An antibody against human IgM and a kit for detecting human IgM

Abstract

The present application relates to the technical field of antibodies, in particular to an antibody against human IgM and a kit for detecting human IgM. The antibody against human IgM or the antigen binding fragment thereof provided by the present application can specifically bind to human IgM and has no cross reaction with similar proteins; the antibody has high affinity with human IgM and high stability. The double antibody sandwich ELISA detection kit developed based on the antibody has high sensitivity, specificity and accuracy for detecting human IgM, and can quickly, accurately and high-throughput detect the content of IgM in human serum or other serum analogs containing human IgM, and has good application prospect in human IgM detection.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to an antibody against human IgM and a kit for detecting human IgM. Background Technology

[0002] As the earliest immunoglobulin produced in the initial humoral immune response of animals, IgM accounts for only about 10% of serum immunoglobulins. It is mainly produced by B cells in the spleen and lymph nodes and distributed in the blood. IgM can exist as a pentamer or a monomer. In the pentamer, all heavy chains and all light chains are identical, with a molecular weight of approximately 900 kDa, making it the largest of all immunoglobulins, also known as a macroglobulin. IgM monomers can be expressed on the plasma membrane of B lymphocytes, acting as B cell antigen receptors. Compared to IgG, IgM is produced earliest in the body, but its duration is short, and it is not the main force of the body's anti-infective immunity. However, because it is the earliest antibody produced when the body first encounters an antigen, it plays a very important role in the early stages of anti-infective immunity. IgM antibody detection can also be used for early serological diagnosis of diseases.

[0003] The commonly used method for IgM determination is immunoturbidimetry. Immunoturbidimetry utilizes the property that antigens and antibodies can specifically bind, forming a complex of a certain size in a liquid phase. This complex refracts or absorbs light, and the IgM content is calculated by measuring the transmitted or scattered light after refraction or absorption. This method has the following main drawbacks: it requires a large sample volume and a large amount of key reagent (anti-human IgM serum); large-scale testing is difficult, the operation is complex, and specific analytical instruments are required; experimental repeatability is poor; the complex molecules formed in the liquid are not stable enough, requiring sufficiently large and numerous molecules to produce a relatively accurate reading, and accuracy is very poor at low sample concentrations; the buffer system used also requires sodium azide, which has poor safety. Similarly, ultraviolet-spectrophotometry for IgM determination also has the above disadvantages. Other IgM detection methods include immunodiffusion assays, HPLC, and capillary electrophoresis. The disadvantages of immunodiffusion assays are that there are many influencing factors during the experimental operation, the detection time is long, and the sensitivity is poor. They are suitable for roughly estimating the content in a sample. The disadvantages of HPLC and capillary electrophoresis are that they require specialized instruments and experienced personnel to operate, and the instruments are expensive.

[0004] In conclusion, there is still a need to develop efficient antibodies, kits, and detection methods for detecting IgM. Summary of the Invention

[0005] This invention provides an antibody against human IgM and a kit for detecting human IgM.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides an antibody against human IgM or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment are as shown in SEQ ID NO. 10, 11, and 12, respectively; the amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is as shown in SEQ ID NO. 13; the amino acid sequence of CDR2 is SSS; and the amino acid sequence of CDR3 is as shown in SEQ ID NO. 14.

[0008] The antibodies or their antigen-binding fragments described above can specifically bind to human IgM without cross-reacting with similar proteins, exhibiting high affinity and stability. Enzyme-linked immunosorbent assay (ELISA) kits developed based on these antibodies or their antigen-binding fragments can achieve human IgM detection with high sensitivity, specificity, and accuracy.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.15 or has at least 80% similarity to the sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16 or has at least 80% similarity to the sequence shown in SEQ ID NO.16.

[0010] The sequence similarity described above is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, and more preferably at least 99.9%.

[0011] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16.

[0012] The antibodies or their antigen-binding fragments mentioned above may be monoclonal antibodies, Fab, Fab', F(ab')2, Fv, or single-chain antibodies.

[0013] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0014] Secondly, the present invention provides a nucleic acid molecule that encodes the antibody or its antigen-binding fragment described above.

[0015] Based on the amino acid sequences of the antibodies or their antigen-binding fragments provided above, those skilled in the art can obtain the nucleotide sequences of nucleic acid molecules encoding the aforementioned antibodies or their antigen-binding fragments. Due to the degeneracy of codons, the nucleotide sequence encoding a nucleic acid molecule of an antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the aforementioned antibodies or their antigen-binding fragments are within the scope of protection of this invention.

[0016] In some specific embodiments of the present invention, the sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.17, and the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.18.

[0017] Thirdly, the present invention provides a biological material containing the nucleic acid molecules described above; the biological material is an expression cassette, a vector, or a host cell.

[0018] The aforementioned expression cassette can be obtained by linking a promoter or other transcriptional or translational regulatory element upstream of the nucleic acid molecule and / or a terminator or other transcriptional or translational regulatory element downstream of it.

[0019] The aforementioned vectors include, but are not limited to, plasmid vectors, bacteriophage vectors, viral vectors, and artificial chromosome vectors.

[0020] The host cells mentioned above include microbial cells, insect cells, or other mammalian cells. The microbial cells may be bacteria or fungi, including but not limited to *Escherichia coli*, and fungi including but not limited to yeast. The mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO), young hamster embryonic kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero), and human embryonic kidney 293 cells (HEK293).

[0021] Fourthly, the present invention provides antibody conjugates, which are obtained by conjugating the antibodies or their antigen-binding fragments described above with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0022] Fifthly, the present invention provides an antibody composition against human IgM, said antibody composition comprising the antibodies of (1) and (2) below:

[0023] (1) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively. The amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YTS and the amino acid sequence of CDR3 is shown in SEQ ID NO.5.

[0024] (2) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.10, 11 and 12, respectively. The amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is shown in SEQ ID NO.13, the amino acid sequence of CDR2 is SSS and the amino acid sequence of CDR3 is shown in SEQ ID NO.14.

[0025] Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is as shown in SEQ ID NO.6 or has at least 80% similarity to the sequence shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or has at least 80% similarity to the sequence shown in SEQ ID NO.7.

[0026] The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is as shown in SEQ ID NO.15 or has at least 80% similarity to the sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16 or has at least 80% similarity to the sequence shown in SEQ ID NO.16.

[0027] The antibody composition described above can be used as a paired antibody in a double-antibody sandwich ELISA method for detecting human IgM, with the two antibodies in the composition serving as the coating antibody and the labeling antibody, respectively. Using this antibody composition in a double-antibody sandwich ELISA method for detecting human IgM exhibits high specificity, sensitivity, and accuracy.

[0028] In some embodiments of the present invention, the antibody in (2) above is used as a coating antibody, and the antibody in (1) above is used as a labeling antibody.

[0029] In a sixth aspect, the present invention provides any of the following applications of the antibodies described above or their antigen-binding fragments, or the antibody conjugates or antibody compositions:

[0030] (1) Prepare products for detecting the presence or level of human IgM in a sample;

[0031] (2) Detection of the presence or level of human IgM in a sample for purposes other than disease diagnosis;

[0032] (3) Prepare products for early diagnosis of infectious diseases, monitoring of autoimmune diseases or monitoring of transplant rejection;

[0033] (4) Immunogenicity testing of vaccines;

[0034] (5) Evaluation of vaccine immunization efficacy;

[0035] (6) Quality control of products containing human IgM.

[0036] In (1) above, the sample includes biological samples derived from humans (e.g., blood, serum, serum analogues, etc.), and also includes in vitro prepared products containing human IgM (e.g., drugs containing human IgM, etc.). The product can be used for disease diagnosis purposes, such as early diagnosis of infectious diseases by detecting the IgM content in the sample; it can also be used for non-disease diagnosis purposes, such as detecting the human IgM content in in vitro prepared products containing human IgM for product manufacturing, quality control, etc. The above-mentioned products include diagnostic reagents or kits.

[0037] In (2) above, the sample is preferably a product containing human IgM prepared in vitro (e.g., a drug containing human IgM). The non-disease diagnostic purpose of the detection includes detecting the content of human IgM in the product containing human IgM prepared in vitro, for product manufacturing, quality control, etc.

[0038] In (3) above, the product includes a detection reagent or kit, which is used to detect the content of human IgM for early diagnosis of infectious diseases, monitoring of autoimmune diseases or monitoring of transplant rejection.

[0039] In (4) and (5) above, the immunogenicity or immunization effect of the vaccine can be detected by detecting the content of IgM in human serum.

[0040] In this invention, the method for detecting human IgM can be any immunological method such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, immunoturbidimetry, etc. ELISA is preferred, and double-antibody sandwich ELISA is more preferred.

[0041] In a seventh aspect, the present invention provides a kit comprising the antibodies or antigen-binding fragments thereof described above, or comprising the antibody conjugates, or comprising the antibody composition.

[0042] Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0043] In some embodiments of the present invention, the kit is a double-antibody sandwich ELISA detection kit.

[0044] Preferably, the kit comprises a coated antibody and a labeled antibody, wherein the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the labeled antibody are as shown in SEQ ID NO.1, 2, and 3, respectively; the amino acid sequence of the complementarity-determining region (CDR1) of the light chain variable region is as shown in SEQ ID NO.4; the amino acid sequence of CDR2 is YTS; and the amino acid sequence of CDR3 is as shown in SEQ ID NO.5. Similarly, the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the coated antibody are as shown in SEQ ID NO.10, 11, and 12, respectively; the amino acid sequence of the complementarity-determining region (CDR1) of the light chain variable region is as shown in SEQ ID NO.13; the amino acid sequence of CDR2 is SSS; and the amino acid sequence of CDR3 is as shown in SEQ ID NO.14.

[0045] The present invention has no particular limitations on the markers for the above-mentioned labeled antibodies; an exemplary marker is HRP.

[0046] Preferably, the kit may also contain other reagents for ELISA detection, including but not limited to enzyme-labeled plates, human IgM standards, PBST washing buffer, blocking buffer, chromogenic buffer, and stop solution.

[0047] In some embodiments of the present invention, the principle of detecting human IgM content using enzyme-linked immunosorbent assay (ELISA) based on the double-antibody sandwich method is as follows: Anti-human IgM monoclonal antibody is coated onto an ELISA plate; serially diluted standards and pre-diluted samples are added, and the human IgM in the standards and samples binds fully to the coated antibody on the ELISA plate; after washing the plate, HRP-labeled anti-human IgM antibody is added, which specifically binds to the human IgM in the standards and samples captured by the coated antibody on the ELISA plate; after washing the plate, the chromogenic substrate TMB is added. If different concentrations of human IgM are present in the reaction wells, HRP will cause the colorless TMB to turn into different shades (positively correlated) of blue. After adding the stop solution, the reaction wells will turn yellow; finally, the absorbance (OD) of the reaction well sample is measured at λmax = 450 nm (OD = 450 nm). The concentration of human IgM in the sample is directly proportional to the OD, and the concentration of human IgM in the sample can be calculated using a standard curve.

[0048] Eighthly, the present invention provides a method for detecting human IgM, the method comprising: using the antibody or its antigen-binding fragment or the antibody conjugate or the antibody composition or the kit to detect the presence or content of human IgM in the sample to be tested.

[0049] The detection methods described above can be selected from immunological methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, and immunochromatography. ELISA is preferred, and double-antibody sandwich ELISA is more preferred.

[0050] The beneficial effects of the present invention include at least the following: the anti-human IgM antibody or its antigen-binding fragment provided by the present invention can specifically bind to human IgM and has no cross-reactivity with similar proteins such as porcine IgG, mouse IgG, bovine IgG, rabbit IgG, duck IgG, goose IgG, chicken IgY, human IgG, human IgG4, human IgA, and human HSA; it has a high affinity for human IgM and high stability. The double-antibody sandwich ELISA kit developed based on this antibody exhibits high sensitivity, specificity, and accuracy for detecting human IgM. Compared with other methods such as immunoturbidimetry (mg level), ultraviolet-spectrophotometry (mg level), and HPLC (g level), the detection sensitivity (ng level) of this kit ranges from 7.8125 to 500 ng / mL. It can rapidly, accurately, and with high throughput detect the IgM content in human serum or other serum analogs containing human IgM. Compared with other methods such as immunoturbidimetry (approximately 4 hours), ultraviolet-spectrophotometry (approximately 4 hours), and HPLC (approximately 4 hours), the detection time of this kit is significantly shortened (2.75 hours), and it can perform high-throughput detection (comparing the detection time of 50 samples). It has good application prospects in human IgM detection. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is the SDS-PAGE detection result of monoclonal antibody 4G1 in Example 1 of the present invention.

[0053] Figure 2 This is the SDS-PAGE detection result of monoclonal antibody 10C11 in Example 1 of the present invention.

[0054] Figure 3 This is the standard curve for detecting human IgM using the double-antibody sandwich ELISA detection method in Example 2 of the present invention. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] Example 1: Preparation of anti-human IgM monoclonal antibody

[0057] 1. Animal immunization

[0058] Female Balb / c mice aged 6-8 weeks were selected and immunized with a mixture of naturally extracted human IgM antigen and an equal volume of Freund's adjuvant. The immunization cycle was two weeks, and blood was collected after three immunizations to measure the titer. A booster immunization was performed three days before fusion.

[0059] 2. Preparation of immune spleen cells

[0060] Take one BALB / c mouse that has been boosted with immunization, euthanize it by exorbitant blood from the eye socket (collect serum, which is the positive serum), disinfect it by soaking it in 75% alcohol for 5-10 minutes, then fix it on a dissecting board for dissection, remove the spleen and cut it open, and place it in a sterile homogenizer; grind and prepare cell suspension as described in SP2 / 0 below, count the cells and set them aside for later use.

[0061] 3. Cell fusion

[0062] Prepared syngeneic myeloma cells were mixed with mouse spleen cells in a specific ratio, and polyethylene glycol (PEG), a fusion promoter, was added. Under the action of PEG, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific procedure is as follows:

[0063] (1) Activation of myeloma (SP2 / 0) cells

[0064] Thaw and resuscitate commercial SP2 / 0 cells, then resuspend them in nutrient solution (RPMI-1640 basal culture medium with added fetal bovine serum) and incubate at 37°C and 5% CO2; passage them after 3-5 days.

[0065] Collect cells and suspend them in RPMI-1640 basal culture medium. After counting, take 0.5-1×10⁶ cells. 6 One tumor was injected subcutaneously into the back of BALB / c mice and cultured for 9-10 days. When the tumor on the back increased in size to about 0.8 cm in diameter, the mice were euthanized by neck retraction, and the tumors were aseptically removed after soaking in 75% alcohol for 5 minutes.

[0066] Cut off the tumor fragment and place it in a sterile homogenizer. Add RPMI-1640 basal culture medium and homogenize thoroughly. Add another 10 mL of RPMI-1640 basal culture medium and let stand for 2 minutes. Transfer the upper cell suspension to another centrifuge tube and add another 10 mL of RPMI-1640 basal culture medium. Repeat the homogenization process twice. Centrifuge the obtained cell suspension at 1000 r / min for 10 minutes, remove the supernatant, and then resuspend in 30 mL of RPMI-1640 basal culture medium.

[0067] Add 15 mL of lymphocyte separation medium to another centrifuge tube, and carefully place the cell suspension on top of the separation medium; then centrifuge at 1200 r / min for 15 min, aspirate the dense white cell layer at the interface with a pipette, wash the cells twice with RPMI-1640 basal culture medium, resuspend them in 10 mL of RPMI-1640 basal culture medium, count them, and set them aside for later use.

[0068] (2) Preparation of feeder cells

[0069] Take an unimmunized BALB / c mouse, perform orbital bloodletting, and collect negative serum. Inject 2-3 mL of RPMI-1640 basal culture medium into the mouse's peritoneal cavity, pipette and transfer the medium to another centrifuge tube for later use. This medium contains peritoneal macrophages. Prepare a spleen cell suspension using the same procedure and transfer it to the peritoneal macrophage tube. Centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells in HAT medium, and incubate at 37°C in a 5% CO2 incubator until use.

[0070] (3) Integration

[0071] 1-2×10 7 SP2 / 0 and 1×10 8 One immune cell was mixed in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 8 min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37°C water bath. Then, 0.8 mL of 50% PEG (Sigma) pre-warmed to 37°C was added, and the mixture was stirred and allowed to stand for 30 s. After standing, 10 mL of RPMI-1640 basal culture medium pre-warmed to 37°C was added. After mixing, the mixture was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the mixture was incubated at 37°C for 5-8 min. Then, the mixture was mixed with the feeder cell suspension and seeded into 96-well plates at 250 μL / well. The plates were then incubated at 37°C in a 5% CO2 incubator. On the 4th day after fusion, the medium was replaced with HT medium and cultured for another 4 days. When the fused cell colonies reached 1 / 4 of the culture well and the medium turned slightly yellow, antibody detection was performed.

[0072] 4. Screening of hybridoma-positive clones and cell cloning

[0073] The purpose of selective culture is to screen for fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells, lacking hypoxanthine-guanine-phosphoribotransferase, cannot synthesize DNA via the salvage pathway and die. Unfused lymphocytes, although possessing hypoxanthine-guanine-phosphoribotransferase, cannot survive long-term in vitro and gradually die. Only fused hybridoma cells, having acquired hypoxanthine-guanine-phosphoribotransferase from spleen cells and possessing the unlimited proliferation characteristic of myeloma cells, can survive and proliferate in HAT medium. The specific procedures are as follows:

[0074] (1) Screening positive hybridoma cells using indirect ELISA

[0075] Coating with known antigens: Dilute the purified coating antigen to 1-10 μg / mL with coating buffer; add 100 μL to each well, shake gently, and incubate overnight at 4°C or for 1 hour at 37°C; discard the liquid in the wells (pat dry the liquid in the wells as much as possible); wash 3 times, 2-3 minutes each time.

[0076] To block the uncoated areas in the enzyme-labeled wells: Add 200 μL of blocking buffer (5% skim milk powder or 0.1% BSA) to each well, shake gently, and incubate at 37°C for 1 hour; discard the liquid in the wells; fill each well with washing buffer, let stand for 2-3 minutes, discard the liquid in the wells, pat dry, and wash 3 times with washing buffer using this method.

[0077] Sample addition: Add 50 μL of supernatant from each well of the hybridoma cells to be tested to the enzyme-labeled wells in sequence, shake gently, incubate at 37°C for 1 h, wash, and pat dry.

[0078] Add enzyme-labeled anti-antibody: First, dilute the enzyme-labeled second antibody to the appropriate working concentration according to the instructions using diluent. Add 100 μL to each well, shake gently, and incubate at 37°C for 1 hour. Then wash and pat dry.

[0079] Add colorimetric reagent: Add 100 μL of freshly prepared colorimetric reagent to each well, shake gently, and incubate at 37°C for 10 min.

[0080] Termination of reaction: Add 50 μL of stop solution to each well.

[0081] Result: ELISA reader OD 450nm The reading is taken below; if it is greater than 3 times that of the negative well, it can be determined as positive.

[0082] (2) Cloning of hybridoma cells (limiting dilution method)

[0083] Prepare mouse feeder cell layers before cloning; gently blow the hybridoma cells to be cloned out of the culture wells and count the number of viable cells using a hemocytometer; dilute the cells with complete culture medium to 5, 10, and 30 cells / mL.

[0084] The three cell suspensions of the above concentrations were added to the prepared feeder cells in 96-well plates at 100 μL / well, so that each well contained 0.5, 1, and 3 cells, respectively. One drop of culture medium was added on day 4. Cell growth in each well was carefully observed and recorded on days 5 and 6.

[0085] (3) Detection of specific antibodies: On days 7-9 after cloning, when the cell clones have grown to fill 1 / 3-1 / 2 of the field of view, they can be detected; the cells in the positive wells can be transferred to 24-well culture plates, and when the cells in the 24-well plates are growing well, mice can be inoculated intraperitoneally to collect ascites fluid.

[0086] 5. Sequencing of the variable regions of monoclonal antibodies 4G1 and 10C11

[0087] Collect more than 10 hybridoma cells 6The samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The gene sequencing results were as follows: The light chain variable region of monoclonal antibody 4G1 is 321 bp long, encoding 107 amino acids; the DNA sequence is shown in SEQ ID NO. 9, and the protein sequence is shown in SEQ ID NO. 7. The heavy chain variable region is 363 bp long, encoding 121 amino acids; the DNA sequence is shown in SEQ ID NO. 8, and the protein sequence is shown in SEQ ID NO. 6. The light chain variable region of monoclonal antibody 10C11 is 321 bp long, encoding 107 amino acids; the DNA sequence is shown in SEQ ID NO. 18, and the protein sequence is shown in SEQ ID NO. 16. The heavy chain variable region is 336 bp long, encoding 112 amino acids; the DNA sequence is shown in SEQ ID NO. 17, and the protein sequence is shown in SEQ ID NO. 15. Analysis revealed that the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of monoclonal antibody 4G1 are shown in SEQ ID NO.1, 2, and 3, respectively; the amino acid sequence of the complementarity-determining region of the light chain variable region, CDR1, is shown in SEQ ID NO.4; the amino acid sequence of CDR2 is YTS; and the amino acid sequence of CDR3 is shown in SEQ ID NO.5. Similarly, the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of monoclonal antibody 10C11 are shown in SEQ ID NO.10, 11, and 12, respectively; the amino acid sequence of the complementarity-determining region of the light chain variable region, CDR1, is shown in SEQ ID NO.13; the amino acid sequence of CDR2 is SSS; and the amino acid sequence of CDR3 is shown in SEQ ID NO.14.

[0088] 6. Large-scale preparation of monoclonal antibodies 4G1 and 10C11

[0089] Hybridoma cells after establishment were injected into the peritoneal cavity of mice. Ascites fluid was collected approximately 7 days later, and antibodies were purified using Proteining affinity chromatography. The SDS-PAGE results of the purified monoclonal antibodies 4G1 and 10C11 are shown below. Figure 1 and Figure 2 As shown.

[0090] 7. Determination of the relative affinity constants of monoclonal antibodies 4G1 and 10C11

[0091] Human IgM antigen was coated onto an ELISA plate and blocked. After washing with PBST, monoclonal antibodies 4G1 and 10C11 were diluted to saturation concentrations and added to each well at 100 μL / well, and incubated at room temperature for 2 h. After washing with PBST, 60 μL / well of NaSCN solution at concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mol / L were added sequentially, and the plates were incubated at room temperature for 15 min. After washing with PBST, HRP-labeled goat anti-mouse IgG was added, and the plates were incubated at room temperature for 45 min for colorimetric detection. The sodium thiocyanate concentration corresponding to a decrease in the OD value at 450 nm to 50% of the uneluted value after elution is the relative affinity constant of the antibody, expressed as M (mol / L). The results (Table 1) show that the relative affinity constants of monoclonal antibodies 4G1 and 10C11 are both greater than 2.5M, indicating good affinity.

[0092] Table 1

[0093]

[0094] Example 2: Establishment of a double-antibody sandwich ELISA kit and detection method for human IgM content.

[0095] 1. HRP labeling of monoclonal antibody 4G1

[0096] Add antibody 4G1 to a dialysis bag and dialyze overnight at 4°C in 0.01M CB buffer. Dissolve 14M ghRP in 2.8mL of water. Prepare a fresh 0.1M NaIO4 solution, add 0.56mL of 0.1M NaIO4 to 2.8mL of POD solution, mix well, incubate at room temperature in the dark for 20min, and dialyze overnight at 4°C in 1M NaAc buffer. Remove the overnight dialyzed antibody and POD solution and add 0.168mL of 0.2M pH 9.5 carbonate buffer, then immediately add the removed antibody to the POD solution and gently stir at room temperature in the dark for 2 hours. Weigh 0.04g NaBH4 and dissolve in 10mL of water, add 0.02mL to the reaction solution, mix well, and incubate at 4°C for 2 hours. Remove and place in a dialysis bag, dialyze in 0.01M PBS, change the solution after two hours, and dialyze overnight at 4°C. Remove the labeled solution from the overnight dialysis, add an equal volume of glycerol, and store at -20°C. The enzyme-labeled antibody is named HRP-4G1.

[0097] 2. Preparation of monoclonal antibody 10C11 ELISA plate

[0098] The monoclonal antibody 10C11 was diluted to 8 μg / mL with 0.05 M pH 9.6 carbonate coating buffer. 100 μL was added to each well of a 96-well polystyrene reagent plate and incubated overnight at 4°C. The next day, the solution in the wells was discarded, and the plate was washed three times with washing buffer for 3 minutes each time. After the above steps, each well of the reaction plate was blocked with 250 μL of 2% BSA solution. μ L, 2h. Discard the solution in the well, dry in a drying room, then vacuum and store at 4℃.

[0099] 3. Establishment of a double-antibody sandwich ELISA method

[0100] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 10C11, allow it to reach room temperature, wash it three times, and agitate it dry. Add 100 μL of human IgM standards at different dilutions: 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, and 7.8125 ng / mL, and include a blank control. After sealing the plate, incubate it at room temperature for 2 hours, wash it four times, and agitate it dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate, incubate it at room temperature for 45 minutes, wash it five times, and agitate it dry. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm using a microplate reader (within 5 minutes). Plot a standard curve with different concentrations of human IgM standards on the x-axis and the corresponding OD values ​​on the y-axis to establish a regression equation. The results show that the detection range is 7.8125-500 ng / mL, R... 2 It is 0.99992 ( Figure 3 ).

[0101] 4. Sensitivity detection of double-antibody sandwich ELISA

[0102] The average OD of 20 zero standard concentrations was added to two standard deviations to calculate the corresponding detectable concentration. The results (Table 2) show that the sensitivity is 2.29 ng / mL.

[0103] Table 2

[0104]

[0105] 5. Specificity detection of double-antibody sandwich ELISA

[0106] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 10C11, allow it to reach room temperature, wash it three times, and agitate it dry. Add 100 μL of human IgM standards at different dilutions or high concentrations of human IgM structural analog proteins, including porcine IgG, mouse IgG, bovine IgG, rabbit IgG, duck IgG, goose IgG, chicken IgY, human IgG, human IgG4, human IgA, and human HSA. After sealing the plate, incubate it at room temperature for 2 hours, wash it four times, and agitate it dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate, and incubate it at room temperature for 45 minutes, wash it five times, and agitate it dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, and develop the color at room temperature in the dark for 15 minutes. Add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm using an ELISA reader (within 5 minutes). The results (Table 3) showed that the antibody pair composed of monoclonal antibodies 10C11 and 4G1 did not react with other similar proteins.

[0107] Table 3

[0108]

[0109] 6. Stability testing of the double-antibody sandwich ELISA kit

[0110] The stability of the double-antibody sandwich ELISA kit containing the monoclonal antibody pair 10C11 and 4G1 was investigated using a 37℃ accelerated assay, as follows:

[0111] The ELISA plate coated with monoclonal antibody 10C11, HRP-labeled monoclonal antibody 4G1 (HRP-4G1), and human IgM standard were placed at 37°C for accelerated stability testing for 11 days (equivalent to approximately 16.5 months at 4°C). Afterward, the plate was removed for testing. The testing method involved washing the plate three times and patting it dry. 100 μL of different dilutions of standard were added: 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, and 7.8125 ng / mL, with a blank control included. The plate was sealed and incubated at room temperature for 2 hours, washed four times, and then dried. 100 μL of HRP-4G1 working solution was added to the reaction wells, the plate was sealed, and incubated at room temperature for 45 minutes, washed five times, and then dried. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm using a microplate reader (within 5 minutes). Plot a standard curve with different concentrations of human IgM standards on the x-axis and the corresponding OD values ​​on the y-axis to establish a regression equation. The results (Table 4) show that on day 11, the OD values ​​of the standard curve did not change significantly, and the OD value gradient of the standard curve was good.

[0112] Table 4

[0113]

[0114] Example 3: Determination of human serum IgM content

[0115] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 10C11, allow it to reach room temperature, wash it three times, and then agitate it dry. Select 16 serum samples from volunteer blood donors (numbered 1, 2, 3, ..., 16), and add 100 μL of human serum samples / standards at different dilutions. The sample dilution was 10,000 times, and the human IgM standard dilutions were 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, and 7.8125 ng / mL. A blank control was also included. After sealing the plate, incubate it at room temperature for 2 hours, wash it four times, and agitate it dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate, and incubate it at room temperature for 45 minutes, washing it five times and agitating it dry. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm using an ELISA reader (within 5 minutes). According to literature reports, the serum IgM content in normal individuals is 0.5–2 mg / mL (IgM content varies depending on age and individual). The detection results of this invention (Table 5) are basically consistent with the reports, indicating that the double-antibody sandwich ELISA kit and detection method of this invention can accurately determine the serum IgM content.

[0116] Table 5

[0117]

[0118] Example 4: Determination of recovery rate and evaluation of linearity of human IgM spiking

[0119] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 10C11, allow it to reach room temperature, wash it three times, and agitate it dry. Select three volunteer blood donor serum samples, one volunteer blood donor plasma sample, and one RPMI 1640 complete medium containing 10% fetal bovine serum supplemented with human IgM standard as samples. Add 100 μL of samples and standards at different dilutions to each well. The human IgM standard dilutions were 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, and 7.8125 ng / mL, and a blank control was included. After sealing the plate, incubate it at room temperature for 2 hours, wash it four times, and agitate it dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate, incubate it at room temperature for 45 minutes, wash it five times, and agitate it dry. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm using a microplate reader (within 5 minutes). The detection results (Tables 6 and 7) show that the double-antibody sandwich ELISA detection method of the present invention has a high recovery rate (a range of 70%-130% indicates good results) and exhibits good linearity within a certain range (a range of 70%-130% indicates good results).

[0120] Table 6

[0121]

[0122] Table 7

[0123]

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody against human IgM or an antigen-binding fragment thereof, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NO.10, 11, and 12, respectively. The amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is shown in SEQ ID NO.13, the amino acid sequence of CDR2 is SSS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

14.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody, Fab, Fab', F(ab')2 or Fv.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A biomaterial, characterized in that, The biomaterial contains the nucleic acid molecule as described in claim 4; The biomaterial is an expression cassette, vector, or host cell.

6. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the antibody or its antigen-binding fragment according to any one of claims 1 to 3 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

7. An antibody composition against human IgM, characterized in that, The antibody composition comprises the antibodies in (1) and (2) below: (1) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively. The amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YTS and the amino acid sequence of CDR3 is shown in SEQ ID NO.

5. (2) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.10, 11 and 12, respectively. The amino acid sequence of the complementarity-determining region CDR1 of the light chain variable region is shown in SEQ ID NO.13, the amino acid sequence of CDR2 is SSS and the amino acid sequence of CDR3 is shown in SEQ ID NO.

14.

8. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 6, or the antibody composition according to claim 7: (1) Prepare products for detecting the presence or level of human IgM in a sample; (2) Detection of the presence or level of human IgM in a sample for purposes other than disease diagnosis; (3) Prepare products for early diagnosis of infectious diseases, monitoring of autoimmune diseases or monitoring of transplant rejection; (4) Prepare products for vaccine immunogenicity testing; (5) Prepare products for evaluating the immunization efficacy of vaccines; (6) Quality control of products containing human IgM.

9. A reagent kit, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or the antibody conjugate as described in claim 6, or the antibody composition as described in claim 7.

10. The reagent kit according to claim 9, characterized in that, The kit is an enzyme-linked immunosorbent assay (ELISA) kit.

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