An antibody against human IgM and its preparation method and application

Abstract

The application provides an antibody against human IgM, a preparation method and application thereof, and relates to the field of antibody detection. Specifically, the antibody provided by the application has HCDR1-3 as shown in SEQ ID NO: 1, 2 and 3 respectively, and LCDR1-3 as shown in SEQ ID NO: 4, 5 and 6 respectively. The antibody provided can specifically recognize human IgM, and the positive coincidence rate and negative coincidence rate and total coincidence rate of a detection reagent constructed using the antibody and a positive control reagent are all 100%.

Description

Technical Field

[0001] This invention belongs to the field of antibody detection technology, and specifically relates to an anti-human IgM antibody, its preparation method, and its application. Background Technology

[0002] Immunoglobulin M (IgM) is the first type of antibody produced in the body's humoral immune response, appearing rapidly in the early stages of infection or after initial antigen stimulation. Therefore, detecting serum IgM antibody levels has become an important serological indicator for the early diagnosis of various acute infectious diseases (such as viral hepatitis, EBV, and cytomegalovirus infection). Anti-human IgM monoclonal antibodies can specifically recognize IgM molecules and are widely used as core detection reagents in in vitro diagnostic platforms such as enzyme-linked immunosorbent assays (ELISA), chemiluminescent immunoassays, and immunochromatographic test strips.

[0003] Monoclonal antibodies targeting human IgM can be divided into two categories based on their epitopes: one type recognizes the entire IgM molecule (including multiple epitopes in the light chain and μ chain constant region), while the other type specifically recognizes the IgM-specific μ chain (especially the CH3 or CH4 domains). In practical applications, the former is prone to non-specific binding with high concentrations of IgG and IgA in serum or interference from rheumatoid factor, leading to false positives or elevated background. The latter, because it binds only to the characteristic IgM μ chain, has a significantly reduced risk of cross-reactivity and is more suitable as a labeling secondary antibody or capture antibody for IgM antibody detection.

[0004] However, although most commercially available anti-human IgM μ-chain monoclonal antibodies are produced using hybridoma technology, they generally suffer from the following technical defects: First, some hybridoma cell lines secrete antibodies with insufficient affinity, resulting in limited detection sensitivity and making it difficult to meet the detection requirements of low-concentration IgM antibodies (such as samples from the early infection window period); Second, the antibody specificity is not ideal, exhibiting varying degrees of cross-reactivity with denatured IgM or other immunoglobulin degradation fragments, affecting the accuracy of detection results; Third, the production process lacks systematic stability optimization, resulting in significant fluctuations in antibody titer and purity between different batches, and a short shelf life under liquid storage conditions (usually only 12-18 months), increasing the difficulty of diagnostic reagent production and quality control; Fourth, high-quality hybridoma cell lines targeting μ-chain epitopes are scarce, and existing cell lines are at risk of decreased or even lost antibody secretion capacity during long-term passage, making it difficult to achieve a stable, large-scale, and low-cost supply.

[0005] Therefore, developing an anti-human IgM monoclonal antibody based on hybridoma technology that specifically recognizes the IgM μ chain, and possesses high affinity, high purity, excellent thermal stability, and long shelf life, and establishing a stable and controllable production process, is of significant clinical importance and market value for improving the performance of early diagnostic reagents for infectious diseases. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-human IgM antibody, its preparation method, and its application.

[0007] On the one hand, this application provides a binding molecule that resists human IgM.

[0008] In some embodiments, the binding molecule is an antibody against human IgM or an antigen-binding fragment thereof.

[0009] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region.

[0010] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:10; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:7. In some specific embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:10; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:7.

[0011] In some specific implementations, the CDR shown is defined according to the numbering system of Kabat, IMGT, Chothia, AbM, or Contact.

[0012] In some specific implementations, the CDR shown is defined according to Kabat's numbering system.

[0013] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:1, 2, and 3, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6; in some specific embodiments, HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively.

[0014] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

[0015] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:10, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:7; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:10, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7; in some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0016] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a recombinant antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0017] In some embodiments, the antibody or its antigen-binding fragment further includes an immunoglobulin Fc region. In some specific embodiments, the Fc is selected from IgG1, IgG2, IgG3, or IgG4.

[0018] In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 95% identity with it, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 95% identity with it. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:12, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:9. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:12, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9.

[0019] The present invention also provides a biomaterial.

[0020] In some embodiments, the biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any of the places above; (2) Expression vectors / vector groups containing any of the nucleic acid molecules described above; (3) A host cell containing any of the nucleic acid molecules or expression vectors / vector groups described above.

[0021] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in any of the above claims.

[0022] In some embodiments, the method includes culturing host cells as described in any of the preceding embodiments. In some specific embodiments, the cultured host cells express an antibody or an antigen-binding fragment thereof. In some specific embodiments, the method further includes isolating the antibody or the antigen-binding fragment thereof. In some specific embodiments, the method further includes purifying the antibody or the antigen-binding fragment thereof.

[0023] The present invention also provides a composition for resisting human IgM.

[0024] In some embodiments, the composition comprises the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.

[0025] The present invention also provides the use of an antibody or antigen-binding fragment thereof as described in any of the preceding claims, or a composition as described in any of the preceding claims.

[0026] In some implementations, the application includes: for preparing products for the preparation of anti-human IgM, or for detecting human IgM not for the purpose of disease diagnosis and treatment; said products are detection kits, test strips, or detection chips.

[0027] In some specific implementations, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip. Attached Figure Description

[0028] Figure 1 The molecular sieve separation spectrum is shown.

[0029] Figure 2 The electrophoresis diagram of the sample separated by molecular sieve is shown. In the diagram, M corresponds to the protein marker; lanes 1-4 correspond to the Protein L elution fraction, molecular sieve peak 1, molecular sieve peak 2, and molecular sieve peak 3, respectively.

[0030] Figure 3 The image shows a reducing SDS-PAGE electrophoresis result of the IgM 3D9 antibody. M corresponds to the protein marker; 1 corresponds to the IgM 3D9 antibody. Detailed Implementation

[0031] The present invention will now be described in detail with reference to embodiments, but the embodiments provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0033] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Nevertheless, this invention still intends to provide a more detailed description and explanation of these terms and phrases. In the event of any inconsistency between the terms and phrases mentioned and their known meanings, the meanings expressed in this invention shall prevail.

[0034] Example 1: Preparation of naturally extracted IgM antigen (1) Sample pretreatment Take 200ml of human serum, centrifuge at 10000 rpm at 4℃ for 10min, and filter through a 0.45um filter membrane.

[0035] (2) Ammonium sulfate precipitation Add saturated ammonium sulfate at a volume ratio of 1:1 (serum to saturated ammonium sulfate), stir at 4°C for 30 min, centrifuge at 10,000 rpm at 4°C for 30 min, dissolve the precipitate in 20 mM PBS solution at pH 7.4, filter and set aside.

[0036] (3) Gel filtration chromatography Pack 20 ml of Qianchun Protein L packing material into a column 15 cm high. Equilibrate with PBS. Load the reconstituted protein solution from step 2 onto the L column at a flow rate of 1 ml / min. After loading, wash with PBS and then elute with 0.1 M glycine at pH 3.0. Immediately after elution, dialyze into 1X PBS and change the solution twice.

[0037] (4) Molecular sieve separation After collecting the antibody from step 3, concentrate it to 5 ml. Equilibrate the Purose 6 Fast Flow molecular sieve column (16 / 60) with PBS. Separate the concentrated antibody using a molecular sieve at a flow rate of 1 ml / min, collecting a portion. The separation pattern is shown below. Figure 1 As shown in the figure.

[0038] (5) Sample collection The three peaks received in step 4 are combined and concentrated, then subjected to gel electrophoresis. For example... Figure 2 The electrophoresis diagram shown indicates that peak 1 is the target protein peak based on the peak elution time and the molecular weight of the heavy and light chains. Peak 1 was then preserved with sodium azide and analyzed using OD. 280 / 1.2 Quantitative analysis was performed, and the sample was stored at -20℃.

[0039] Example 2: Preparation of mouse monoclonal antibody against human IgM antigen 2.1 Animal Immunization Five female Balb / c mice aged 6-8 weeks were used. The purified IgM antigen was used as an immunogen and mixed with Freund's complete adjuvant at a dose of 50 μg per mouse. The mixture was emulsified to a water-in-oil state using a double-syringe method. The mice were then immunized for the first time by subcutaneous injection at multiple points along the spine on the back, bilaterally in the groin, and bilaterally in the axilla, at an injection volume of 200 μL per mouse. On day 14 after the first immunization, the same dose of the IgM antigen was emulsified with Freund's incomplete adjuvant and administered to the mice via the same injection dose and route as the first immunization. On day 15 after the second immunization, the same dose of the IgM antigen was emulsified with Freund's incomplete adjuvant and administered to the mice via intraperitoneal injection at the same injection dose as the first immunization. On day 7 after the third immunization, blood was collected from the immunized mice by tail amputation, and serum was separated. The titer of anti-human IgM antibodies in the serum was detected by ELISA.

[0040] A method for detecting the titer of anti-human IgM antibodies in serum using an indirect ELISA method includes the following steps: First, an ELISA plate is coated with human IgM antigen at a concentration of 5 μg / mL and incubated overnight at 4°C. After washing, PBST blocking buffer containing 5% skim milk powder is added and the plate is blocked at 37°C for 2 hours. Then, the serum sample to be tested is serially diluted from 1:100, and each dilution is added to the coated wells and incubated at 37°C for 30 minutes. After thorough washing with PBST, horseradish peroxidase-labeled goat anti-mouse IgG (1:20000 dilution) is added and the plate is incubated at 37°C for 30 minutes. After washing again, TMB substrate solution is added and the plate is developed in the dark at 37°C for 15 minutes. The reaction is then terminated by adding stop solution, and the OD value of each well is measured at dual wavelengths of 450 nm and 630 nm. The mean absorbance of the negative control wells was multiplied by 2.1 as the cutoff value. The reciprocal of the highest serum dilution factor with an absorbance value greater than or equal to the cutoff value was determined as the anti-human IgM antibody titer of the serum sample.

[0041] The test results are shown in Table 1. A titer of 1:100,000 or higher was used as the criterion for qualification. Immunized mice with a serum titer of 1:100,000 or higher were selected. Three days before cell fusion, the IgM antigen was dissolved in sterile phosphate buffer at a dose of 100 μg per mouse without adjuvant and administered intraperitoneally to boost the immunization of the mice in preparation for cell fusion.

[0042] Table 1. Results of valence determination

[0043] 2.2 Construction of B cells (1) Preparation of myeloma cells In this embodiment, the SP2 / 0 myeloma cell line was used and adaptively passaged before fusion to bring it into the logarithmic growth phase. On the day of fusion, the cells were gently blown off the wall of the flask with a bent dropper and collected in a 50 mL centrifuge tube. The tube was centrifuged at 1000 r / min for 5-10 min. The supernatant was discarded, and the pellet was resuspended in 30 mL of culture medium and centrifuged and washed once using the same method. After discarding the supernatant, the pellet was resuspended in 20 mL of culture medium for later use.

[0044] (2) Preparation of spleen cells Immunized BALB / c mice were enucleated, and blood was collected. Serum was separated and used as a positive control. The mice were then euthanized by cervical dislocation and immersed in 75% alcohol for 5 min. The spleen was aseptically removed in a laminar flow hood and washed in a petri dish containing 10 mL of culture medium to remove surrounding connective tissue. The spleen was punctured with a sterile syringe needle and repeatedly perfused until the spleen tissue turned white. The spleen cell suspension in the petri dish was collected into a 50 mL centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, and the precipitate was resuspended in 10 mL of culture medium. A small amount was diluted and counted.

[0045] (3) Cell fusion and cloning The spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1 and fused using polyethylene glycol (PEG) as a fusion agent. After fusion, the cells were cultured in HAT selective medium. Ten days later, the hybridoma cell supernatant was screened by ELISA. Positive clones were cloned using the limiting dilution method. After five rounds of screening, 15 stable positive hybridoma cell lines secreting specific antibodies were finally obtained.

[0046] (4) Screening of hybridoma cells (ELISA method) IgM antigen was coated at 5 μg / mL, 50 μL / well in a 96-well ELISA plate and incubated overnight at 4°C. The next day, the liquid in the wells was discarded, the plates were washed three times, and the plates were blotted dry. 100 μL / well blocking buffer was added, and the plates were blocked at 37°C for 2 h, washed twice, and blotted dry. 100 μL of the supernatant of the hybridoma cells to be tested (15 strains in total) was added to each well, along with positive, negative, and blank controls. The plates were incubated at 37°C for 30 min, washed four times, and blotted dry. 100 μL / well of horseradish peroxidase-labeled goat anti-mouse IgG (1:20000 dilution) was added, and the plates were incubated at 37°C for 30 min, washed four times, and blotted dry. 100 μL of TMB substrate chromogenic solution was added to each well, and the plates were incubated at room temperature for 15 min. The reaction was terminated by adding 50 μL / well of 2 mol / L dilute hydrochloric acid. The OD values ​​of each well were measured at dual wavelengths of 450 nm and 630 nm.

[0047] The results are shown in Table 2. The OD value of the blank wells was <0.02, and the OD value of the negative wells was <0.1. The positive wells were clearly distinguishable from the negative controls, indicating that the obtained hybridoma cells can secrete antibodies that specifically recognize IgM antigens. Using the same method, the serum titer of mouse ocular blood reached over 1:100,000, meeting the fusion requirements.

[0048] Table 2. ELISA test results

[0049] 2.3 Production and purification of monoclonal antibodies From the aforementioned 15 positive hybridoma cell lines, one with strong antibody secretion capacity was selected for ascites fluid preparation. Five Balb / c mice weighing approximately 25 g were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin; two weeks later, they were further sensitized with 1×10⁻⁶... 6 Hybridoma cells were intraperitoneally inoculated into mice at a dose of 1 per mouse. After the abdomen of the mice became significantly distended, ascites fluid was collected, centrifuged at 10,000 rpm for 10 min to remove cells and precipitates, and the supernatant was collected and stored at -20℃ for later use.

[0050] For purification, the frozen ascites fluid was thawed and centrifuged. The supernatant was filtered through a 0.22 μm filter membrane and purified using a Protein A affinity chromatography column, following the manufacturer's instructions. The eluted antibody fraction was collected and dialyzed against 20 mM PBS (pH 7.4) for 72 h. After dialyzing, the fraction was sterilely filtered through a 0.22 μm filter membrane, aliquoted, and stored at -20°C for subsequent identification.

[0051] 2.4 Characterization of Monoclonal Antibodies Antibody concentration determination: The absorbance of the purified antibody at wavelengths of 280 nm and 260 nm was measured using ultraviolet spectrophotometry. 280 A 260 According to the formula: Protein content (mg / mL) = (A 280 Calculate the antibody concentration using (× dilution factor) / 1.35.

[0052] Antibody molecular mass identification results as follows Figure 3 As shown: The molecular weight of the purified antibody was determined by SDS-PAGE, with the heavy chain at approximately 55 kD and the light chain at approximately 25 kD.

[0053] The purified ascites monoclonal antibody was measured by indirect ELISA. The ELISA titer results are shown in Table 3. The purified titer was greater than 1:100000.

[0054] Table 3. Results of ELISA titer assay

[0055] Example 3: Application of anti-human IgM 3D9 antibody in chromatographic test strips for Mycoplasma pneumoniae IgM detection (1) Preparation of coating membrane Lung bronchitis antigen and goat anti-mouse IgG were diluted to 1 mg / mL with 10 mM phosphate buffer (pH 7.4), and then streaked onto the T and C lines of a nitrocellulose membrane at a volume of 1 μL / cm. The membranes were then dried in a 37°C oven for 20 hours before use.

[0056] (2) Preparation of colloidal gold Add 1 ml of 2% HAuCl4 to 100 ml of purified water, heat to boiling, then add 1 ml of 2% trisodium citrate, heat until the color turns wine red, and continue heating for 10 minutes. After natural cooling, measure the maximum absorption wavelength and OD value at 400-600 nm using a spectrophotometer.

[0057] (3) Anti-human IgM 3D9 colloidal gold labeling: Take 60 ml of colloidal gold solution (λ=530±5 nm, OD=2), adjust the pH to 8.0 with 0.1 M K2CO3; after thorough mixing, add 600 μg of anti-human IgM 3D9 and react for 45 min; then add 600 μl of 10% BSA solution to block for 30 min; centrifuge at 9000 rpm at 4℃ for 30 min, take the supernatant, redissolve the precipitate with 3 ml of gold redissolving solution, and store at 2-8℃ in the dark for later use.

[0058] (4) Preparation of anti-human IgM 3D9 gold-labeled pad: Take 3 ml of the gold marker prepared above, concentrate it 20 times, and spray it onto the treated conjugate pad at a rate of 2 μl / cm using an XYZ three-dimensional gold spraying instrument. Dry it at 37°C for 24 h. Then cut it into single strips using a strip cutter, seal and dry it for later use.

[0059] (5) Reagent performance testing After combining the prepared pulmonary bronchial MP coating membrane and the anti-human IgM 3D9 gold-labeled pad, and attaching the sample pad, the strips were cut into 3mm test strips. The test strips were then used to test the company's reference material and 503 clinical samples collected from the hospital using the YHLO MP-IgM chemiluminescence reagent.

[0060] (6) The test results for the enterprise reference product are as follows: 1) Negative compliance rate: The compliance rate (- / -) of 10 negative reference samples N1 to N10 from the enterprise is 10 / 10, which meets the requirements; 2) Positive compliance rate: The compliance rate (+ / +) of the 5 positive national reference samples P1 to P5 is 5 / 5, which meets the requirements; 3) Precision: The precision reference sample was repeatedly tested 10 times, and all results were positive with uniform color development, meeting the requirements; 4) Minimum detection limit: For the detection sensitivity reference sample, L1 and L2 are positive, and L3 is negative, which meets the requirements.

[0061] (7) The results of the clinical sample test are shown in Table 4 below. The results show that the consistency rate of the Mycoplasma pneumoniae IgM detection reagent made with anti-human IgM 3D9 with the control reagent is 100%, which indicates that the consistency is good.

[0062] Table 4. Immunoassay results of test reagent and control reagent ; Example 4: Application of IgM-3D9 antibody in chemiluminescent reagent for Mycoplasma pneumoniae IgM 1. Preparation of monoclonal antibodies coated with magnetic microparticles: Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 37°C, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with Mycoplasma pneumoniae antigen at a ratio of 10 μg of antigen to 1 mg of magnetic microparticles. The conjugation process was carried out at 25°C for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25°C for 2 h and then stored at 2-8°C for later use.

[0063] 2. Preparation of alkaline phosphatase-labeled monoclonal antibodies (1) Take 1.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing 0.15 M sodium chloride, and add 0.04 mg TR to it. React at 37 °C for 1 h.

[0064] (2) Take 1 mg of IgM-3D9 antibody, adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC, and react at 37℃ for 1 h.

[0065] (3) Add the alkaline phosphatase that has completed the reaction to the SMCC-treated IgM-3D9 antibody, mix them evenly at a mass ratio of 1:1, and react at 25°C for 1 hour. After the reaction is complete, add 0.1 mL of phosphate buffer containing 0.15 M sodium chloride, 2% cysteine, and pH 7.4, let it stand for 1 hour, then add 1 mL of glycerol and store at -20°C for later use.

[0066] 3. Reagent performance testing The magnetic microparticles coated with the antigen were diluted 20 times with phosphate buffer containing 0.15M sodium chloride, and the alkaline phosphatase-labeled IgM-3D9 antibody was diluted 1000 times with phosphate buffer containing 0.15M sodium chloride before being used for testing. The test samples were 491 clinical samples tested using the Mycoplasma pneumoniae IgM detection kit from Shenzhen Yahuilong Biotechnology Co., Ltd.

[0067] The test results are shown in Table 5 below. The positive concordance rate, negative concordance rate, and total concordance rate of the reagent described in this invention and the YHLO control reagent are all 100%. All indicators show that the results of the "test reagent" and the "YHLO reagent" are completely consistent with each other, with no difference. Kappa=1, which is perfect consistency. Moreover, the signal value of the test reagent is 30% higher than that of the YHLO reagent, and the CV is also significantly better than that of YHLO, which is more conducive to detection and can fully meet the needs of clinical testing.

[0068] Table 5. Comparison of test results between the test reagent and YHLO. ; The relevant sequences of this invention (HCDR1-3 and LCDR1-3 are defined according to the kabat numbering convention) are as follows: 3D9-HCDR1 NSWIN SEQ ID NO:1 3D9-HCDR2 RIYPGDGDSNYNGKFKG SEQ ID NO:2 3D9-HCDR3 SGGLGKGDFDY SEQ ID NO:3 3D9-LCDR1 TANSSVSSSYLH SEQ ID NO:4 3D9-LCDR2 STSNLAS SEQ ID NO:5 3D9-LCDR3 HQYHRSPWT SEQ ID NO:6 Heavy chain variable region QVQLQQSGPELVKPGASVKISCKTSGYDFSNSWINWVKQRPGQGLEWIGRIYPGDGDSNYNGKFKGKATLTADFSSSTAYMQLSSLTSVDSAVYFCARSGGLGKGDFDYWGQGTTLTVSS SEQ ID NO:7 >Heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:8 >Heavy chain QVQLQQSGPELVKPGASVKISCKTSGYDFSNSWINWVKQRPGQGLEWIGRIYPGDGDSNYNGKFKGKATLTADFSSSTAYMQLSSLTSVDSAVYFCARSGGLGKGDFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:9 Light chain variable region DIQMTQSPAIMSASLGERVTMTCTANSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTITSMEAEDAATYYCHQYHRSPWTFGGGTKLEIK SEQ ID NO:10 Light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:11 Light chain full length DIQMTQSPAIMSASLGERVTMTCTANSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTITSMEAEDAATYYCHQYHRSPWTFGGGTKLEI KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:12 The specific embodiments are merely illustrative of the present invention and are not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An antibody against human IgM or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown below: The light chain variable region includes LCDR1, LCDR2, and LCDR3, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv, or dsFv.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, It also includes the immunoglobulin Fc region, wherein the Fc is selected from IgG1, IgG2, IgG3 or IgG4.

5. A biomaterial, characterized in that, The biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-4; (2) An expression vector / vector group containing the nucleic acid molecules described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the expression vector / vector group described in (2).

6. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, characterized in that, The method includes culturing the host cells as described in claim 5 to express an antibody or an antigen-binding fragment thereof; and optionally includes isolating or purifying the antibody or the antigen-binding fragment thereof.

7. A composition for resisting human IgM, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

8. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or the composition as described in claim 7, characterized in that, Products used to prepare anti-human IgM products, or for detecting human IgM not for the purpose of disease diagnosis and treatment; said products are detection kits, test strips, or detection chips.

9. The application according to claim 8, characterized in that, The detection kit is an ELISA detection kit, immunochromatographic kit, immunoturbidimetric detection kit, magnetic particle detection kit, chemiluminescence detection kit, immunofluorescence detection kit, or radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip.

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