Anti-human IgM antibody and its preparation method and use

Abstract

The present disclosure relates to an anti-human IgM antibody, its preparation method, and use. The anti-human IgM monoclonal antibody prepared in the present disclosure has high reactivity to human IgM and has good blocking and immune interference elimination effects in immunoassays, providing an important source of blocking agent raw materials.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-human IgM antibody and a preparation method and use thereof. Background Art

[0002] Immunoassays based on antigen-antibody reactions are widely used and are categorized by antibody marker, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and chemiluminescence. In clinical practice, the accuracy of immunoassay results is often affected to varying degrees by interfering substances in patient serum, leading to erroneous results. Serum interfering substances can be categorized as endogenous and exogenous. Exogenous interferences include hemolysis, specimen contamination, incomplete blood coagulation, and sample stability and storage conditions. Endogenous interferences include rheumatoid factor (RF), heterophilic antibodies (HA), autoantibodies, complement, jaundice, and hyperlipidemia. RF and HA are significant interfering factors. Studies have demonstrated that approximately 3-15% of healthy individuals harbor endogenous interfering factors, while heterophilic antibodies (HA) are found in over 10% (30-40%) of patients, and HAMA (heterophilic antigen-binding protein) (HAMA) interference in 10-40% of the population. Among endogenous interfering substances, RF and HA are the most common. Therefore, researching effective methods to reduce or eliminate interference from rheumatoid factor and heterophilic antibodies (HA) is crucial for ensuring the reliability of medical immunoassay results and protecting the interests of doctors and patients. The simplest and most effective way to eliminate interference from rheumatoid factor and heterophilic antibodies (HA) in immunodiagnosis is to add blocking agents to the test system to directly block the binding of interfering substances to antibodies or antigens in the test system.

[0003] Blocking agents are biological agents that are added to the immunoassay system and can react with endogenous antibodies, thereby effectively preventing non-analyte-mediated antibody bridging. Blocking agents can be divided into passive blockers and active blockers. Passive blockers 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 MAK33 and MAB33 in commercial reagents. This type of reagent has limited use and can only block the active reagent of a human anti-specific animal antibody (such as human anti-mouse antibody). The blocking effect depends on the affinity of the human heterologous antibody. Generally, the affinity of human heterologous antibodies is usually around 10. -5 -10 -6 Therefore, passive blockers are often added at high concentrations to reduce interference. Furthermore, heterophilic interference involves many components, and different passive blockers are required to block different types of heterophilic antibodies.

[0004] Active blockers are specific for human immunoglobulins and can specifically, actively, and efficiently neutralize interfering antibody components, thereby preventing unintended binding. Examples include IIR and HBR in commercial reagents. These agents can eliminate various heterophilic interferences and have specific binding affinity for interfering heterophilic antibodies. They require only low concentrations for efficient blocking, minimizing the impact. IIR is a mixed murine McAb produced from HA and HAAA as immunogens and has a high affinity for HAAA. HBR is a mouse anti-human IgM McAb. During active blocking, the effectiveness of interference elimination depends on the active blocker's affinity for heterophilic antibodies. Due to their high affinity, active blockers offer stronger blocking capabilities than passive blockers in some analyses. Therefore, providing a high-performance blocker is highly marketable. Summary of the Invention

[0005] The present application provides an antibody or a functional fragment thereof, which has good binding reactivity to human IgM and has good blocking and immune interference elimination effects for immunoassays, providing an important source of blocking agent raw materials.

[0006] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or a functional fragment thereof is provided, which binds to human IgM protein and comprises three complementary determining regions having a heavy chain variable region of any one of the amino acid sequences of SEQ ID NO: 17, 18, 38, 56, 74, 92, 93, 94, 95, 120, 138, 177, 195, 214, 232, 250, 268, 286, 304, 322 or 340 and three complementary determining regions having a light chain variable region of any one of the amino acid sequences of SEQ ID NO: 19, 39, 57, 75, 96, 121, 139, 140, 141, 142, 143, 144, 145, 146, 178, 196, 215, 233, 251, 269, 287, 305, 323 or 341.

[0007] To achieve the above objectives, according to a second aspect of the present invention, an antibody or a functional fragment thereof 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, 18, 38, 56, 74, 92, 93, 94, 95, 120, 138, 177, 195, 214, 232, 250, 268, 286, 304, 322 or 340; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 19, 39, 57, 75, 96, 121, 139, 140, 141, 142, 143, 144, 145, 146, 178, 196, 215, 233, 251, 269, 287, 305, 323 or 341.

[0008] To achieve the above objectives, according to the third aspect of the present invention, an antibody or a functional fragment thereof 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: 20, 21, 40, 58, 76, 97, 98, 99, 100, 122, 147, 179, 197, 216, 234, 252, 270, 288, 306, 324, 342; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22, 41, 59, 77, 101, 123, 148, 149, 150, 151, 152, 153, 154, 155, 180, 198, 217, 235, 253, 271, 289, 307, 325, 343.

[0009] In order to achieve the above object, according to the fourth aspect of the present invention, a nucleic acid is provided, which encodes any one of the above antibodies or functional fragments thereof.

[0010] In order to achieve the above object, according to a fifth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid.

[0011] To achieve the above object, according to a sixth aspect of the present invention, there is provided a method for preparing any one of the above antibodies or functional fragments thereof, the method comprising culturing the above cells.

[0012] In order to achieve the above-mentioned object, according to the seventh aspect of the present invention, there is provided the use of the above-mentioned antibody or its functional fragment in immunoassay or in the preparation of an immunoblocking agent.

[0013] In order to achieve the above object, according to the eighth aspect of the present invention, a blocking agent is provided, which includes the above-mentioned antibody or a functional fragment thereof.

[0014] In order to achieve the above object, according to the ninth aspect of the present invention, a detection reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or functional fragment thereof or the above-mentioned blocking agent.

[0015] In order to achieve the above object, according to the tenth aspect of the present invention, a method for reducing / eliminating endogenous interference is provided, wherein the above-mentioned antibody or its functional fragment or the above-mentioned blocker is added to the immunoassay system.

[0016] In order to achieve the above object, according to the eleventh aspect of the present invention, there is provided an immunoassay method, comprising: adding the above-mentioned antibody or its functional fragment or the above-mentioned blocker to an immunoassay system.

[0017] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, a method for detecting IgM is provided, which comprises: contacting the antibody or its functional fragment described in any one of the above items with a sample from the subject to perform a binding reaction under conditions sufficient for a binding reaction to occur; and detecting the immune complex produced by the binding reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The results of reducing SDS-PAGE of Anti-hIgM Rmb1 are shown. DETAILED DESCRIPTION

[0020] In a first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, which binds to a human IgM protein and comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NO: 17, 18, 38, 56, 74, 92, 93, 94, 95, 120, 138, 177, 195, 214, 232, 250, 268, 286, 304, 322 or 340 and three complementarity determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NO: 19, 39, 57, 75, 96, 121, 139, 140, 141, 142, 143, 144, 145, 146, 178, 196, 215, 233, 251, 269, 287, 305, 323 or 341.

[0021] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or its functional fragment described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or its functional fragment described in the first aspect.

[0022] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 19.

[0023] Under the Kabat definition, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region SEQ ID NO:17 are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively. Then, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0024] Under the Kabat definition, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region SEQ ID NO:19 are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Then, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

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

[0026] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

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

[0028] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may 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, with slightly different notations in different references. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0029] Table 1: CDR Definition 1

[0030] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0031] 1 The numbering of all CDR definitions in Table 1 is based on 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." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0032] 2"AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0033] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0034] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0035] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

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

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

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

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

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

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

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

[0043] According to an embodiment 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] CDR Kabat AbM IMGT Chothia HCDR1 H31 - H35 H26 - H35 H26 - H33 H26 - H32 HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L{97

[0045] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0046] In an optional embodiment, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a CDR sequence of any of the following numbered combinations:

[0047]

[0048]

[0049] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0050] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0051] In an optional embodiment, the antibody or functional fragment thereof comprises any of the following numbered combinations of FR sequences or has at least 80% identity with any of the numbered combinations of FR sequences:

[0052]

[0053]

[0054] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned corresponding framework regions.

[0055] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, 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, 18, 38, 56, 74, 92, 93, 94, 95, 120, 138, 177, 195, 214, 232, 250, 268, 286, 304, 322 or 340; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 19, 39, 57, 75, 96, 121, 139, 140, 141, 142, 143, 144, 145, 146, 178, 196, 215, 233, 251, 269, 287, 305, 323 or 341.

[0056] In an optional embodiment, the antibody or functional fragment thereof described in the first aspect or the second aspect comprises any combination of the following heavy chain variable regions and light chain variable regions:

[0057]

[0058]

[0059] In an alternative embodiment, the antibody or functional fragment thereof further comprises a constant region.

[0060] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0061] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0062] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0063] In an alternative embodiment, the species origin of the constant region is mouse.

[0064] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO: 15, 119, 213, and the light chain constant region (CL) sequence is shown as SEQ ID NO: 16.

[0065] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0066] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0067] The functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that the functional fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the above-mentioned functional fragments.

[0068] Functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0069] In a third aspect, the present invention provides an antibody or a functional fragment thereof, 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: 20, 21, 40, 58, 76, 97, 98, 99, 100, 122, 147, 179, 197, 216, 234, 252, 270, 288, 306, 324, and 342; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 22, 41, 59, 77, 101, 123, 148, 149, 150, 151, 152, 153, 154, 155, 180, 198, 217, 235, 253, 271, 289, 307, 325, and 343.

[0070] In an optional embodiment, the antibody described in the first, second, and third aspects above comprises any combination of the following heavy chains and light chains:

[0071] Combination Heavy chain Light chain 1 SEQ ID NO:20 SEQ ID NO:22 2 SEQ ID NO:21 SEQ ID NO:22 3 SEQ ID NO:40 SEQ ID NO:41 4 SEQ ID NO:58 SEQ ID NO:59 5 SEQ ID NO:76 SEQ ID NO:77 6 SEQ ID NO:97 SEQ ID NO:101 7 SEQ ID NO:98 SEQ ID NO:101 8 SEQ ID NO:99 SEQ ID NO:101 9 SEQ ID NO:100 SEQ ID NO:101 10 SEQ ID NO:122 SEQ ID NO:123 11 SEQ ID NO:147 SEQ ID NO:148 12 SEQ ID NO:147 SEQ ID NO:149 13 SEQ ID NO:147 SEQ ID NO:150 14 SEQ ID NO:147 SEQ ID NO:151 15 SEQ ID NO:147 SEQ ID NO:152 16 SEQ ID NO:147 SEQ ID NO:153 17 SEQ ID NO:147 SEQ ID NO:154 18 SEQ ID NO:147 SEQ ID NO:155 19 SEQ ID NO:179 SEQ ID NO:180 20 SEQ ID NO:197 SEQ ID NO:198 21 ​ ​ 22 ​ ​ 23 ​ SEQ ID NO:253 24 SEQ ID NO:270 SEQ ID NO:271 25 SEQ ID NO:288 SEQ ID NO:289 26 SEQ ID NO:306 SEQ ID NO:307 27 SEQ ID NO:324 SEQ ID NO:325 28 SEQ ID NO:342 SEQ ID NO:343

[0072] The present invention also provides a nucleic acid encoding any of the above antibodies or functional fragments thereof.

[0073] The present invention also provides a cell comprising the above nucleic acid.

[0074] The present invention also provides a method for preparing any of the above antibodies or functional fragments thereof, the method comprising culturing the above cells.

[0075] The present invention provides the use of the above-mentioned antibody or its functional fragment in immunoassay or in the preparation of immune blockers. The blockers can be used in combination on a chemiluminescence platform, a fluorescent immunochromatography platform, and a colloidal gold platform, and have good blocking effects.

[0076] The present invention provides a blocking agent, which comprises the above-mentioned antibody or a functional fragment thereof.

[0077] Optionally, the concentration of the antibody in the blocking agent is 5-100 μg / ml.

[0078] The present invention provides a detection reagent or a kit, which comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned blocking agent.

[0079] The present invention provides a method for reducing / eliminating endogenous interference, wherein the above-mentioned antibody or its functional fragment or the above-mentioned blocker is added to an immunoassay system.

[0080] The present invention provides an immunoassay method, comprising: adding the above-mentioned antibody or its functional fragment or the above-mentioned blocking agent into an immunoassay system.

[0081] The present invention provides a method for detecting IgM, comprising: contacting any one of the above antibodies or functional fragments thereof with a sample from the subject under conditions sufficient for a binding reaction to occur; and detecting the immune complex generated by the binding reaction.

[0082] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it falls within the scope of protection of the present invention.

[0083] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0085] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0086] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0087] Example 1 Preparation of Anti-IgM Monoclonal Antibody

[0088] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. Hybridoma cell lines secreting anti-IgM monoclonal antibodies were pre-existing hybridoma monoclonal cell lines AbM1 to AbM28, which were revived and used for future use.

[0089] (1) Antibody gene preparation

[0090] mRNA was extracted from hybridoma monoclonal cell lines AbM1 to AbM28, and DNA products were obtained by RT-PCR. The products were subjected to A-addition reaction with rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells, and after colonies grew, the Heavy Chain and Light Chain gene clones were obtained. Four clones each were sent to a gene sequencing company for sequencing.

[0091] (2) Sequence analysis of the variable region gene of anti-IgM antibody

[0092] The gene sequences obtained by the above sequencing were placed in the KABAT antibody database for analysis, and VNTI11.5 software was used for analysis to confirm that the genes amplified by the heavy chain and light chain primer pairs were correct.

[0093] (3) Construction of recombinant antibody expression plasmid

[0094] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify the 0.70-0.75 kb Light Chain gene fragment and the 1.40-1.45 kb Heavy Chain gene fragment.

[0095] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the hybridoma monoclonal cell lines AbM1 to AbM28 Heavy Chain and Light Chain, respectively.

[0096] 2. Recombinant Antibody Production

[0097] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. The 28 plasmid DNAs and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the 28 plasmid DNA diluents, mixed well, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed well, and samples were taken and counted. The viability of the cells after transfection was recorded and observed. The cells were then placed in a 35°C constant temperature incubator for incubation at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0098] The obtained antibodies were named Anti-IgM Rmb1 to Anti-IgM Rmb28. The sequences of the heavy (H) and light (L) chains of the above antibodies are shown in the following table:

[0099] Table 2 Antibody sequences

[0100] Antibody Name Heavy Chain Light Chain Anti-IgM Rmb1 SEQ ID NO:20 SEQ ID NO:22 Anti-IgM Rmb2 SEQ ID NO:21 SEQ ID NO:22 Anti-IgM Rmb3 SEQ ID NO:40 SEQ ID NO:41 Anti-IgM Rmb4 SEQ ID NO:58 SEQ ID NO:59 Anti-IgM Rmb5 SEQ ID NO:76 SEQ ID NO:77 Anti-IgM Rmb6 SEQ ID NO:97 SEQ ID NO:101 Anti-IgM Rmb7 SEQ ID NO:98 SEQ ID NO:101 Anti-IgM Rmb8 SEQ ID NO:99 [[ID= ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ SEQ ID NO:235 Anti-IgM Rmb23 SEQ ID NO:252 SEQ ID NO:253 Anti-IgM Rmb24 SEQ ID NO:270 SEQ ID NO:271 Anti-IgM Rmb25 SEQ ID NO:288 SEQ ID NO:289 Anti-IgM Rmb26 SEQ ID NO:306 SEQ ID NO:307 Anti-IgM Rmb27 SEQ ID NO:324 SEQ ID NO:325 Anti-IgM Rmb28 SEQ ID NO:342 SEQ ID NO:343

[0101] Example 2 Performance testing of antibodies

[0102] 1. Activity and subclass identification

[0103] Coating solution (main component NaHCO3) diluted human IgM (purchased from Feipeng Bio) to 500 or 100ug / ml, 100uL per well, 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+Nacl) and pat dry; add blocking solution (20% BSA+80% PBS), 120uL per well, 37℃, 1h, pat dry; add diluted purified antibody, 100uL / well, 37℃, 30min; wash 5 times with washing solution and pat dry; add goat anti-mouse IgG1-HRP, goat anti-mouse IgG2a-HRP or goat anti-mouse IgG2b-HRP, 100uL per well, 37℃, 30min; wash 5 times with washing solution and pat dry; add color development solution A (50uL / well), add color development solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on microplate reader.

[0104] Table 3 Activity and subclass identification data

[0105]

[0106] 2. Performance Evaluation

[0107] 2.1 The blocking effect of the above anti-hIgM series antibodies (IgG1 subclass) was verified on the chemiluminescence platform HIV p24 antibody pairing

[0108] HIV p24 antibodies (IgG1 subclass, purchased from Feipeng Bio) were paired and coupled to magnetic beads and acridinium ester, respectively. The following reaction protocol was used on an ishine 2910 chemiluminescence instrument: 50 μl of sample, 50 μl of magnetic bead working solution, and 50 μl of acridinium ester working solution were pipetted into a reaction cup. The reaction was allowed to react for 15 minutes before detection. In the experimental group, 5 μg / ml of the aforementioned anti-hIgM antibodies were added to the acridinium ester working solution, while the control group contained an equal volume of anti-hIgM antibody dilution solution. The samples contained interference from heterophilic antibodies or RF. The experimental group demonstrated a significant elimination of false positives, demonstrating that the aforementioned anti-hIgM antibodies demonstrated a significant blocking effect, outperforming the market-leading raw material A.

[0109] Table 4 Chemiluminescence platform IgG1 (HIV P24) blocking data

[0110]

[0111]

[0112] 2.2 Verify the blocking effect of the above anti-hIgM series antibodies (IgG1 subclass) on the chemiluminescence platform cTnI antibody pairing

[0113] cTnI antibodies (IgG1 subclass) were paired and coupled to magnetic beads and acridinium ester, respectively. The following reaction protocol was used on an ishine 2910 chemiluminescence instrument: 50 μl of sample, 50 μl of magnetic bead working solution, and 50 μl of acridinium ester working solution were pipetted into a reaction cup. The reaction was allowed to react for 15 minutes before detection. In the experimental group, 5 μg / ml of the aforementioned anti-hIgM antibodies were added to the acridinium ester working solution, while the control group contained an equal volume of anti-hIgM antibody diluent. Samples containing heterophilic antibodies or RF interference were tested. The experimental group demonstrated a significant elimination effect on false-positive samples, demonstrating that the aforementioned anti-hIgM antibodies demonstrated a significant blocking effect, outperforming the mainstream raw material A on the market.

[0114] Note: The smaller the value, the weaker the reaction and the better the blocking effect.

[0115] Table 5 Chemiluminescence platform IgG1 (cTnI) blocking data

[0116]

[0117] 2.3 The blocking effect of the above anti-hIgM series antibodies (IgG2a subclass) was verified on the IgG2a subtype pairing model of the chemiluminescence platform

[0118] The selected IgG2a antibodies (purchased from Feipeng Bio) were paired and coupled to magnetic beads and acridinium ester, respectively. The following reaction procedure was used on the chemiluminescence instrument ishine2910: 50ul of sample, 50ul of magnetic bead working solution, and 50ul of acridinium ester working solution were pipetted into a reaction cup. The reaction was allowed to react for 15 minutes before detection. In the experimental group, 5ug / ml of the aforementioned anti-hIgM series antibodies (IgG2a subclass) were added to the acridinium ester working solution, while the control group contained an equal volume of anti-hIgM antibody diluent. The samples contained interference from heterophilic antibodies or RF. The experimental results showed that the experimental group had a significant elimination effect on false positive specimens, indicating that the aforementioned anti-hIgM series antibodies had a significant blocking effect, and were also slightly better than the mainstream raw material A on the market.

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

[0120] Table 6 Chemiluminescence platform IgG2a blocking data

[0121] Sample No. HAMA sample 4 HAMA sample 5 HAMA Sample 6 HAMA Sample 7 Control 1: Anti-hIgM antibody diluent blank control 135329 282462 350184 7692 Control 2: Mainstream raw material A in the market 9806 35522 72515 1844 Anti-IgMRmb10 3123 7826 2685 1439 Anti-IgMRmb19 2010 7211 2540 1555 Anti-IgMRmb20 1970 7627 2678 1377

[0122] 2.4 Verification of the blocking effect of the above anti-hIgM series antibodies (IgG2b subclass) on the IgG2b subtype pairing model on the chemiluminescence platform

[0123] The selected IgG2b antibodies (purchased from Feipeng Bio) were paired and coupled to magnetic beads and acridinium ester, respectively. The following reaction procedure was used on the chemiluminescence instrument ishine 2910: 50μl of sample, 50μl of magnetic bead working solution, and 50μl of acridinium ester working solution were pipetted into a reaction cup. The reaction was allowed to react for 15 minutes before detection. In the experimental group, 5μg / ml of the aforementioned anti-hIgM series antibodies were added to the acridinium ester working solution, while the control group contained an equal volume of anti-hIgM antibody diluent. The samples contained interference from heterophilic antibodies or RF. The experimental results showed that the experimental group had a significant effect on eliminating false positive specimens, indicating that the aforementioned anti-hIgM had a significant blocking effect and was slightly better than the mainstream raw material A on the market.

[0124] Note: The smaller the value, the weaker the reaction and the better the blocking effect.

[0125] Table 7 Chemiluminescence platform IgG2b blocking data

[0126]

[0127] 2.5 Verification of blocking effect on cTnI pairing on fluorescence immunochromatography platform

[0128] The CTNI antibodies (IgG1 subclass antibodies, purchased from Feipeng Bio) were paired, and the membranes were smeared and labeled separately. The experimental group used the mouse IgG1 series, and the sample pads were treated with the above-mentioned anti-hIgM antibody and the mainstream raw material B on the market, respectively. The sample pads of the control group were not treated. False positive specimens were tested separately. The experimental results showed that the experimental group had a significant elimination effect on false positive specimens, indicating that the above-mentioned anti-hIgM had a significant blocking effect, and was also slightly better than the mainstream raw material B on the market.

[0129] Note: The smaller the value, the weaker the reaction and the better the blocking effect.

[0130] Table 8 IgG1 blocking data of fluorescence immunochromatography platform

[0131]

[0132] 2.6 Verification of blocking effect on HIV p24 antigen detection pairing on immunocolloidal gold platform

[0133] HIV p24 antibodies (purchased from Feipeng Bio, IgG1 subclass) were paired and then stripped and labeled. The experimental group used a mouse IgG1 series. Sample pads were treated with anti-hIgM series antibodies and mainstream raw material B, respectively, while the control group received no treatment. False-positive specimens were tested, and the experimental group demonstrated a significant elimination effect, demonstrating that the anti-hIgM series antibodies demonstrated a significant blocking effect, slightly superior to mainstream raw material B.

[0134] Note: Color intensity is expressed in numbers. The smaller the number, the stronger the color. B means no color. "+" means the color intensity is 0.5 color cards.

[0135] Table 9 IgG1 blocking data of colloidal gold platform

[0136]

[0137] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0138] Some of the amino acid sequences involved in this application are as follows:

[0139] Table 10 Amino acid sequence

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

Claims

1. An anti-human IgM antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 17 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 19; or The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 38 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 39; or The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 92 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 96; or The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 177 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 178; or The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 232 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 233; or The antibody or antigen-binding fragment thereof has three complementarity determining regions identical to the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 340 and three complementarity determining regions identical to the light chain variable region shown in the amino acid sequence of SEQ ID NO: 341; The complementarity determining regions of the heavy chain variable region and the light chain variable region are defined by any one of the systems of Kabat, Chothia, IMGT, AbM or Contact, and the numbering system is Kabat.

2. An anti-human IgM antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises any combination of the following CDR sequences: The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 1 to 6, respectively; or The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 24 to 29, respectively; or The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 78 to 83; or The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 78 to 83; or The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 163 to 168, respectively; or The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 218 to 223; or The amino acid sequences are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 326 to 331.

3. An anti-human IgM antibody or antigen-binding fragment thereof, 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: 38, 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: 92, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 96; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 177, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 178; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 232, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 233; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 340, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

341.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof further comprises a constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The constant region includes a heavy chain constant region and a light chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

8. The antibody or antigen-binding fragment thereof according to claim 4, wherein The species origin of the constant region is mouse.

9. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The heavy chain constant region sequence is shown in any one of SEQ ID NO: 15, 119 or 213 or has at least 80% identity thereto, and the light chain constant region sequence is shown in SEQ ID NO: 16 or has at least 80% identity thereto.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 5 to 9, wherein: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

11. An anti-human IgM antibody comprising a heavy chain and a light chain, characterized in that: The heavy chain amino acid sequence is shown in SEQ ID NO: 20, and the light chain amino acid sequence is shown in SEQ ID NO: 22; or The heavy chain amino acid sequence is shown in SEQ ID NO: 40, and the light chain amino acid sequence is shown in SEQ ID NO: 41; or The heavy chain amino acid sequence is shown in SEQ ID NO: 97, and the light chain amino acid sequence is shown in SEQ ID NO: 101; or The heavy chain amino acid sequence is shown in SEQ ID NO: 179, and the light chain amino acid sequence is shown in SEQ ID NO: 180; or The heavy chain amino acid sequence is shown in SEQ ID NO: 234, and the light chain amino acid sequence is shown in SEQ ID NO: 235; or The heavy chain amino acid sequence is shown in SEQ ID NO: 342, and the light chain amino acid sequence is shown in SEQ ID NO:

343.

12. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11.

13. A cell, characterized in that The cell comprises the nucleic acid of claim 12.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11, characterized in that: The method comprises culturing the cell of claim 13.

15. A blocking agent, characterized in that The blocking agent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11.

16. A detection reagent, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the blocking agent according to claim 15.

17. A detection kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the blocking agent according to claim 15.

18. A method for reducing / eliminating endogenous interference, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the antibody according to claim 11, or the blocking agent according to claim 15 is added to the immunoassay system.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11 in the preparation of an immunoblocking agent, characterized in that: The method comprises: contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the antibody according to claim 11 with a sample from the subject under conditions sufficient for a binding reaction to occur; and detecting an immune complex generated by the binding reaction.

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