Anti-human IgM antibody and application thereof

Abstract

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

Description

Technical Field

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

[0002] Immunoassay methods based on antigen-antibody reactions are widely used and are divided into different detection methods according to antibody markers, such as enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence, etc. In clinical applications, the accuracy of immunoassay results is often affected to varying degrees by interferences in patient serum, resulting in erroneous test results. Interferences in serum can be divided into endogenous interference and exogenous interference. Exogenous interference includes hemolysis, specimen contamination, incomplete blood coagulation, sample stability and storage conditions, etc.; endogenous interference includes rheumatoid factor (RF), heterophilic antibody (HA), autoantibodies, complement, jaundice, high fat, etc. Among them, rheumatoid factor (RF) and heterophilic antibody (HA) are important interference factors. Studies have confirmed that about 3-15% of healthy people have endogenous interference factors in their bodies, more than 10% (30-40%) of patients have heterophilic antibodies (HA), and 10-40% of the population have HAMA interference. Among endogenous interferences, rheumatoid factor and heterophilic antibody (HA) are the most common. Therefore, researching and developing effective means to reduce and eliminate the interference of rheumatoid factors and heterophilic antibodies (HA) is an important issue to ensure the reliability of medical immunoassay results and protect the interests of doctors and patients. The simplest and most effective way to eliminate the interference of rheumatoid factors and heterophilic antibodies (HA) in immunodiagnosis is to add blocking agents to the detection system to directly block the binding of interfering substances with antibodies or antigens in the detection system.

[0003] Blockers are biological agents that are added to the immunoassay system and can react with endogenous antibodies, thereby effectively preventing non-analyte-mediated antibody bridging. Blockers 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 an active reagent of human anti-specific animal antibodies (such as human anti-mouse antibodies). The blocking effect depends on the affinity of human heterologous antibodies. Usually, the affinity of human heterologous antibodies is usually around 10. -5 -10 -6 Therefore, during the use of passive blockers, high concentrations are often added to reduce interference. In addition, 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 components of interfering antibodies, thereby blocking the generation of unintended binding. This preparation can eliminate various heterophilic interferences, has specific binding force to heterophilic antibodies that cause interference, and can effectively block at low concentrations, minimizing the impact. Active blockers are, for example, mixed mouse McAbs produced from HA and HAAA as immunogens. Or they can also be mouse anti-human IgM McAbs. Those skilled in the art have a strong demand for active blockers with good performance. Summary of the invention

[0005] The present application provides an anti-human IgM antibody or blocker, which has good binding reaction activity to human IgM and has good blocking and immune interference elimination effects when used in immunoassays, and provides an important source of blocker raw materials.

[0006] In order to achieve the above-mentioned object, according to one aspect of the present invention, an anti-human IgM antibody or blocker is provided, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 17, 37, 57, and 77 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19, 39, 59, and 79.

[0007] In order to achieve the above object, according to the second aspect of the present invention, an anti-human IgM antibody or blocking agent is provided, wherein the anti-human IgM antibody or blocking agent comprises 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] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, an anti-human IgM antibody or blocker 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 shown in any one of SEQ ID NOs: 17, 37, 57, and 77; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 19, 39, 59, and 79.

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

[0011] In order to achieve the above object, according to the fifth aspect of the present invention, a blocking agent is provided, wherein the blocking agent comprises the above anti-human IgM antibody.

[0012] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above anti-human IgM antibody or the above blocking agent.

[0013] In order to achieve the above object, according to the seventh aspect of the present invention, a method for reducing / eliminating endogenous interference is provided, the method comprising: adding the above antibody or the above blocking agent to the immunoassay system.

[0014] In order to achieve the above object, according to an eighth aspect of the present invention, a method of immunoassay is provided, the method comprising: adding the above antibody or the above blocking agent to an immunoassay system.

[0015] In order to achieve the above object, according to the ninth aspect of the present invention, a nucleic acid is provided, wherein the nucleic acid encodes the above anti-human IgM antibody or blocker.

[0016] In order to achieve the above object, according to the tenth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0017] In order to achieve the above object, according to the eleventh aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above anti-human IgM antibody or blocking agent.

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

[0019] In order to achieve the above-mentioned purpose, according to the thirteenth aspect of the present invention, there is provided a use of the above-mentioned anti-human IgM antibody, blocker, reagent or kit in the preparation of a product for detecting IgM or blocking IgM interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The results of reducing SDS-PAGE of Anti-IgM 15D2, Anti-IgM 10E2, Anti-IgM 5H9, and Anti-IgM 15G4 are shown. DETAILED DESCRIPTION

[0022] In the first aspect, an embodiment of the present invention provides an anti-human IgM antibody or blocker, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 17, 37, 57, and 77, and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19, 39, 59, and 79.

[0023] 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 anti-human IgM antibody or blocker 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 anti-human IgM antibody or blocker described in the first aspect.

[0024] For example, the 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; the 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 the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies or antigen-binding fragments, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0026] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region 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] The definition methods of CDR are well known in the art, and the CDR definition methods 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., US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that 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 extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person 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 Table 1 also belong to the scope of protection of the present disclosure.

[0029] Table 1: CDR Definition 1

[0030] CDR Kabat AbM2 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 the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the 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 by Kabat et al., U.S. Patent. 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] 3If 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 system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[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 the 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 - L97

[0045] According to an embodiment of the present invention, the antibody or blocking agent comprises a complementary determining region of any of the following groups:

[0046] 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 .

[0047] In a second aspect, an embodiment of the present invention provides an anti-human IgM antibody or a blocking agent, wherein the anti-human IgM antibody or the blocking agent comprises a complementary determining region of any one of the following groups:

[0048] Group No. 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 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 other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; 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.

[0051] 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.

[0052] In an optional embodiment, the anti-human IgM antibody or blocking agent of the first aspect or the second aspect further has 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 framework region of the anti-human IgM antibody or blocker 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 above-mentioned corresponding framework region.

[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 IgM with high affinity.

[0056] There are many methods for determining antibody affinity (KD), which can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).

[0057] In an alternative embodiment, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example by using a kinetic assay such as Biosensor system of the system.

[0058] In a third aspect, an embodiment of the present invention provides an anti-human IgM antibody or blocker, 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 shown in any one of SEQ ID NOs: 17, 37, 57, and 77, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 19, 39, 59, and 79.

[0059] In an optional embodiment, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect 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 an optional embodiment, the antibody or blocking agent described in the first aspect, the second aspect or the third aspect comprises any combination of the following heavy chain variable regions and light chain variable regions:

[0062]

[0063]

[0064] In an optional embodiment, the anti-human IgM antibody or blocker described in the first aspect, the second aspect, and the third aspect further comprises a constant region.

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

[0066] In an optional embodiment, 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 alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

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

[0069] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0070] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0071] In an alternative embodiment, the species origin of the constant region is mouse.

[0072] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in any one of SEQ ID NO:15, 35, 55, 75, and the light chain constant region (CL) sequence is as shown in any one 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 above-mentioned constant region (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 F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0075] The antigen-binding fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, it is easy for those skilled in the art to obtain the above antigen-binding fragments.

[0076] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0077] In a fourth aspect, the present invention provides an anti-human IgM antibody or blocker, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 18, 38, 58, and 78, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 20, 40, 60, and 80.

[0078] In an optional embodiment, the antibody or blocking agent described in the first aspect, the second aspect, the third aspect or the fourth aspect comprises any combination of the following heavy chains 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] In a fifth aspect, the present invention provides a blocking agent, which includes the above-mentioned anti-human IgM antibody.

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

[0082] As mentioned above, the anti-human IgM antibodies or blockers in some embodiments or examples of the present invention can effectively bind to IgM, so the reagents or kits containing the anti-human IgM antibodies or blockers can effectively block the binding interference of IgM. The reagents or kits provided by the present invention can be used, for example, for detection involving the use of IgM and its antibody-specific binding properties such as immunoblotting and immunoprecipitation. As mentioned above, the anti-human IgM antibodies or blockers in some embodiments or examples of the present invention have higher binding activity or affinity with human IgM, so the reagents or kits containing the anti-human IgM antibodies or blockers 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 immunoassay system.

[0084] In an eighth aspect, the present invention provides an immunoassay method, comprising: adding the above-mentioned antibody or blocking agent to an immunoassay system.

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

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

[0087] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

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

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

[0090] In a fourteenth aspect, the present invention provides the use of the above-mentioned antibody or blocker or the above-mentioned 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 the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-human IgM antibody or blocker, for example, separating and purifying the anti-human IgM antibody or blocker from the culture product of a recombinant cell that can recombinantly express the anti-human IgM antibody or blocker as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-human IgM antibody or blocker of the present invention, it belongs to the protection scope of the present invention.

[0092] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they 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 those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0094] 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 skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (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 (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). 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.

[0095] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

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

[0097] 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 purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting anti-IgM monoclonal antibody was prepared in this laboratory and revived for future use.

[0098] (1) Antibody gene preparation

[0099] mRNA was extracted from the hybridoma cell line secreting anti-IgM monoclonal antibodies, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

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

[0101] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 309bp-333bp, and there was a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 351bp-369bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

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

[0103] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced 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; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.7kb-0.71kb Light Chain gene fragment and the 1.38kb-1.40kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0104] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0105] 2. Recombinant Antibody Production

[0106] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a proteinA affinity chromatography column. Take 6ug layer of purified antibodies for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

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

[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 Performance testing of antibodies

[0111] 1. Affinity Analysis

[0112] Purified antibodies were diluted in advance, and human IgM antigen (from Feipeng Bio) was diluted in gradients. The binding and dissociation curves of antigen and antibody were tested on the Biacore 8K+ device using a CM5 chip that had been pre-coupled with goat anti-mouse IgG. The instrument automatically fitted 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 to IgM antigen was 1.0×10 -12 M≤KD<1.0×10 -7 M, and the affinity of the control antibody to human IgM antigen is better than that of the control antibody. (KD represents the equilibrium dissociation constant, i.e., 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 a blocker in CKMB (creatine kinase isoenzyme, Creatine Kinase-MB) to analyze the blocking effect of the antibody in actual detection.

[0115] The detection process includes: aspirating 1.5ul of the sample on the sample plate, and at the same time aspirating 200ul of R1 (Tris buffer) containing 0.1mg / ml blocking agent, adding them to the colorimetric cup, mechanically stirring, and incubating at 37°C for 5 minutes; adding 50ul of R2 (latex coupled with mouse antibody to be analyzed) to the colorimetric cup, mechanically stirring; the detection wavelength is 570nm, and the instrument records the absorbance values ​​at 7 minutes and 30 seconds and 12 minutes from the addition of the sample. The difference is the reactivity of the sample, and the reactivity is proportional to the concentration of the sample to be tested in the sample.

[0116] In the CKMB project, the specific responsiveness data are shown in Table 3 below.

[0117] Table 3 CKMB project blocking effect table

[0118] Clinical sample information Without 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, in the latex enhanced turbidimetry platform CKMB project, the blocker described in the present invention can significantly reduce the abnormal test results caused by false positive interference, the test results are close to those of negative samples, and do not affect the test results of positive samples, which greatly improves the accuracy of clinical test results.

[0121] In the PGⅡ (pepsinogen II) project, the specific reactivity data are shown in Table 4 below.

[0122] Table 4 PGⅡ project blocking effect table

[0123]

[0124]

[0125] For the PCT (procalcitonin) project, the specific response data are shown in Table 5 below.

[0126] Table 5 PCT project blocking effect table

[0127]

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

[0129] Table 6 D-dimer project blocking effect table

[0130] Clinical sample information Without blocking agent 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 sample 5 1285.3 385.59 102.32 514.12 668.36

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0132] Some of the amino acid sequences involved in this application are shown in Table 7:

[0133] Table 7 Amino Acid Sequence Listing

[0134]

[0135]

[0136]

[0137]

Claims

1. An anti-human IgM antibody or blocker, comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 17, 37, 57, and 77 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19, 39, 59, and 79.

2. The anti-human IgM antibody or blocking agent according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-human IgM antibody or blocking agent, characterized in that: The anti-human IgM antibody or blocking agent comprises a complementary determining region of any of the following groups: Optionally, the anti-human IgM antibody or blocking agent comprises any of the following groups of framework regions or has at least 80% identity with any of the following groups of framework regions: 。 4. An anti-human IgM antibody or blocking agent, 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 any one of SEQ ID NOs: 17, 37, 57, and 77; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 19, 39, 59, and 79; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: Optionally, the anti-human IgM antibody or blocking agent further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, 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; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in any one of SEQ ID NO: 15, 35, 55, 75 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in any one of SEQ ID NOs: 16, 36, 56, 76 or has at least 80% identity thereto; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. An anti-human IgM antibody or blocking agent, comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 18, 38, 58, and 78; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 20, 40, 60, and 80.

6. A reagent or a kit, characterized in that: The reagent or kit comprises the anti-human IgM antibody or blocking agent according to any one of claims 1 to 5.

7. A method for reducing / eliminating endogenous interference, characterized in that: The method comprises: adding the anti-human IgM antibody or blocking agent according to any one of claims 1 to 5 to an immunoassay system.

8. A method of immunoassay, characterized in that: The method comprises: adding the antibody or blocking agent according to any one of claims 1 to 5 to an immunoassay system.

9. A nucleic acid, a vector, a cell or a method for preparing the antibody or the blocking agent according to any one of claims 1 to 5, characterized in that: The nucleic acid encodes the antibody or blocking agent according to any one of claims 1 to 5; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method comprises culturing the above-mentioned cell.

10. Use of the anti-human IgM antibody or blocker according to any one of claims 1 to 5, or the reagent or kit according to claim 6 in the preparation of a product for detecting IgM or blocking IgM interference.

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