Anti-human igm antibody and use thereof

Abstract

The application discloses an anti-IgM antibody and application thereof, and relates to the field of antibodies.The anti-human IgM antibody provided by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, has a good blocking and immune interference eliminating effect, and provides an important blocking agent raw material.

Description

Technical Field

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

[0002] Immunoassay methods based on antigen-antibody reactions are widely used and are categorized into different methods according to the antibody markers, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and chemiluminescence immunoassay (CIA). In clinical applications, the accuracy of immunoassay results is often affected to varying degrees by interfering substances in the patient's serum, leading to erroneous results. Interfering substances in serum can be divided into endogenous and exogenous interferences. Exogenous interferences include hemolysis, bacterial contamination of the specimen, incomplete blood clotting, sample stability, and storage conditions. Endogenous interferences include rheumatoid factor (RF), heterophile antibodies (HA), autoantibodies, complement, jaundice, and hyperlipidemia. Among these, rheumatoid factor (RF) and heterophile antibodies (HA) are significant interfering factors. Studies have confirmed that approximately 3-15% of healthy individuals contain endogenous interfering factors, more than 10% (30-40%) of patients have heterophile antibodies (HA), and HAMA interference is present in 10-40% of the population. Among endogenous interferences, rheumatoid factor and heterophile antibodies (HA) are the most common. Therefore, researching and developing effective methods to reduce and eliminate interference from rheumatoid factor and heterophile antibodies (HA) is an important issue for ensuring the reliability of medical immunological test results and protecting the interests of doctors and patients. The simplest and most effective method for eliminating interference from rheumatoid factor and heterophile antibodies (HA) in immunodiagnostics is to add a blocking agent to the detection system to directly block the binding of interfering substances to antibodies or antigens in the detection system.

[0003] An inhibitor is a biological agent added to an immunoassay system that reacts with endogenous antibodies, effectively preventing non-analyte-mediated antibody bridging. Inhibitors can be divided into passive and active inhibitors. Passive inhibitors use non-specific substances (such as mouse IgG, mouse serum, non-specific monoclonal antibodies, aggregated IgG, etc.) to block the binding of human heterologous antibodies, such as commercially available reagents like MAK33 and MAB33. These reagents have limited applications, only blocking the activity of one type of human anti-specific animal antibody (such as human anti-mouse antibody). The blocking effect depends on the affinity of the human heterologous antibody, which is typically around 10. -5 -10 -6 The K value is within the range. Therefore, passive blockers are often added at high concentrations to reduce interference during use. Furthermore, heterophilic interference involves many components, and different passive blockers are required to block different types of heterophilic antibodies.

[0004] Active blocking agents are specifically targeted at human immunoglobulins and can specifically, actively, and efficiently neutralize components of interfering antibodies, thereby blocking the formation of unintended binding. Such formulations can eliminate various heterophilic interferences, have specific binding affinity to the heterophilic antibodies causing the interference, and require only low concentrations for efficient blocking, minimizing the impact. Examples of active blocking agents include mixed mouse McAbs produced using HA and HAAA as immunogens, or mouse anti-human IgM McAbs. There is a strong demand in those skilled in the art for active blocking agents with good performance. Summary of the Invention

[0005] This application provides an anti-human IgM antibody or blocking agent that has good binding activity to human IgM and can effectively block and eliminate immune interference in immunoassays, thus providing an important source of raw materials for blocking agents.

[0006] To achieve the above objectives, according to one aspect of the present invention, an anti-human IgM antibody or blocking agent is provided, the anti-human IgM antibody or blocking agent comprising three complementary determining regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:17, 37, 57, 77 and three complementary determining regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:19, 39, 59, 79.

[0007] To achieve the above objective, according to a second aspect of the present invention, an anti-human IgM antibody or blocking agent is provided, said anti-human IgM antibody or blocking agent comprising a complementary determining region of any one of the following groups:

[0008] Group number HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 2 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 3 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 4 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65 SEQ ID NO:66

[0009] To achieve the above objectives, according to a third aspect of the present invention, an anti-human IgM antibody or blocking agent is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 17, 37, 57, 77; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 19, 39, 59, 79.

[0010] To achieve the above objectives, according to a fourth aspect of the present invention, an anti-human IgM antibody or blocking agent is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 18, 38, 58, 78; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 20, 40, 60, 80.

[0011] To achieve the above objectives, according to a fifth aspect of the present invention, an inhibitor is provided, the inhibitor comprising the above-described anti-human IgM antibody.

[0012] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the above-described anti-human IgM antibody or the above-described blocking agent.

[0013] To achieve the above objectives, according to a seventh aspect of the present invention, a method for reducing / eliminating endogenous interference is provided, the method comprising: adding the above-described antibody or the above-described blocking agent to an immune detection system.

[0014] To achieve the above objectives, according to an eighth aspect of the present invention, an immunoassay method is provided, the method comprising: adding the above-described antibody or the above-described blocking agent to an immunoassay system.

[0015] To achieve the above objectives, according to a ninth aspect of the present invention, a nucleic acid is provided that encodes the aforementioned anti-human IgM antibody or blocking agent.

[0016] To achieve the above objectives, according to a tenth aspect of the present invention, a carrier is provided, the carrier comprising the above-described nucleic acid.

[0017] To achieve the above objectives, according to the eleventh aspect of the present invention, a cell is provided, said cell comprising the above-described nucleic acid, vector, or expressing the above-described anti-human IgM antibody or blocking agent.

[0018] To achieve the above objectives, according to a twelfth aspect of the present invention, a method for preparing the above-described anti-human IgM antibody or blocking agent is provided, the method comprising culturing the above-described cells.

[0019] To achieve the above objectives, according to the thirteenth aspect of the present invention, the use of the above-described anti-human IgM antibody, blocking agent, reagent or kit in the preparation of products for detecting IgM or blocking IgM interference is provided. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Results of SDS-PAGE for the reducing properties of Anti-IgM 15D2, Anti-IgM 10E2, Anti-IgM 5H9, and Anti-IgM 15G4. Detailed Implementation

[0022] In a first aspect, embodiments of the present invention provide an anti-human IgM antibody or blocking agent, the anti-human IgM antibody or blocking agent comprising three complementary determining regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:17, 37, 57, 77 and three complementary determining regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:19, 39, 59, 79.

[0023] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the anti-human IgM antibody or blocking agent described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the anti-human IgM antibody or blocking agent described in the first aspect.

[0024] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:17; LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:19.

[0025] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific, multispecific antibodies or antigen-binding fragments, as well as chimeric antibodies, as long as they exhibit the desired biological activity.

[0026] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0027] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0028] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0029] Table 1: CDR Definition 1

[0030] CDR Kabat AbM2 IMGT Chothia [[ID= <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0031] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0032] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0033] 3If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0034] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0035] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0036] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0037] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0038] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0039] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0040] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0041] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0042] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0043] According to embodiments of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0044] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0045] According to embodiments of the present invention, the antibody or blocking agent includes a complementary determinant region from any of the following groups:

[0046] ​ ​ ​ ​ ​ ​ ​ 1 ​ ​ ​ ​ ​ ​ 2 ​ ​ ​ ​ ​ ​ 3 ​ ​ ​ ​ ​ ​ 4 ​ SEQ ID NO:62 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65 SEQ ID NO:66

[0047] Secondly, embodiments of the present invention provide an anti-human IgM antibody or blocking agent, wherein the anti-human IgM antibody or blocking agent comprises a complementary determining region of any one of the following groups:

[0048] Group number HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 2 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 3 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 4 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65 SEQ ID NO:66

[0049] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0050] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0051] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0052] In an optional embodiment, the anti-human IgM antibody or blocking agent described in the first or second aspect further comprises a framework region of any one of the following groups:

[0053]

[0054] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the anti-human IgM antibody or blocking agent provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions described above.

[0055] In an optional embodiment, the anti-human IgM antibody or blocking agent has a KD ≤ 10. -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 M, KD≤10 -13 M, KD≤10 -14 M or KD≤10 -15 M binds to IgM with affinity.

[0056] There are many methods for determining antibody affinity (KD), which can be categorized into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods based on their detection principles. Common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biomembrane optical interferometry (BLI); and common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA).

[0057] In an optional implementation, KD is measured using a kinetic detection method; alternatively, surface plasmon resonance, for example, by using a method such as The system's biosensor system.

[0058] Thirdly, embodiments of the present invention provide an anti-human IgM antibody or blocking agent comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO:17, 37, 57, 77, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO:19, 39, 59, 79.

[0059] In an optional implementation, the heavy chain variable region and the light chain variable region described in the first or third aspect above are selected from any combination of the following:

[0060] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:17 SEQ ID NO:19 2 SEQ ID NO:37 SEQ ID NO:39 3 SEQ ID NO:57 SEQ ID NO:59 4 SEQ ID NO:77 SEQ ID NO:79

[0061] In optional embodiments, the antibody or blocking agent described in the first, second, or third aspect above includes a heavy chain variable region and a light chain variable region in any combination of the following:

[0062]

[0063]

[0064] In optional embodiments, the anti-human IgM antibody or blocking agent described in the first, second, and third aspects above further includes a constant region.

[0065] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0066] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0067] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0068] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0069] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0070] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0071] In an optional implementation, the species source of the constant region is mice.

[0072] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in any of SEQ ID NO: 15, 35, 55, 75, and the light chain constant region sequence (CL) is as shown in any of SEQ ID NO: 16, 36, 56, 76.

[0073] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant regions (SEQ ID NO: 15, 35, 55, 75, 16, 36, 56, or 76).

[0074] In an optional embodiment, the antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0075] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0076] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0077] Fourthly, the present invention provides an anti-human IgM antibody or blocking agent, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:18, 38, 58, 78, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:20, 40, 60, 80.

[0078] In an optional embodiment, the antibody or blocking agent described in the first, second, third, or fourth aspect above includes any combination of the heavy and light chains:

[0079] combination Heavy chain Light chain 1 SEQ ID NO:18 SEQ ID NO:20 2 SEQ ID NO:38 SEQ ID NO:40 3 SEQ ID NO:58 SEQ ID NO:60 4 SEQ ID NO:78 SEQ ID NO:80

[0080] Fifthly, the present invention provides an inhibitor comprising the above-mentioned anti-human IgM antibody.

[0081] In a sixth aspect, the present invention provides a reagent or kit comprising the above-described anti-human IgM antibody or the above-described blocking agent.

[0082] As previously stated, the anti-human IgM antibodies or blocking agents in some embodiments or examples of the present invention can effectively bind to IgM. Therefore, reagents or kits containing the aforementioned anti-human IgM antibodies or blocking agents can effectively block IgM binding interference. The reagents or kits provided by the present invention can be used, for example, in detections involving the specific binding properties of IgM and its antibodies, such as immunoblotting and immunoprecipitation. As previously stated, the anti-human IgM antibodies or blocking agents in some embodiments or examples of the present invention have higher binding activity or affinity for human IgM. Therefore, reagents or kits containing the aforementioned anti-human IgM antibodies or blocking agents have higher blocking performance.

[0083] In a seventh aspect, the present invention provides a method for reducing / eliminating endogenous interference, comprising: adding the above-mentioned antibody or blocking agent to an immune detection system.

[0084] In an eighth aspect, the present invention provides a method for immune detection, comprising: adding the aforementioned antibody or blocking agent to an immune detection system.

[0085] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned anti-human IgM antibody or blocking agent.

[0086] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0087] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0088] In a twelfth aspect, the present invention provides a method for preparing an anti-human IgM antibody or blocking agent, comprising: culturing cells as described above.

[0089] In a thirteenth aspect, the present invention provides the use of the above-described anti-human IgM antibody or blocking agent or the above-described reagent or kit in the preparation of products for detecting IgM or blocking IgM interference.

[0090] In a fourteenth aspect, the present invention provides the use of the above-described antibody or blocking agent or the above-described reagent or kit in detecting IgM or indicating IgM-related diseases or blocking IgM interference.

[0091] Based on the amino acid sequence of the anti-human IgM antibody or blocker disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-human IgM antibody or blocker using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the anti-human IgM antibody or blocker can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the anti-human IgM antibody or blocker as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-human IgM antibody or blocker of this invention, it falls within the protection scope of this invention.

[0092] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0094] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0095] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0096] Example 1: Preparation of anti-human IgM monoclonal antibody

[0097] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit and pMD-18T vector were purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting anti-IgM monoclonal antibody was a hybridoma cell line prepared in our laboratory and was revived for later use.

[0098] (1) Antibody gene preparation

[0099] mRNA was extracted from hybridoma cell lines secreting anti-IgM monoclonal antibodies, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0100] (2) Sequence analysis of the variable region genes of Anti-IgM 15D2, Anti-IgM 10E2, Anti-IgM 5H9, and Anti-IgM 15G4 antibodies

[0101] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pairs, the VL gene sequence was 309bp-333bp, with a 57bp leader peptide sequence preceding it. Among the gene fragments amplified by the Heavy Chain primer pairs, the VH gene sequence was 351bp-369bp, belonging to the VH1 gene family, with a 57bp leader peptide sequence preceding it.

[0102] (3) Construction of recombinant antibody expression plasmid

[0103] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.7kb-0.71kb Light Chain gene fragment and the 1.38kb-1.40kb Heavy Chain gene fragment were amplified by PCR.

[0104] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0105] 2. Recombinant antibody production

[0106] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0107] The resulting antibodies were named Anti-IgM 15D2, Anti-IgM 10E2, Anti-IgM 5H9, and Anti-IgM 15G4. The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in the table below:

[0108] Table 2 Antibody Sequences

[0109] Antibody name Heavy chain Light chain Anti-IgM 15D2 SEQ ID NO:18 SEQ ID NO:20 Anti-IgM 10E2 SEQ ID NO:38 SEQ ID NO:40 Anti-IgM 5H9 SEQ ID NO:58 SEQ ID NO:60 Anti-IgM 15G4 SEQ ID NO:78 SEQ ID NO:80

[0110] Example 2: Antibody Performance Detection

[0111] 1. Affinity Analysis

[0112] Antibodies were pre-diluted and purified, while human IgM antigen (from Phytobio) was serially diluted. Using a CM5 chip pre-coupled with goat anti-mouse IgG, the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ instrument. The instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate. The results showed that the affinity of antibodies Anti-IgM 15D2, Anti-IgM10E2, Anti-IgM 5H9, and Anti-IgM 15G4 for the IgM antigen was 1.0 × 10⁻⁶. -12 M≤KD<1.0×10 -7 M, and it has a better affinity for human IgM antigen than the control antibody. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0113] 2. Performance evaluation of blocking agents

[0114] One of the antibodies Anti-IgM 15D2, Anti-IgM 10E2, Anti-IgM 5H9, and Anti-IgM 15G4 was used as an inhibitor in CKMB (creatine kinase-MB) to analyze the blocking effect of the antibody in actual detection.

[0115] The detection procedure includes: aspirating 1.5 μL of sample from the sample tray, and simultaneously aspirating 200 μL of R1 (Tris buffer) containing 0.1 mg / mL blocking agent, adding them to a cuvette, mechanically stirring, and incubating at 37°C for 5 minutes; adding 50 μL of R2 (latex conjugated with the analyte mouse antibody) to the cuvette, and mechanically stirring; detecting at a wavelength of 570 nm, and recording the absorbance values ​​at 7 minutes and 30 seconds and 12 minutes from the time the sample was added. The difference between these values ​​is the reactivity of the sample, which is directly proportional to the concentration of the analyte in the sample.

[0116] The specific reactivity data for the CKMB project are shown in Table 3 below.

[0117] Table 3. Blocking Effect of CKMB Project

[0118] Clinical sample information No blocking agent Anti-IgM 15D2 Anti-IgM 10E2 Anti-IgM 5H9 Anti-IgM 15G4 Abnormal Sample 1 504.54 151.36 151.36 252.27 100.91 Abnormal Sample 2 627.57 251.03 313.79 188.27 251.03 Abnormal Sample 3 35.82 14.33 10.75 17.91 14.33 Abnormal Sample 4 108.99 21.80 10.90 43.59 10.90 Abnormal Sample 5 440.68 220.34 44.07 88.14 176.27 Abnormal Sample 6 474.47 94.89 189.79 237.24 189.79 Abnormal Sample 7 457.12 137.14 137.14 228.56 228.56 Abnormal Sample 8 406.39 81.28 81.28 121.92 162.56 Abnormal Sample 9 196.81 78.72 59.04 59.04 78.72 Abnormal Sample 10 904.39 361.76 452.20 452.20 452.20 Abnormal sample 11 82.15 24.64 32.86 16.43 16.43 Abnormal sample 12 1154.80 230.96 115.48 346.44 230.96 Abnormal sample 13 200.29 20.03 40.06 100.14 60.09 negative control 55.2 27.60 27.60 27.60 27.60 Positive control 2235.2 20787.36 19110.96 18105.12 20116.80

[0119] Note: The smaller the value, the weaker the reaction and the better the blocking effect.

[0120] As shown in the table above, on the latex-enhanced turbidimetric platform CKMB project, the blocking agent described in this invention can significantly reduce the abnormal detection results caused by false positive interference. The test results are close to the test results of negative samples and do not affect the test results of positive samples, thus greatly improving the accuracy of clinical test results.

[0121] The specific reactivity data for the PGⅡ (pepsinogen II) test are shown in Table 4 below.

[0122] Table 4. Blocking Effect of PG II Project

[0123]

[0124]

[0125] The specific responsiveness data for the PCT (procalcitonin) test are shown in Table 5 below.

[0126] Table 5. Blocking Effect of PCT Project

[0127]

[0128] The specific reactivity data for the D-dimer project are shown in Table 6 below.

[0129] Table 6. Blocking Effect of D-dimer Project

[0130] Clinical sample information No blocking agent added Anti-IgM 15D2 Anti-IgM 10E2 Anti-IgM 5H9 Anti-IgM 15G4 negative control 30.5 15.25 30.50 15.25 30.50 Positive control 4236.8 41520.64 41944.32 40249.60 41096.96 False positive sample 1 589.5 58.95 176.85 47.54 141.48 False positive sample 2 412.3 4.88 181.41 247.38 41.23 False positive sample 3 865.3 86.53 207.67 64.86 432.65 False positive sample 4 952.8 285.84 95.28 419.23 2.50 False positive samples 5 1285.3 385.59 102.32 514.12 668.36

[0131] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0132] The partial amino acid sequences involved in this application are shown in Table 7:

[0133] Table 7 Amino acid sequence list

[0134]

[0135]

[0136]

[0137]

Claims

1. An anti-human IgM antibody, characterized in that, The anti-human IgM antibody comprises: The three complementarity-determining regions of the heavy chain variable region as shown in SEQ ID NO:17 and the three complementarity-determining regions of the light chain variable region as shown in SEQ ID NO:19; or The anti-human IgM antibody comprises three complementarity-determining regions of the heavy chain variable region as shown in SEQ ID NO:37 and three complementarity-determining regions of the light chain variable region as shown in SEQ ID NO:39; or The anti-human IgM antibody comprises three complementarity-determining regions of the heavy chain variable region as shown in SEQ ID NO:57 and three complementarity-determining regions of the light chain variable region as shown in SEQ ID NO:59; or The anti-human IgM antibody contains three complementary determinant regions of the heavy chain variable region as shown in SEQ ID NO:77 and three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:

79. The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An anti-human IgM antibody, characterized in that, The anti-human IgM antibody includes the following complementarity-determining region: The amino acid sequences are as follows: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, as shown in SEQ ID NO:1~6; or The amino acid sequences are as shown in SEQ ID NO:21~26: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3; or The amino acid sequences are as follows: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, as shown in SEQ ID NO:41~46; or The amino acid sequences are as shown in SEQ ID NO:61~66: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3.

3. The anti-human IgM antibody according to claim 1 or 2, characterized in that, The anti-human IgM antibody includes the following framework region: HFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:7; HFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:8; HFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:9; HFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:10; LFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:11; LFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:12; LFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:13; and LFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:14; or HFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:27, HFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:28, HFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:29, HFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:30, LFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:31, LFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:32, LFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:33, and LFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:34; or HFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:47, HFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:48, HFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:49, HFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:50, LFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:51, LFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:52, LFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:53, and LFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:54; or HFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:67, HFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:68, HFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:69, HFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:70, LFR1 is an amino acid sequence with at least 80% identity to SEQ ID NO:71, LFR2 is an amino acid sequence with at least 80% identity to SEQ ID NO:72, LFR3 is an amino acid sequence with at least 80% identity to SEQ ID NO:73, and LFR4 is an amino acid sequence with at least 80% identity to SEQ ID NO:

74.

4. An anti-human IgM antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:39; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:57, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:59; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

79.

5. The anti-human IgM antibody according to any one of claims 1, 2, and 4, characterized in that, The anti-human IgM antibody also includes a constant region.

6. The anti-human IgM antibody according to claim 5, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

7. The anti-human IgM antibody according to claim 6, characterized in that, The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

8. The anti-human IgM antibody according to claim 6, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

9. The anti-human IgM antibody according to claim 5, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

10. The anti-human IgM antibody according to claim 5, characterized in that, The species source of the constant region is mice.

11. The anti-human IgM antibody according to claim 6, characterized in that, The heavy chain constant region is an amino acid sequence with at least 80% identity to SEQ ID NO:15, and the light chain variable region is an amino acid sequence with at least 80% identity to SEQ ID NO:16; or The heavy chain constant region is an amino acid sequence with at least 80% identity to SEQ ID NO:35, and the light chain variable region is an amino acid sequence with at least 80% identity to SEQ ID NO:36; or The heavy chain constant region is an amino acid sequence with at least 80% identity to SEQ ID NO:55, and the light chain variable region is an amino acid sequence with at least 80% identity to SEQ ID NO:56; or The heavy chain constant region is an amino acid sequence with at least 80% identity to SEQ ID NO:75, and the light chain variable region is an amino acid sequence with at least 80% identity to SEQ ID NO:

76.

12. The anti-human IgM antibody according to any one of claims 1, 2, and 4, characterized in that, The anti-human IgM antibody includes an antigen-binding fragment of the antibody, wherein the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

13. An anti-human IgM antibody, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:18, and the amino acid sequence of the light chain is shown in SEQ ID NO:20; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:38, and the amino acid sequence of the light chain is shown in SEQ ID NO:40; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:58, and the amino acid sequence of the light chain is shown in SEQ ID NO:60; or The amino acid sequence of the heavy chain is shown in SEQ ID NO:78, and the amino acid sequence of the light chain is shown in SEQ ID NO:

80.

14. A blocking agent, characterized in that, The blocking agent includes the anti-human IgM antibody according to any one of claims 1 to 13.

15. A reagent or kit, characterized in that, The reagent or kit comprises the anti-human IgM antibody as described in any one of claims 1 to 13 or the blocking agent as described in claim 14.

16. A method for reducing / eliminating endogenous interference, characterized in that, The method comprises adding an anti-human IgM antibody according to any one of claims 1 to 13 or an inhibitor according to claim 14 to an immune detection system, the method being used for non-direct diagnostic or therapeutic purposes.

17. A nucleic acid, characterized in that, The nucleic acid encodes the anti-human IgM antibody according to any one of claims 1 to 13.

18. A cell characterized in that, The cell contains the nucleic acid as described in claim 17.

19. A method for preparing the antibody according to any one of claims 1 to 13, characterized in that, The method includes culturing the cells of claim 18.

20. Use of the anti-human IgM antibody according to any one of claims 1 to 13 in the preparation of products for detecting IgM or blocking IgM interference.

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