISA plate washer, patted dry, 100. mu.L of 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer, incubated at 37 ℃ for 10 minutes, and 50. mu.L of 2M sulfuric acid solution was added to each well to terminate the reaction, and the absorbance at 450nm was measured (multifunctional reader, manufacturer Thermo, model Varioskan Flas). The following table shows the results of a cross-reactivity assay with IgM-Ab10, with no cross-reactivity with the four IgGs.
TABLE 5 Cross-reaction assay
| Enzyme-labeled IgM-Ab10 dilution | 1:500 | 1：1000 | 1：2000 | 1：4000 | 1:8000 |
| Human IgM | 1.981 | 1.866 | 1.853 | 1.637 | 1.319 |
| Human IgG | 0.09 | 0.048 | 0.048 | 0.058 | 0.082 |
| Mouse IgG | 0.085 | 0.063 | 0.084 | 0.077 | 0.057 |
| Bovine IgG | 0.084 | 0.061 | 0.135 | 0.066 | 0.054 |
Example 8 clinical diagnosis of pathogens
(1) Preparation of detection reagent
The kit adopts a magnetic particle chemiluminescence immunoassay method, utilizes the capture method principle to label an anti-human IgM monoclonal antibody IgM-Ab10 and biotin with a ratio of 1:4-1:8, uses PBS buffer (containing 0.5% BSA) containing sodium dihydrogen phosphate and having a pH of 7.2 and a concentration of 0.02M to coat the magnetic beads with a coating amount of 15-50 mu g/mg, incubate for 30min at 37 ℃, wash for 3 times with the PBS buffer and dilute with a ratio of 1:25 to prepare R1. HRP-labeled hepatitis B virus core antigen was diluted with PBS buffer (containing 0.5% BSA) at an appropriate ratio (1: 1000-1: 5000) to prepare R2. The prepared reagent was measured by an IS1200 full-automatic chemiluminescence apparatus (Mike Bio Inc.).
A two-step process was used with a sample size of 50. mu.l, 50. mu.l R1 and 100. mu.l R2. And (3) calculating the multiple ratio of the relative luminous value of the sample to a critical value (Cut off value) through calibration of the calibrator, qualitatively judging the result, and judging whether the hepatitis B virus core IgM antibody is detected in the sample.
(2) Sensitivity detection
The detection kit prepared by the method detects 200 positive samples and 300 negative samples respectively. Table 6 shows the comparison experiment between the kit prepared by the monoclonal antibody IgM-Ab10 of the present invention and a commercial kit (Beijing Korea biotechnology Co., Ltd.), and the test results show that the detection kit prepared by the monoclonal antibody IgM-Ab10 of the present invention has higher sensitivity and specificity than the commercial kit, and the detection results are shown in Table 6.
TABLE 6 Positive and negative sample testing
Example 9 stability verification
Stability of monoclonal antibodies of the invention in harsh environments monoclonal antibody IgM-Ab10 of the invention was treated under the following conditions: repeatedly freezing and thawing at a-20 ℃ for 2 times; repeated freeze thawing at the temperature of between 20 and b for 3 times; repeatedly freezing and thawing at c-20 deg.C for 4 times; repeated freeze thawing at d-20 deg.c for 5 times; e-20 deg.C for 7 months; f thermal acceleration at 37 ℃ for 7 days; g, accelerating the heating at 37 ℃ for 14 days, and preparing the detection reagent according to the method to respectively detect 15 parts of positive reference substances and 15 parts of negative reference substances, wherein the coincidence rate is 100%. The antibody is good in stability, and the detection reagent prepared by the antibody is good in precision. Selecting commercial anti-human IgM monoclonal antibody B (Xiamen Bosheng) with higher potency, processing the monoclonal antibody B under the same conditions to prepare a detection reagent for respectively detecting 15 parts of positive reference substances and 15 parts of negative reference substances, wherein the result shows that the detection reagent starts to be partially degraded after being repeatedly frozen and thawed for 5 times or being stored for 7 months at-20 ℃ or being thermally accelerated for 14 days at 37 ℃, and the detection result is not completely consistent with the reference substances. The results of the tests are listed in table 7.
TABLE 7 stability results
Therefore, the monoclonal antibody of the invention has high stability, and the IgM detection reagent of the hepatitis B virus core antibody prepared by the monoclonal antibody can also keep high stability and reliability under severe environmental conditions, which is a valuable progress in clinical and laboratory applications.
Claims (12)
1. A hybridoma cell strain IgM-10 which is preserved in China center for type culture Collection with the preservation number of CCTCCNO C201852.
2. A monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. A kit comprising the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2.
4. The kit of claim 3, which is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunosorbent kit or a fluoroimmunoassay kit, or which is a microfluidic chip kit.
5. The kit of claim 3, which is a chemiluminescent kit.
6. Use of the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2 in the preparation of a kit.
7. The use according to claim 6, said kit being an immunoassay-based kit.
8. The use according to claim 7, wherein the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluoroimmunoassay kit, or the kit is a microfluidic chip kit.
9. The use according to claim 8, said kit being a chemiluminescent kit.
10. The use of claim 6, wherein the kit is for detecting human IgM.
11. The use of claim 10, wherein the human IgM is a hepatitis b virus core IgM antibody.
12. Use of the hybridoma cell line of claim 1 or the monoclonal antibody of claim 2 in the preparation of a kit for diagnosis of hepatitis b virus.
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