Anti-human IgM antibody or antigen binding fragment thereof and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-human IgM antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] IgM is the first immunoglobulin produced in the initial humoral immune response of an animal body. Its content 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, in which all heavy chains are the same, all light chains are the same, and the molecular weight is about 900kDa. It is the largest molecular weight of 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 as a B cell antigen receptor. Compared with IgG, IgM is produced the earliest in the body, but the duration is short, so it is not the main force of the body's anti-infection immunity. However, since it is the earliest antibody produced in the body when the body first contacts the antigenic substance, it plays a very important role in the early stage of anti-infection immunity. The detection of IgM antibodies can also be used for serological early diagnosis of epidemic diseases.

[0003] At present, the commonly used method for determining IgM is immunoturbidimetry, which uses the property that antigens and antibodies can specifically bind to each other to produce a complex of a certain size in the liquid phase, forming light refraction or absorption, and measuring the transmitted light or scattered light after refraction or absorption to infer the content of IgM. This method has the following main disadvantages: it requires a large amount of sample, and a large amount of key reagents (anti-human IgM serum) are used; large-scale detection is difficult, the operation is complicated, and specific analytical instruments are required; the experimental repeatability is poor; the complex molecules formed in the liquid are not stable enough, and they need to be large enough and sufficient to produce a more accurate reading. When the sample content is low, the accuracy will be very poor; sodium azide is also needed in the buffer system used, and the safety is poor. Similarly, the UV-spectrophotometric method for determining IgM content also has the above disadvantages.

[0004] Other IgM detection methods include immunodiffusion test, HPLC and capillary electrophoresis. The disadvantages of immunodiffusion test are that there are many factors affecting the experimental operation, long detection time, poor sensitivity, and it is suitable for roughly estimating the content in the sample; the disadvantages of HPLC and capillary electrophoresis are that they require special instruments and experienced personnel to operate, and the instruments are expensive.

[0005] In summary, there is still a need to develop antibodies and kits and methods for efficiently detecting IgM. Summary of the invention

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

[0007] Specifically, the present invention provides the technical solution described below.

[0008] In the first aspect, the present invention provides an anti-human IgM antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown as 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 as SEQ ID NO.4, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.5.

[0009] The above-mentioned antibodies or their antigen-binding fragments can specifically bind to human IgM without cross-reacting with similar proteins, and have high affinity and stability. The enzyme-linked immunosorbent assay (ELISA) kit developed based on the above-mentioned antibodies or their antigen-binding fragments can achieve human IgM detection with high sensitivity, specificity and accuracy.

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

[0011] The above 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%, 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 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.

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

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

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

[0016] According to the amino acid sequence of the antibody or its antigen-binding fragment provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding an antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the above antibody or its antigen-binding fragment are within the scope of protection 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 its antigen-binding fragment is shown as SEQ ID NO.8, and the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.9.

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

[0019] The above-mentioned expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0020] The above-mentioned vectors include but are not limited to plasmid vectors, phage vectors, virus vectors, artificial chromosome vectors and the like.

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

[0022] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or antigen-binding fragment thereof with a marker, and the marker 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 anti-human IgM antibody composition, wherein the antibody composition comprises the antibodies of (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.

[0024] 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.

[0025] 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.

[0026] The above antibody composition can be used as a paired antibody for detecting human IgM by double antibody sandwich ELISA method, and the two antibodies in the antibody composition are used as coating antibody and labeling antibody respectively. The above antibody composition is used to detect human IgM by double antibody sandwich ELISA method, which has high specificity, sensitivity and accuracy.

[0027] 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.

[0028] In a sixth aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the antibody conjugate, or the antibody composition: (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.

[0029] In (1) above, 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, such as early diagnosis of infectious diseases by detecting the content of IgM in the sample; it can also be used for non-disease diagnosis purposes, such as 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 kits.

[0030] In the above (2), the sample is preferably a product containing human IgM prepared in vitro (e.g., a drug containing human IgM, etc.). The detection for non-disease diagnosis purposes includes detecting the content of human IgM in a product containing human IgM prepared in vitro for product production, quality control, etc.

[0031] In the above (3), the product includes a detection reagent or a kit, and the product performs early diagnosis of infectious diseases, monitoring of autoimmune diseases or monitoring of transplant rejection reactions by detecting the content of human IgM.

[0032] 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.

[0033] In the present invention, the method for detecting human IgM can be an immunological method such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, immunoturbidimetry, etc. Preferably, it is ELISA, and more preferably, it is a double antibody sandwich ELISA.

[0034] In a seventh aspect, the present invention provides a kit comprising the above-described antibody or antigen-binding fragment thereof, or the antibody conjugate, or the antibody composition.

[0035] Preferably, the kit is an enzyme-linked immunosorbent assay kit.

[0036] In some embodiments of the present invention, the kit is a double antibody sandwich ELISA detection kit.

[0037] Preferably, the kit comprises a coating antibody and a labeled antibody, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the labeled antibody are shown as 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 as SEQ ID NO.4, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.5; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the coating antibody are shown as SEQ ID NO.10, 11, and 12, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown as SEQ ID NO.13, the amino acid sequence of CDR2 is SSS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.14.

[0038] The present invention has no particular limitation on the label of the above-mentioned labeled antibody, and an exemplary label is HRP.

[0039] Preferably, the kit may also contain other reagents for ELISA detection, including but not limited to ELISA plate, human IgM standard, PBST washing solution, blocking solution, color developing solution, stop solution and the like.

[0040] In some embodiments of the present invention, the principle of detecting the content of human IgM by the enzyme-linked immunosorbent assay technology based on the double antibody sandwich method is as follows: coating an anti-human IgM monoclonal antibody on an ELISA plate; adding gradiently diluted standards and pre-diluted samples respectively, and the human IgM in the standards and samples will fully bind to the coated antibodies on the ELISA plate; adding HRP-labeled anti-human IgM antibodies after washing the plate, and the antibodies will specifically bind to the human IgM in the standards and samples captured by the coated antibodies on the ELISA plate; adding the color developer substrate TMB after washing the plate, if there are different concentrations of human IgM in the sample in the reaction well, HRP will turn the colorless TMB into different shades (positively correlated) of blue substances, and the reaction well will turn yellow after adding the stop solution; finally, measuring the absorbance (OD) of the reaction well sample at λmax=450nm (OD=450nm), the concentration of human IgM in the sample is proportional to OD, and the concentration of human IgM in the sample can be calculated through the standard curve.

[0041] In an eighth aspect, the present invention provides a method for detecting human IgM, comprising: using the antibody or its antigen-binding fragment or the antibody conjugate or the antibody composition or the kit to detect whether human IgM exists or its content in a sample to be tested.

[0042] The above detection method can be selected from immunological methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc. Preferably, it is ELISA, and more preferably, it is double antibody sandwich ELISA.

[0043] The beneficial effects of the present invention include at least: 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 pig IgG, mouse 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 with human IgM and has 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), UV-spectrophotometry (mg level), HPLC (g level), etc., the detection sensitivity (ng level) of this kit ranges from 7.8125-500ng / 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 4h), UV-spectrophotometry (about 4h), HPLC (about 4h), etc., the detection time of this kit is significantly shortened (2.75h), and high-throughput detection can be performed (compared with the detection time of 50 samples), which has good application prospects in human IgM detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 This is the SDS-PAGE test result of the monoclonal antibody 4G1 in Example 1 of the present invention.

[0046] Figure 2 This is the SDS-PAGE test result of the monoclonal antibody 10C11 in Example 1 of the present invention.

[0047] 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 DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1 Preparation of anti-human IgM monoclonal antibody 1. Animal immunization Female Balb / c mice aged 6-8 weeks were selected and immunized with naturally extracted human IgM antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks. Blood was collected after three immunizations to measure the titer, and the immunization was boosted again three days before fusion.

[0050] 2. Preparation of immune spleen cells Take a BALB / c mouse that has been boosted with immunity, bleed it from its orbits to kill it (collect serum, i.e., positive serum), and immerse it in 75% alcohol for 5-10 minutes for disinfection. Then fix it on a dissecting board for dissection, remove the spleen, cut it, and place it in a sterile homogenizer; the grinding and cell suspension preparation methods are the same as those described in SP2 / 0 below, and set aside after counting.

[0051] 3. Cell fusion The prepared syngeneic myeloma cells are mixed with mouse spleen cells in a certain ratio, and the fusion promoter polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is as follows: (1) Myeloma (SP2 / 0) cell activation Commercial SP2 / 0 cells were thawed and revived, then resuspended in nutrient solution (RPMI-1640 basal culture medium supplemented with calf serum), and cultured in an incubator at 37°C and 5% CO2; passaged after 3-5 days.

[0052] The cells were collected and suspended in RPMI-1640 basal culture medium, and 0.5-1×10 6 The cells were injected subcutaneously into the back of BALB / c mice and cultured for 9-10 days. When the back tumor volume increased to about 0.8 cm in diameter, the mice were killed by pulling the neck, and the tumors were removed by aseptic operation after soaking in 75% alcohol for 5 minutes.

[0053] The tumor pieces were cut off and placed in a sterilized homogenizer. After adding RPMI-1640 basal culture medium and grinding thoroughly, 10 mL of RPMI-1640 basal culture medium was added and allowed to stand for 2 min. The cell suspension on the upper layer was aspirated and placed in another centrifuge tube. 10 mL of RPMI-1640 basal culture medium was added and grinding was repeated twice. The cell suspension obtained above was centrifuged at 1000 r / min for 10 min to remove the supernatant, and then resuspended in 30 mL of RPMI-1640 basal culture medium.

[0054] Add 15 mL of lymphocyte separation fluid to another centrifuge tube, and carefully place the above cell suspension on the separation fluid; 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 fluid, and resuspend them in 10 mL of RPMI-1640 basal culture fluid, count and set aside.

[0055] (2) Preparation of feeder cells Take an unimmunized BALB / c mouse, bleed from the eye socket, and collect the negative serum. Inject 2-3mL RPMI-1640 basal culture medium into the mouse's peritoneal cavity, aspirate after blowing, and place in another centrifuge tube for standby use. The liquid contains peritoneal macrophages. Prepare spleen cell suspension in the same way as above, and place in the peritoneal macrophage tube. Centrifuge at 1000r / min for 10min to remove the supernatant, suspend the cells in HAT medium, and place in a 37℃, 5% CO2 incubator for standby use.

[0056] (3) Integration 1-2×10 7 SP2 / 0 and 1×10 8 Mix the immune cells in a 50mL centrifuge tube, centrifuge at 1000r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 0.8mL of 50% PEG (Sigma) preheated to 37℃, stir and let stand for 30 s. After standing, add 10mL of RPMI-1640 basal culture medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, seed in a 96-well culture plate, 250μL / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HT medium and continue to culture. When the fused cell colony grows to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0057] 4. Screening of hybridoma positive clones and cell cloning The purpose of selective culture is to screen fused hybridoma cells, using HAT selective culture medium. In HAT culture medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT culture medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells that can proliferate indefinitely. The specific operation is as follows: (1) Screening of positive hybridoma cells using indirect ELISA Coating with known antigens: dilute the purified coating antigen to 1-10 μg / mL with coating buffer; add 100 μL to each microwell, shake gently, and incubate at 4°C overnight or at 37°C for 1 hour; shake off the liquid in the well (tap dry as much as possible); wash 3 times, 2-3 minutes each time.

[0058] Block the areas in the enzyme-labeled wells that are not coated with antigens: add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each microwell, shake gently, and incubate at 37°C for 1 hour; shake off the liquid in the wells; fill each well with washing buffer, let it stand for 2-3 minutes, shake off the liquid in the wells, pat dry, and wash three times with washing buffer using this method.

[0059] Sample addition: Take 50 μL of supernatant of the hybridoma cells to be tested and add them to the enzyme-labeled wells in order, shake gently, incubate at 37°C for 1 hour, wash, and pat dry.

[0060] Add enzyme-labeled anti-antibody: first dilute the enzyme-labeled secondary antibody to an appropriate working concentration using diluent according to the instructions, add 100 μL to each well, shake gently, and incubate at 37°C for 1 hour; then wash and pat dry.

[0061] Add color developing solution: Add 100 μL of freshly prepared color developing solution to each well, shake gently, and incubate at 37°C for 10 min.

[0062] Stop reaction: add 50 μL of stop solution to each well.

[0063] Determination result: OD of microplate reader 450nm The reading was performed at 3 times that of the negative well, which was considered positive.

[0064] (2) Cloning of hybridoma cells (limiting dilution method) Prepare a mouse feeder cell layer before cloning; gently blow the hybridoma cells to be cloned from the culture well and count the number of live cells using a hemocytometer; dilute the cells to 5, 10, or 30 cells / mL with complete culture medium.

[0065] Add the three concentrations of cell suspension to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 0.5, 1 and 3 cells, respectively. Add one drop of liquid on the 4th day of culture, and carefully observe the growth of cells in each well on the 5th-6th day and record it.

[0066] (3) Detection of specific antibodies: On the 7th to 9th day after cloning, when the cell clones have grown to cover 1 / 3 to 1 / 2 of the field of view, they can be detected; the cells in the positive wells can be moved to a 24-well culture plate. When the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0067] 5. Variable region sequence determination of monoclonal antibodies 4G1 and 10C11 The number of hybridoma cells collected was greater than 10 6 , and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The gene sequencing results were obtained: the light chain variable region sequence of monoclonal antibody 4G1 was 321 bp long, encoding 107 amino acids, the DNA sequence was shown in SEQ ID NO.9, and the protein sequence was shown in SEQ ID NO.7; the heavy chain variable region sequence was 363 bp long, encoding 121 amino acids, the DNA sequence was shown in SEQ ID NO.8, and the protein sequence was shown in SEQ ID NO.6. The light chain variable region sequence of monoclonal antibody 10C11 was 321 bp long, encoding 107 amino acids, the DNA sequence was shown in SEQ ID NO.18, and the protein sequence was shown in SEQ ID NO.16; the heavy chain variable region sequence was 336 bp long, encoding 112 amino acids, the DNA sequence was shown in SEQ ID NO.17, and the protein sequence was shown in SEQ ID NO.15. After analysis, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 4G1 are shown as SEQ ID NOs.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.5; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 10C11 are shown as 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 as SEQ ID NO.13, the amino acid sequence of CDR2 is SSS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.14.

[0068] 6. Large-scale preparation of monoclonal antibodies 4G1 and 10C11 The hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected after 7 days. The antibodies were purified by protein affinity chromatography. The SDS-PAGE test results of the purified monoclonal antibodies 4G1 and 10C11 were as follows: Figure 1 and Figure 2 shown.

[0069] 7. Determination of relative affinity constants of monoclonal antibodies 4G1 and 10C11 Human IgM antigen was coated on the ELISA plate and blocked. After washing the plate with PBST, monoclonal antibodies 4G1 and 10C11 were diluted to saturation concentration and added to the ELISA plate, 100 μL / well, and incubated at room temperature for 2 hours. After washing the plate with PBST, 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 Mol / L NaSCN solution was added in sequence at 60 μL / well, and incubated at room temperature for 15 minutes. After washing the plate with PBST, HRP-labeled goat anti-mouse IgG was added, and the plate was incubated at room temperature for 45 minutes for color development detection. The sodium thiocyanate concentration corresponding to the OD value at 450nm after elution dropped to 50% of that without elution is the relative affinity constant of the antibody, expressed in M ​​(mol / L). The results showed (Table 1) that the relative affinity constants of monoclonal antibodies 4G1 and 10C11 were both greater than 2.5 M, indicating good affinity.

[0070] Table 1

[0071] Example 2 Establishment of a double antibody sandwich ELISA kit and detection method for human IgM content 1. HRP labeling of monoclonal antibody 4G1 Add antibody 4G1 to the dialysis bag and immerse it in 0.01M CB buffer at 4℃ for overnight dialysis. Take 14mg hRP and dissolve it in 2.8mL water. Prepare a fresh 0.1M NaIO4 solution, add 0.56mL 0.1M NaIO4 to 2.8mL POD solution, mix well, stand at room temperature and avoid light for 20min, immerse in 1MM NaAc buffer and dialyze at 4℃ overnight. Take out the antibody and POD solution that have been dialyzed overnight and add 0.168mL 0.2M pH 9.5 carbonate buffer, then immediately add the antibody that has been taken out to the POD solution, and stir gently at room temperature and avoid light for 2 hours. Weigh 0.04g NaBH4 and dissolve it in 10mL water, take 0.02mL and add it to the reaction solution, mix well, and place it at 4℃ for 2 hours. Take it out and put it in the dialysis bag, dialyze it in 0.01M PBS, change the solution once after two hours, and dialyze it at 4℃ overnight. Take out the labeling solution dialyzed overnight, add an equal volume of glycerol, and store at -20°C. The enzyme-labeled antibody is named HRP-4G1.

[0072] 2. Preparation of monoclonal antibody 10C11 ELISA plate Dilute monoclonal antibody 10C11 to 8 μg / mL with 0.05M pH 9.6 carbonate coating buffer. 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 three times with washing buffer for 3 minutes each. After the above steps, block each well of the reaction plate with 2% BSA solution, 250 μL per well. μ L, 2h. Discard the solution in the wells, place in a dry room to dry, evacuate and store at 4℃.

[0073] 3. Establishment of double antibody sandwich ELISA method 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human IgM standard with different dilution concentrations, the human IgM dilution concentrations 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 up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction well, seal the plate, incubate at room temperature for 45 minutes, wash the plate 5 times and spin dry. Add 100μL of chromogenic substrate TMB to the reaction wells, seal the plate and color at room temperature in the dark for 15 minutes, add 50μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450nm wavelength using an ELISA reader (within 5 minutes). Draw a standard curve with different concentrations of human IgM standards as the horizontal axis and the corresponding OD value as the vertical axis, and establish a regression equation. The results show that its detection range is 7.8125-500ng / mL, R 2 is 0.99992 ( Figure 3 ).

[0074] 4. Double antibody sandwich ELISA sensitivity test The average value of the OD of 20 zero standard concentrations was added with two standard deviations to calculate the corresponding detectable concentration. The results showed (Table 2) that the sensitivity was 2.29 ng / mL.

[0075] Table 2

[0076] 5. Double antibody sandwich ELISA specific detection 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human IgM standard or high concentration of human IgM structural similar proteins with different dilutions, including pig IgG, mouse 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 in a room temperature incubator for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction well, incubate in a room temperature incubator for 45 minutes after sealing the plate, wash the plate 5 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 15 minutes after sealing the plate, add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450nm with an ELISA reader (within 5 minutes). The results showed (Table 3) that the antibody pair consisting of monoclonal antibodies 10C11 and 4G1 did not react with other similar proteins.

[0077] Table 3

[0078] 6. Stability test of double antibody sandwich ELISA kit The stability of the double antibody sandwich ELISA kit containing the monoclonal antibody 10C11 and 4G1 antibody pair was investigated by 37°C accelerated experiment. The specific method is as follows: 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 test and placed for 11 days (equivalent to about 16.5 months at 4°C). Then take out the test. The test method is to take out the ELISA plate, wash the plate 3 times and pat dry. Add 100μL of standards with different dilution concentrations, with concentrations of 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.625ng / mL, 7.8125ng / mL, and set up a blank control. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and spin dry. Add 100μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction well, seal the plate, incubate at room temperature for 45min, wash the plate 5 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 minutes). Draw a standard curve with different concentrations of human IgM standards as the horizontal axis and the corresponding OD values ​​as the vertical axis to establish a regression equation. The results show (Table 4) that on the 11th day, the OD value of the standard curve did not change much, and the OD value gradient of the standard curve was good.

[0079] Table 4

[0080] Example 3 Determination of human serum IgM content 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, return to room temperature, wash the plate 3 times and spin dry. Select 16 sera from volunteer blood donors (numbered 1, 2, 3, ..., 16), add 100μL of human serum samples / standards with different dilution multiples, the sample dilution multiple is 10,000 times, the dilution concentration of human IgM standard is 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.625ng / mL, 7.8125ng / mL, and set up a blank control. After sealing the plate, incubate it in a room temperature incubator for 2h, wash the plate 4 times and spin dry. Add 100μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction well, seal the plate, incubate it in a room temperature incubator for 45min, wash the plate 5 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450 nm using an ELISA reader (within 5 minutes). According to literature reports, the normal human serum IgM content is 0.5~2 mg / mL (depending on age and individual, the IgM content is different), and the test results of the present invention (Table 5) are basically consistent with the report, indicating that the double antibody sandwich ELISA kit and detection method of the present invention can accurately measure the content of human serum IgM.

[0081] Table 5

[0082] Example 4 Human IgM spike recovery determination and linearity assessment 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 10C11, return to room temperature, wash the plate 3 times and spin dry. Select 3 voluntary blood donor sera, 1 voluntary blood donor plasma and 1 RPMI 1640 complete medium containing 10% fetal bovine serum with human IgM standard as samples, add 100 μL of samples and standards with different dilution multiples to each well, and the dilution concentrations of human IgM standard are 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.625ng / mL, 7.8125ng / mL, and set up a blank control. After sealing the plate, incubate in a room temperature incubator for 2h, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4G1 working solution to the reaction well, incubate in a room temperature incubator for 45min after sealing the plate, wash the plate 5 times and spin dry. 100 μL of chromogenic substrate TMB was added to the reaction wells, the plate was sealed and color was developed at room temperature in the dark for 15 min, 50 μL of stop solution (2M sulfuric acid solution) was added, and the OD value was immediately measured 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 high (the range of 70%-130% indicates a good result), and has a good linear relationship within a certain range (the range of 70%-130% indicates a good result).

[0083] Table 6

[0084] Table 7

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 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.

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, Fv or a single-chain antibody.

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