An antibody against human IgM or its antigen-binding fragment and its application

Abstract

The present invention relates to the technical field of antibodies, and particularly to an antibody against human IgM or its antigen-binding fragment and its application. The antibody against human IgM or its antigen-binding fragment provided by the present invention can specifically bind to human IgM without cross-reaction with similar proteins; it has a high affinity for human IgM and high stability. The double-antibody sandwich ELISA detection kit developed based on this 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 prospects in the detection of human IgM.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an antibody against human IgM or its antigen-binding fragment and its application. Background Art

[0002] IgM is the immunoglobulin produced earliest in the primary humoral immune response of animal bodies. Its content only accounts for about 10% of serum immunoglobulins. It is mainly produced by B cells in the spleen and lymph nodes and is distributed in the blood. IgM can exist as a pentamer, in which all heavy chains are the same and all light chains are the same, with a molecular weight of about 900 kDa, which is the largest among all immunoglobulins and is also called macroglobulin. IgM also exists in the form of a monomer, for example, it is expressed on the plasma membrane of B lymphocytes and serves as a B cell antigen receptor. Compared with IgG, IgM is produced earliest in the body but has a short duration, so it is not the main force in the body's anti-infection immunity. However, since it is the earliest antibody produced in the body when the body first comes into contact with antigenic substances, it plays a very important role in the early stage of anti-infection immunity and can also be used for serological early diagnosis of diseases by detecting IgM antibodies.

[0003] Currently, the commonly used method for IgM determination is immunoturbidimetry. Immunoturbidimetry utilizes the property that antigens and antibodies can specifically bind to produce a complex of a certain size in the liquid phase, forming light refraction or absorption, and the content of IgM therein is calculated by measuring the transmitted light or scattered light after refraction or absorption. This method mainly has the following disadvantages: it requires a relatively large sample volume and a relatively large amount of the key reagent (anti-human IgM serum); it is difficult to detect in large batches, with complex operations and the need for specific analytical instruments; the experimental repeatability is poor; the complex molecules formed in the liquid are not stable enough and need to reach a sufficient size and quantity to produce a relatively accurate reading, and the accuracy is very poor when the sample content is low; sodium azide is also required in the buffer system used, and the safety is poor. Similarly, the ultraviolet-spectrophotometry method for IgM content determination also has the above-mentioned disadvantages.

[0004] Other IgM detection methods also include immunodiffusion experiments, HPLC, and capillary electrophoresis. The disadvantage of immunodiffusion experiments is that there are many influencing factors in the experimental operation, the detection time is long, and the sensitivity is poor, which is suitable for roughly estimating the content in the sample; the disadvantages of HPLC and capillary electrophoresis are that they require specialized instruments and experienced personnel to operate, and the instrument prices are expensive.

[0005] In summary, there is still a need to develop antibodies for efficient IgM detection, as well as their kits and detection methods. Summary of the Invention

[0006] The present invention provides an antibody against human IgM or its antigen-binding fragment and its application.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] 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 shown in SEQ ID NOs. 1, 2, and 3, respectively, and 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.

[0009] The above-mentioned antibody or its antigen-binding fragment can specifically bind to human IgM without cross-reacting with similar proteins, and has high affinity and stability. An enzyme-linked immunosorbent assay (ELISA) kit developed based on the above-mentioned antibody or its antigen-binding fragment can achieve highly sensitive, specific, and accurate detection of human IgM.

[0010] Preferably, the amino acid sequence of the heavy-chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO. 6 or has at least 80% similarity (Identities) with the sequence shown in SEQ ID NO. 6, and the amino acid sequence of the light-chain variable region is shown in SEQ ID NO. 7 or has at least 80% similarity with the sequence shown in SEQ ID NO. 7.

[0011] The above-mentioned sequence similarity 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%.

[0012] 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. 6, and the amino acid sequence of the light-chain variable region is shown in SEQ ID NO. 7.

[0013] The above-mentioned antibody or its antigen-binding fragment can be a monoclonal antibody, Fab, Fab', F(ab') 2 , Fv or single-chain antibody.

[0014] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described above.

[0016] Based on the amino acid sequence of the antibody or antigen-binding fragment thereof provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding an antibody or antigen-binding fragment thereof is not unique, and all nucleic acid molecules capable of encoding and producing the above antibody or antigen-binding fragment are within the protection scope of the present invention.

[0017] 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 antigen-binding fragment thereof is as shown in SEQ ID NO.8, and the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.9.

[0018] In a third aspect, the present invention provides a biomaterial containing the nucleic acid molecule described above; the biomaterial is an expression cassette, a vector or a host cell.

[0019] The above expression cassette can be obtained by connecting transcriptional or translational regulatory elements such as promoters upstream of the nucleic acid molecule and / or connecting transcriptional or translational regulatory elements such as terminators downstream thereof.

[0020] The above vectors include but are not limited to plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, etc.

[0021] The above host cells include microbial cells, insect cells or other mammalian cells. Among them, the microbial cells can be bacteria or fungi. The bacteria include but are not limited to Escherichia coli, and the fungi include but are not limited to yeast. The mammalian cells include but are not limited to Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero), and human embryonic kidney 293 cells (HEK293), etc.

[0022] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the antibody or antigen-binding fragment thereof described above with a label, and 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.

[0023] In a fifth aspect, the present invention provides an antibody composition against human IgM, and the antibody composition contains the antibodies in the following (1) and (2):

[0024] (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;

[0025] (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.

[0026] Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is 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 shown in SEQ ID NO.7 or has at least 80% similarity to the sequence shown in SEQ ID NO.7.

[0027] The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is 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 shown in SEQ ID NO.16 or has at least 80% similarity to the sequence shown in SEQ ID NO.16.

[0028] The above antibody composition can be used as a paired antibody for detecting human IgM by the double antibody sandwich ELISA method, and the two antibodies in the antibody composition serve as the coating antibody and the labeled antibody respectively. Using the above antibody composition to detect human IgM by the double antibody sandwich ELISA method has high specificity, sensitivity, and accuracy.

[0029] In some embodiments of the present invention, the antibody in (2) above serves as the coating antibody, and the antibody in (1) above serves as the labeled antibody.

[0030] In the sixth aspect, the present invention provides any one of the following applications of the above-mentioned antibody or its antigen-binding fragment or the antibody conjugate or the antibody composition:

[0031] (1)Preparing a product for detecting the presence or level of human IgM in a sample;

[0032] (2)Detecting the presence or level of human IgM in a sample for non-disease diagnosis purposes;

[0033] (3) Preparing products for early diagnosis of infectious diseases, monitoring of autoimmune diseases, or monitoring of transplant rejection reactions;

[0034] (4) Detecting the immunogenicity of vaccines;

[0035] (5) Evaluating the immune effect of vaccines;

[0036] (6) Quality control of products containing human IgM.

[0037] In the above (1), the sample includes biological samples derived from humans (such as blood, serum, serum analogs, etc.), and also includes products containing human IgM prepared in vitro (such as drugs containing human IgM, etc.). The product can be used for disease diagnosis purposes. For example, early diagnosis of infectious diseases can be carried out by detecting the content of IgM in the sample; it can also be used for non-disease diagnosis purposes. For example, detecting the content of human IgM in products containing human IgM prepared in vitro for product production, quality control, etc. The above products include detection reagents or reagent kits.

[0038] In the above (2), the sample is preferably a product containing human IgM prepared in vitro (such as drugs containing human IgM, etc.). The detection for non-disease diagnosis purposes includes detecting the content of human IgM in products containing human IgM prepared in vitro for product production, quality control, etc.

[0039] In the above (3), the product includes a detection reagent or a reagent kit, and the product is used for early diagnosis of infectious diseases, monitoring of autoimmune diseases, or monitoring of transplant rejection reactions by detecting the content of human IgM.

[0040] In the above (4) and (5), the immunogenicity or immune effect of the vaccine can be detected by detecting the content of IgM in human serum.

[0041] In the present invention, the method for detecting human IgM can be immunological methods such as ELISA, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, immunoturbidimetry, etc. ELISA is preferred, and sandwich ELISA is more preferred.

[0042] In a seventh aspect, the present invention provides a reagent kit, which contains the above-mentioned antibody or its antigen-binding fragment, or contains the antibody conjugate, or contains the antibody composition.

[0043] Preferably, the reagent kit is an enzyme-linked immunosorbent assay kit.

[0044] In some embodiments of the present invention, the reagent kit is a sandwich ELISA kit.

[0045] Preferably, the kit comprises a coated antibody and a labeled antibody. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the labeled antibody are shown in SEQ ID NO.1, 2, and 3 respectively, the amino acid sequence of CDR1 in 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. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the coated antibody are shown in SEQ ID NO.10, 11, and 12 respectively, the amino acid sequence of CDR1 in 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.

[0046] There is no particular limitation on the label of the above-mentioned labeled antibody. Exemplary labels are HRP.

[0047] Preferably, the kit may further comprise other reagents for ELISA detection, including but not limited to enzyme-linked immunosorbent assay plates, human IgM standards, PBST washing solutions, blocking solutions, chromogenic solutions, termination solutions, etc.

[0048] In some embodiments of the present invention, the principle of detecting the content of human IgM by enzyme-linked immunosorbent assay technology based on the double antibody sandwich method is as follows: Coating the anti-human IgM monoclonal antibody on the enzyme-linked immunosorbent assay plate; adding gradient-diluted standards and pre-diluted samples respectively. The human IgM in the standards and samples will fully bind to the coated antibody on the enzyme-linked immunosorbent assay plate; after washing the plate, adding the HRP-labeled anti-human IgM antibody, which will specifically bind to the human IgM in the standards and samples captured by the coated antibody on the enzyme-linked immunosorbent assay plate; after washing the plate, adding the chromogenic substrate TMB. If there are different concentrations of human IgM in the sample in the reaction wells, HRP will turn the colorless TMB into blue substances of different shades (positively correlated). After adding the termination solution, the reaction wells will turn yellow; finally, measuring the absorbance (OD) of the sample in the reaction wells at λmax = 450 nm (OD = 450 nm). The concentration of human IgM in the sample is proportional to the OD, and the concentration of human IgM in the sample can be calculated through the standard curve.

[0049] In the eighth aspect, the present invention provides a method for detecting human IgM, the method comprising: detecting whether human IgM or its content exists in a sample to be tested by using the antibody or its antigen-binding fragment or the antibody conjugate or the antibody composition or the kit.

[0050] The above detection methods can be selected from immunological methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, etc. ELISA is preferred, and sandwich ELISA is more preferred.

[0051] The beneficial effects of the present invention at least include: 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-reaction with similar proteins such as porcine IgG, murine IgG, bovine IgG, rabbit IgG, duck IgG, goose IgG, chicken IgY, human IgG, human IgG4, human IgA, human HSA, etc.; it has a high affinity for human IgM and high stability. The double-antibody sandwich ELISA detection kit developed based on this antibody has high sensitivity, specificity, and accuracy for detecting human IgM. Compared with other methods such as immunoturbidimetry (mg level), ultraviolet-spectrophotometry (mg level), HPLC method (g level), etc., the detection sensitivity (ng level) range of this kit is 7.8125 - 500 ng / mL, and it can quickly, accurately, and high-throughput detect the content of IgM in human serum or other serum analogs containing human IgM. Compared with other methods such as immunoturbidimetry (about 4 h), ultraviolet-spectrophotometry (about 4 h), HPLC method (about 4 h), etc., the detection time of this kit is significantly shortened (2.75 h), and it can be detected in high throughput (compared with the detection time of 50 samples), and it has good application prospects in the detection of human IgM. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0053] Figure 1 It is the SDS-PAGE detection result of monoclonal antibody 4G1 in Example 1 of the present invention.

[0054] Figure 2 It is the SDS-PAGE detection result of monoclonal antibody 10C11 in Example 1 of the present invention.

[0055] Figure 3 It is the standard curve for detecting human IgM by the double-antibody sandwich ELISA detection method in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0056] To make the objectives, technical solutions and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0057] Example 1 Preparation of anti-human IgM monoclonal antibody

[0058] 1. Animal immunization

[0059] Select 6-8 week-old female Balb / c mice. Immunize them with natural human IgM antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle is two weeks. After immunizing 3 times, take blood to measure the titer, and boost the immunization again three days before fusion.

[0060] 2. Preparation of immune spleen cells

[0061] Take one BALB / c mouse that has been boosted immunized, sacrifice it by orbital bleeding (collect the serum, which is the positive serum), soak it in 75% alcohol for 5-10 minutes for disinfection, then fix it on the dissection board for dissection, take out the spleen and cut it open, and place it in a sterilized homogenizer; the method of grinding and preparing the cell suspension is the same as that described for SP2 / 0 below. After counting, it is reserved for use.

[0062] 3. Cell fusion

[0063] Mix the prepared syngeneic myeloma cells and mouse spleen cells in a certain proportion, and add the fusogen polyethylene glycol (PEG). Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is as follows:

[0064] (1) Activation of myeloma (SP2 / 0) cells

[0065] Thaw and resuscitate commercial SP2 / 0 cells, then resuspend them in a nutrient solution (RPMI-1640 basal medium supplemented with calf serum), and place them in an incubator at 37°C and 5% CO 2 for culturing; passage after 3-5 days.

[0066] Collect the cells and suspend them in RPMI-1640 basal medium. After counting, take 0.5-1×10 6 cells and inject them subcutaneously into the back of BALB / c mice, and continuously culture for 9-10 days. When the tumor volume on the back increases to a diameter of about 0.8 cm, sacrifice the mice by cervical dislocation. After soaking in 75% alcohol for 5 minutes, take the tumor under sterile conditions.

[0067] Cut the tumor mass and place it in a sterilized homogenizer. Add RPMI-1640 basal medium and grind thoroughly. Then add 10 mL of RPMI-1640 basal medium, let it stand for 2 min, aspirate the upper cell suspension and place it in another centrifuge tube. Add another 10 mL of RPMI-1640 basal medium and repeat the grinding twice. Centrifuge the obtained cell suspension at 1000 r / min for 10 min to remove the supernatant, and then resuspend it in 30 mL of RPMI-1640 basal medium.

[0068] Add 15 mL of lymphocyte separation medium to another centrifuge tube, and carefully place the above cell suspension on top of the separation medium. Then centrifuge at 1200 r / min for 15 min. Use a pipette to aspirate the dense white cell layer at the interface, wash the cells twice with RPMI-1640 basal medium, and resuspend them in 10 mL of RPMI-1640 basal medium. After counting, set aside for use.

[0069] (2)Preparation of feeder cells

[0070] Take an unimmunized BALB / c mouse, collect blood from the orbital cavity, and collect the serum as negative serum. Inject 2 - 3 mL of RPMI-1640 basal medium into the abdominal cavity of the mouse, aspirate it after pipetting and place it in another centrifuge tube for standby. This liquid contains peritoneal macrophages. Prepare a spleen cell suspension in the same way as above and put it into the tube containing peritoneal macrophages. Centrifuge at 1000 r / min for 10 min to remove the supernatant. Suspend the cells in HAT medium and place them in an incubator at 37℃ and 5% CO 2 for standby.

[0071] (3)Fusion

[0072] Mix 1 - 2×10 7 SP2 / 0 cells with 1×10 8 immunized cells in a 50 mL centrifuge tube, centrifuge at 1000 r / min for 8 min. After discarding the supernatant completely, place the centrifuge tube containing the cell mixture in a 37℃ water bath. Then add 0.8 mL of pre-warmed 50% PEG (Sigma) at 37℃, stir and let it stand for 30 s. After standing, add 10 mL of pre-warmed RPMI-1640 basal medium at 37℃. Mix well and centrifuge at 1000 r / min for 5 min, discard the supernatant and place it at 37℃ for 5 - 8 min. Then mix it with the feeder cell suspension, seed it into a 96-well culture plate, 250 μL / well, and culture it in an incubator at 37℃ and 5% CO 2 for culture. On the 4th day after fusion, change to HT medium and continue culturing. When the fused cell colonies grow to 1 / 4 of the culture well and the medium turns slightly yellow, perform antibody detection.

[0073] 4. Screening of hybridoma positive clones and cloning of cells

[0074] The purpose of selective cultivation is to screen for fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase and cannot utilize the salvage pathway to synthesize DNA, so they die. Unfused lymphocytes, although they have hypoxanthine-guanine phosphoribosyltransferase, cannot survive in vitro for a long time on their own and also gradually die. Only the fused hybridoma cells can survive and proliferate in HAT medium because they obtain hypoxanthine-guanine phosphoribosyltransferase from spleen cells and have the characteristic of unlimited proliferation of myeloma cells. The specific operation is as follows:

[0075] (1)Screen positive hybridoma cells by indirect ELISA

[0076] Coat with known antigen: Dilute the purified coating antigen with coating buffer to 1 - 10 μg / mL; add 100 μL to each well in the microplate, shake gently, incubate overnight at 4°C or for 1 h at 37°C; discard the liquid in the wells (try to pat dry the liquid in the wells); wash 3 times, 2 - 3 minutes each time.

[0077] Block the positions in the enzyme-labeled wells that are not coated with antigen: Add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each well in the microplate, shake gently, incubate for 1 h at 37°C; discard the liquid in the wells; fill each well with washing buffer, let stand for 2 - 3 min, discard the liquid in the wells, pat dry, and wash 3 times with washing buffer in this way first.

[0078] Add sample: Take 50 μL of the supernatant of the hybridoma cells to be tested and add them to the enzyme-labeled wells in sequence, shake gently, incubate for 1 h at 37°C, wash, and pat dry.

[0079] Add enzyme-labeled anti-antibody: First, dilute the enzyme-labeled secondary antibody with diluent to the appropriate working concentration according to the instructions, add 100 μL to each well, shake gently, incubate for 1 h at 37°C; then wash and pat dry.

[0080] Add chromogenic solution: Add 100 μL of freshly prepared chromogenic solution to each well, shake gently, incubate for 10 min at 37°C.

[0081] Terminate the reaction: Add 50 μL of termination solution to each well.

[0082] Judge the result: Read the OD value with an enzyme-labeled instrument 450nm and read at the following. If it is more than 3 times that of the negative wells, it can be judged as positive.

[0083] (2)Clonalization of hybridoma cells (limiting dilution method)

[0084] Prepare a mouse feeder cell layer 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 to 5, 10, and 30 cells / mL with complete medium.

[0085] Add the cell suspensions at the above three concentrations to a 96-well culture plate with prepared feeder cells, 100 μL / well, so that each well contains 0.5, 1, and 3 cells respectively. Add one drop of additional medium on the 4th day of culture, and carefully observe the cell growth in each well on the 5th - 6th day and record.

[0086] (3)Detection of specific antibodies: On the 7th - 9th day after cloning, when the cell clones cover 1 / 3 - 1 / 2 of the field of view, detection can be carried out; the cells in the positive wells can be transferred to a 24-well culture plate. When the cells in the 24-well plate grow well, mice can be inoculated intraperitoneally to collect ascites.

[0087] 5. Sequencing of the variable region sequences of monoclonal antibodies 4G1 and 10C11

[0088] Collect hybridoma cells with a quantity greater than 10 6, sent to BGI (Shanghai) Co., Ltd. for sequencing. The gene sequencing results were obtained as follows: the light chain variable region sequence of monoclonal antibody 4G1 is 321 bp in length, encoding 107 amino acids, the DNA sequence is as shown in SEQ ID NO.9, and the protein sequence is as shown in SEQ ID NO.7; the heavy chain variable region sequence is 363 bp in length, encoding 121 amino acids, the DNA sequence is as shown in SEQ ID NO.8, and the protein sequence is as shown in SEQ ID NO.6. The light chain variable region sequence of monoclonal antibody 10C11 is 321 bp in length, encoding 107 amino acids, the DNA sequence is as shown in SEQ ID NO.18, and the protein sequence is as shown in SEQ ID NO.16; the heavy chain variable region sequence is 336 bp in length, encoding 112 amino acids, the DNA sequence is as shown in SEQ ID NO.17, and the protein sequence is as shown in SEQ ID NO.15. After analysis, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 4G1 are as shown in SEQ ID NOs.1, 2, and 3 respectively, the amino acid sequence of 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; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 10C11 are as shown in SEQ ID NOs.10, 11, and 12 respectively, the amino acid sequence of 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.

[0089] 6. Large-scale preparation of monoclonal antibodies 4G1 and 10C11

[0090] The hybridoma cells after establishing the cell line were injected into the abdominal cavity of mice, and ascites was collected after about 7 days. The antibodies were purified by Protein G affinity chromatography. The SDS-PAGE detection results of the purified monoclonal antibodies 4G1 and 10C11 are shown respectively in Figure 1 and Figure 2 as shown.

[0091] 7. Determination of the relative affinity constants of monoclonal antibodies 4G1 and 10C11

[0092] Coat the human IgM antigen onto an ELISA plate and block it. After washing the plate with PBST, dilute the monoclonal antibodies 4G1 and 10C11 to the saturation concentration and add them to the ELISA plate respectively, 100 μL per well, and incubate at room temperature for 2 h. After washing the plate with PBST, add 60 μL per well of NaSCN solutions with concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 Mol / L in sequence, and incubate at room temperature for 15 min. After washing the plate with PBST, add HRP-labeled goat anti-mouse IgG, and incubate at room temperature for 45 min for color development detection. The sodium thiocyanate concentration corresponding to the OD value at 450 nm dropping to 50% of that without elution after elution is the relative affinity constant of the antibody, expressed in M (mol / L). The results show (Table 1) that the relative affinity constants of the monoclonal antibodies 4G1 and 10C11 are both greater than 2.5 M, indicating good affinity.

[0093] Table 1

[0094]

[0095] Example 2 Establishment of a Double-Antibody Sandwich ELISA Kit and Detection Method for Human IgM Content

[0096] 1. HRP Labeling of Monoclonal Antibody 4G1

[0097] Add antibody 4G1 into a dialysis bag and dialyze it overnight at 4℃ in 0.01 M CB buffer. Take 14 mg HRP and dissolve it in 2.8 mL of water. Prepare a fresh 0.1 M NaIO 4 solution, add 0.56 mL of 0.1 M NaIO 4 to 2.8 mL of POD solution, mix well, let it stand in the dark at room temperature for 20 min, and dialyze it overnight at 4℃ in 1 M NaAc buffer. Take out the antibody and POD solution that have been dialyzed overnight, add 0.168 mL of 0.2 M carbonate buffer with pH 9.5, and then immediately add the taken-out antibody to the POD solution, and gently stir in the dark at room temperature for 2 hours. Weigh 0.04 g of NaBH 4 dissolve it in 10 mL of water, take 0.02 mL and add it to the reaction solution, mix well, place it at 4℃ for 2 hours. Take it out and put it in a dialysis bag, dialyze it in 0.01 M PBS, change the solution once after 2 hours, and dialyze it overnight at 4℃. Take out the labeled solution that has been dialyzed overnight, add an equal volume of glycerol, and store it at -20℃. The enzyme-labeled antibody is named HRP-4G1.

[0098] 2. Preparation of ELISA Plate Coated with Monoclonal Antibody 10C11

[0099] Dilute monoclonal antibody 10C11 to 8 μg / mL with 0.05 M carbonate coating buffer at pH 9.6. Add 100 μL to the reaction wells of a 96-well polystyrene reagent plate and incubate overnight at 4°C. The next day, discard the solution in the wells and wash 3 times with washing buffer, 3 minutes each time. After the above steps, block each well of the reaction plate with 2% BSA solution, 250 μ μL per well, for 2 h. Discard the solution in the wells, place in a drying room to dry and then evacuate, and store at 4°C.

[0100] 3. Establishment of double antibody sandwich ELISA method

[0101] Thirty minutes before the experiment, take out the enzyme-labeled plate coated with monoclonal antibody 10C11, allow to return to room temperature, wash the plate 3 times and shake dry. Add 100 μL of human IgM standards at different dilution concentrations. The dilution concentrations of human IgM are 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, and set a blank control. After sealing the plate, incubate at room temperature for 2 h, wash the plate 4 times and shake dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate and incubate at room temperature for 45 min, wash the plate 5 times and shake dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and develop color in the dark at room temperature for 15 min, add 50 μL of stop solution (2 M sulfuric acid solution), and immediately measure the OD value at 450 nm wavelength with an enzyme-linked immunosorbent assay reader (within 5 minutes). Plot a standard curve with different concentrations of human IgM standards as the abscissa and the corresponding OD values as the ordinate, and establish a regression equation. The results show that the detection range is 7.8125 - 500 ng / mL, and R 2 is 0.99992 ( Figure 3 )

[0102] 4. Detection of the sensitivity of double antibody sandwich ELISA

[0103] Detect the average value of the OD of 20 zero standard concentrations plus two standard deviations, and calculate the corresponding detectable concentration. The results show (Table 2) that the sensitivity is 2.29 ng / mL.

[0104] Table 2

[0105]

[0106] 5. Specificity detection of double antibody sandwich ELISA

[0107] 30 min before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, restore it to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human IgM standards or high-concentration human IgM structural analog proteins with different dilution factors, including porcine IgG, murine IgG, bovine IgG, rabbit IgG, duck IgG, goose IgG, chicken IgY, human IgG, human IgG4, human IgA, human HSA, etc. After sealing the plate, incubate it in an incubator at room temperature for 2 h, wash the plate 4 times and spin dry. Add 100 μL of the working solution of enzyme-labeled antibody HRP-4G1 to the reaction wells, seal the plate and incubate it in an incubator at room temperature for 45 min, wash the plate 5 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and develop color in the dark at room temperature for 15 min, add 50 μL of stop solution (2 M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450 nm with an ELISA reader (within 5 minutes). The results showed (Table 3) that the antibody pair composed of monoclonal antibodies 10C11 and 4G1 did not react with other similar proteins.

[0108] Table 3

[0109]

[0110] 6. Stability Detection of Double Antibody Sandwich ELISA Kit

[0111] The stability of the double antibody sandwich ELISA kit containing the antibody pair of monoclonal antibodies 10C11 and 4G1 was investigated by an accelerated experiment at 37 °C. The specific method is as follows:

[0112] The enzyme-labeled plate coated with monoclonal antibody 10C11, HRP-labeled monoclonal antibody 4G1 (HRP-4G1), and human IgM standard were placed at 37 °C for an accelerated stability test for 11 days (equivalent to about 16.5 months at 4 °C). Then, they were taken out for detection. The detection method was as follows: the enzyme-labeled plate was taken out, washed 3 times and patted dry. 100 μL of standard products with different dilution concentrations, namely 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, were added, and a blank control was set. After sealing the plate, it was incubated at room temperature for 2 h, washed 4 times and spun dry. 100 μL of the working solution of enzyme-labeled antibody HRP-4G1 was added to the reaction wells. After sealing the plate, it was incubated at room temperature for 45 min, washed 5 times and spun dry. 100 μL of chromogenic substrate TMB was added to the reaction wells. After sealing the plate, it was developed color at room temperature in the dark for 15 min. 50 μL of the termination solution (2 M sulfuric acid solution) was added, and the OD value was immediately measured at a wavelength of 450 nm with an enzyme-labeled instrument (within 5 minutes). A standard curve was plotted with different concentrations of human IgM standard as the abscissa and the corresponding OD values as the ordinate, and a regression equation was established. The results showed (Table 4) that on the 11th day, the OD values of the standard curve changed little and the OD value gradient of the standard curve was good.

[0113] Table 4

[0114]

[0115] Example 3 Determination of Human Serum IgM Content

[0116] 30 minutes before the experiment, take out the enzyme-labeled plate coated with monoclonal antibody 10C11, restore it to room temperature, wash the plate 3 times and centrifuge to dry. Select 16 serum samples from volunteer blood donors (numbered 1, 2, 3, ……, 16), add 100 μL of human serum samples / standards with different dilution factors. The dilution factor of the sample is 10,000 times, and the dilution concentrations of human IgM standards are 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, and set a blank control. After sealing the plate, incubate it in an incubator at room temperature for 2 h, wash the plate 4 times and centrifuge to dry. Add 100 μL of the enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate and incubate it in an incubator at room temperature for 45 min, wash the plate 5 times and centrifuge to dry. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate and develop color in the dark at room temperature for 15 min, add 50 μL of the termination solution (2 M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450 nm with an enzyme-labeled instrument (within 5 minutes). According to the literature reports, the content of IgM in normal human serum is 0.5 - 2 mg / mL (the IgM content varies according to age and individual). The detection results of the present invention (Table 5) are basically consistent with the reports, indicating that the double-antibody sandwich ELISA kit and detection method of the present invention can accurately determine the content of human serum IgM.

[0117] Table 5

[0118]

[0119] Example 4 Determination of the recovery rate of added human IgM and evaluation of linearity

[0120] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, restore it to room temperature, wash the plate 3 times and centrifuge to dry. Select 3 serum samples from volunteer blood donors, 1 plasma sample from a volunteer blood donor, and 1 RPMI 1640 complete medium containing 10% fetal bovine serum supplemented with human IgM standard as samples. Add 100 μL of samples and standards with different dilution multiples to each well. The dilution concentrations of the human IgM standard are 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, and set a blank control. After sealing the plate, incubate it in an incubator at room temperature for 2 h, wash the plate 4 times and centrifuge to dry. Add 100 μL of the enzyme-labeled antibody HRP-4G1 working solution to the reaction wells, seal the plate, and incubate it in an incubator at room temperature for 45 min, wash the plate 5 times and centrifuge to dry. Add 100 μL of the chromogenic substrate TMB to the reaction wells, seal the plate, and develop color in the dark at room temperature for 15 min. Add 50 μL of the stop solution (2 M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450 nm using an ELISA reader (within 5 minutes). The test results (Tables 6 and 7) show that the recovery rate of the double-antibody sandwich ELISA detection method of the present invention is relatively high (a result is considered good within the range of 70% - 130%), and it has a good linear relationship within a certain range (a result is considered good within the range of 70% - 130%).

[0121] Table 6

[0122]

[0123] Table 7

[0124]

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements 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 anti-human IgM antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary 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.1, 2 and 3, respectively; the amino acid sequence of the complementary 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 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof according to any one of claims 1 to 3.

5. Biomaterial, characterized in that The biological material contains the nucleic acid molecule according to claim 4; The biological material is an expression cassette, a vector or a host cell.

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

7. An anti-human IgM antibody composition, characterized in that The antibody composition comprises the following antibodies (1) and (2): (1) The amino acid sequences of the complementary 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 complementary 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 complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs. 10, 11 and 12, respectively; the amino acid sequence of the complementary 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) Preparing 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 non-disease diagnosis purposes; (3) Preparation of products for early diagnosis of infectious diseases, monitoring of autoimmune diseases or monitoring of transplant rejection; (4) Vaccine immunogenicity testing; (5) Evaluation of vaccine immune efficacy; (6) Quality control of products containing human IgM.

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

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

Images