WO2023088443A1

WO2023088443A1 - Anti-human igm antibody and preparation method therefor and use thereof

Info

Publication number: WO2023088443A1
Application number: PCT/CN2022/132980
Authority: WO
Inventors: 孟媛; 唐丽娜; 钟冬梅
Original assignee: 东莞市朋志生物科技有限公司
Priority date: 2021-11-20
Filing date: 2022-11-18
Publication date: 2023-05-25
Also published as: CN116143931A; CN116143931B

Abstract

The present invention relates to an anti-human IgM antibody and a preparation method therefor and a use thereof. The anti-human IgM monoclonal antibody prepared by the present invention has high affinity, high reaction activity, high sensitivity and specificity for human IgM. The anti-human IgM monoclonal antibody of the present invention is used for immunodetection and has good effects of blocking and eliminating immune interference, and an important blocking agent raw material source is provided.

Description

A kind of anti-human IgM antibody and its preparation method and application

priority information

This application claims the priority and benefit of the patent application No. 202111410598.8 filed with the State Intellectual Property Office of China on November 20, 2021, which is hereby incorporated by reference in its entirety.

technical field

The invention belongs to the technical field of antibodies. More specifically, it relates to an anti-human IgM antibody and its preparation method and use.

Background technique

Antigen-antibody reaction-based immunoassay methods are widely used, and are divided into different detection methods according to different antibody markers, such as: enzyme-linked immunosorbent immunoassay, radioimmunoassay, chemiluminescence, etc. In clinical applications, the accuracy of immunoassay results is often affected to varying degrees by interfering substances in patient serum, resulting in false test results. Interfering substances in serum can be divided into endogenous interference and exogenous interference. Among endogenous interferences, rheumatoid factor and heterophile antibody (HA) are the most common. Therefore, the research and development of effective means to reduce and eliminate the interference of rheumatoid factor and heterophile antibody (HA) is an important topic to ensure the reliability of medical immunological test results and protect the interests of doctors and patients. In order to eliminate the interference of rheumatoid factor and heterophilic antibody (HA) in immunodiagnosis, the simplest and most effective method is to add blocking agents to the detection system to directly block the combination of interfering substances and antibodies or antigens in the detection system.

A blocking agent is a biological agent added to an immunoassay system that reacts with endogenous antibodies to effectively prevent non-analyte-mediated antibody bridging. Blockers can be divided into passive blockers and active blockers. Passive blocking agents 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 reagents have limited uses and can only block one kind of human anti-specific animal antibody active reagents (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 in the range of 10 ^5-106 K value range ^. Therefore, passive blockers are often added at high concentrations to reduce interference during use. In addition, heterophilic interference involves many components, and different passive blocking agents are required to block different kinds of heterophilic antibodies.

Active blocking agents are specific to human immunoglobulins, and can specifically, actively, and efficiently neutralize the components of interfering antibodies, thereby blocking the generation of unintended binding, such as IIR and HBR in commercial reagents. This preparation can eliminate all kinds of heterophilic interference, has specific binding ability to the interfering heterophilic antibody, and only needs a low concentration to block it efficiently, minimizing the impact. During active blocking, the effect of eliminating interference depends on the affinity of the active agent for the heterophilic antibody. Due to the high affinity of active blockers, the blocking ability in some assays is stronger than that of passive blockers.

Although there are many blocker products on the market, they all have certain performance defects. In addition, the amount of blocker itself is very large, so the market urgently needs blockers with better performance and lower cost.

Contents of the invention

The invention utilizes recombinant antibody technology to develop an anti-human IgM antibody, which has high affinity, high reactivity, high sensitivity or specificity to human IgM. The anti-human IgM monoclonal antibody has good effects of blocking and eliminating immune interference when used in immunodetection, and provides an important source of blocking agent raw materials for immunodiagnosis.

The present disclosure provides an anti-human IgM antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or are any of SEQ ID NO:15, SEQ ID NO:24 An amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region shown; the LCDR1-LCDR3 includes or is the same as that of the light chain variable region shown in any one of SEQ ID NO:16 and SEQ ID NO:25-27 Consensus amino acid sequence of LCDR1-LCDR3.

Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (CDRs for short) are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

The present disclosure provides an anti-human IgM antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising the following complementarity determining regions:

HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;

HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;

HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of it;

LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of it;

LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of it;

LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.

Optionally, the antibody or its functional fragments further comprise the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region;

Wherein, HFR1 includes an amino acid sequence as shown in any one of SEQ ID NO:7, 21, or an amino acid sequence having at least 80% homology therewith;

HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto;

HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto;

HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;

LFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 22, or an amino acid sequence having at least 80% homology therewith;

LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto;

LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 13, 23, or an amino acid sequence having at least 80% homology therewith;

LFR4 comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

The present disclosure provides an anti-human IgM antibody or a functional fragment thereof, the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region comprises the above-mentioned HCDR1- HCDR3 and the above-mentioned HFR1-HFR4, the light chain variable region includes the above-mentioned LCDR1-LCDR3 and the above-mentioned LFR1-LFR4.

Optionally, the amino acid sequence of the heavy chain variable region is shown in any of SEQ ID NO: 15, 24; the amino acid sequence of the light chain variable region is shown in any of SEQ ID NO: 16, 25, 26, 27 Show.

Optionally, the antibody or functional fragment thereof further comprises a constant region.

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region.

Optionally, 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 .

Optionally, the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man.

Optionally, the species source of the constant region is mouse.

Optionally, the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17; the light chain constant region is SEQ ID NO: 18 or an amino acid sequence with SEQ ID NO: 17 NO:18 is an amino acid sequence with more than 80% homology.

Optionally, the functional fragment is selected from any one of F(ab') ₂ , Fab', Fab, Fv and scFv of the antibody.

The present disclosure provides an anti-human IgM antibody or a functional fragment thereof, comprising a heavy chain and/or a light chain, the heavy chain comprising the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain comprising The above-mentioned light chain variable region and the above-mentioned light chain constant region.

Optionally, the amino acid sequence of the heavy chain is shown in any of SEQ ID NO: 19, 28; the amino acid sequence of the light chain is shown in any of SEQ ID NO: 20, 29, 30, 31.

The present disclosure provides an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.

Optionally, the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioactive isotope , fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, One or more of glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label, drug.

Optionally, the coupling moiety is selected from solid phase supports, such as magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

The present disclosure also provides a nucleic acid encoding the antibody or a functional fragment thereof according to any one of the above.

The present disclosure also provides a cell comprising the above-mentioned nucleic acid.

The present disclosure also provides a method for preparing any of the above-mentioned antibodies or functional fragments thereof, the method comprising culturing the above-mentioned cells.

The present disclosure provides the application of the above-mentioned antibody or its functional fragment in immunoassay or in the preparation of immunoblocking agent.

The present disclosure provides a blocking agent, which includes the above-mentioned antibody or a functional fragment thereof.

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

The present disclosure provides a detection reagent or kit, which includes the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate or the above-mentioned blocking agent.

The present disclosure provides a method for reducing/eliminating endogenous interference, adding the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate, or the above-mentioned blocking agent to the immune detection system.

The present disclosure provides an immunoassay method, comprising: adding the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate, or the above-mentioned blocking agent to an immunoassay system.

The present disclosure provides a method for detecting IgM, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof, the above-mentioned antibody conjugates, the above-mentioned contacting said nucleic acid, said cell or said reagent or kit with a sample from said subject to carry out a binding reaction; and B) detecting an immune complex generated by the binding reaction.

The amino acid sequence involved in the disclosure of this application is as follows:

Description of drawings

Figure 1 is the reduced SDS-PAGE results of 8G5MRAb1 to 8G5MRAb6 antibodies (the lanes are from left to right the reduced SDS-PAGE results of 8G5MRAb1, 8G5MRAb2, 8G5MRAb3, 8G5MRAb4, 8G5MRAb5 and 8G5MRAb6).

Detailed ways

Some embodiments of the present disclosure provide an anti-human IgM antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or are the same as SEQ ID NO: 15, SEQ ID The amino acid sequence consistent with HCDR1-HCDR3 of any heavy chain variable region shown in NO:24; said LCDR1-LCDR3 includes or is the light chain shown in any one of SEQ ID NO:16, SEQ ID NO:25-27 The amino acid sequence consistent with LCDR1-LCDR3 of the variable region. Herein, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

As used herein, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

Herein, 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. Commonly used CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This disclosure uses the Kabat annotation standard to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present invention.

The system for defining the CDR is not particularly limited, and the CDR sequences defined by conventional systems in the art are within the protection scope of the present application. For example, the CDR definition method is referred to such as Kabat et al., U.S.Dept.of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) or Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.

Table 1: CDR Definition ¹

¹ Numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below).

² "AbM" as used in Table 1 has a lowercase "b" and refers to the CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can unambiguously map this Kabat numbering system to any variable region sequence, without reliance 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. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully able to convert the sequence numbers of the sequence listing into Kabat numbers.

In an optional embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (CDRs for short) are defined by the Kabat, Chothia, IMGT, AbM or Contact systems. In an optional embodiment, the HCDR1, HCDR2, and HCDR3 sequentially comprise amino acid sequences at positions 31-35, 50-65, and 94-99 of the heavy chain variable region, or sequentially such as 31 amino acid sequences of the heavy chain variable region. ～35, 50～65, 94～99 amino acid sequences;

The LCDR1, LCDR2 and LCDR3 sequentially comprise amino acid sequences at positions 24-34, 50-56, and 89-95 of the light chain variable region, or sequentially such as 24-34 and 50-56 of the light chain variable region , 89-95 amino acid sequence.

Moreover, the numbering of the amino acid positions is based on the Kabat numbering system.

In an alternative embodiment, the antibody or functional fragment thereof comprises the following complementarity determining regions:

As used herein, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to an antibody heavy chain variable region (which may be denoted as VH) and a light chain variable region (which may be denoted as VL) The regions other than the CDRs in ; where the heavy chain framework region is denoted by "HFR" and can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region Regions are indicated by "LFR" and can be further subdivided into contiguous regions separated by CDRs, comprising the LFR1, LFR2, LFR3 and LFR4 framework regions.

In an optional embodiment, the antibody or its functional fragments further comprise the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and HFR3 of the light chain variable region. LFR4;

In an alternative embodiment,

The amino acid sequence of HFR1 is as shown in any of SEQ ID NO: 7, 21, or has at least 80% homology therewith;

The amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;

The amino acid sequence of HFR3 is as shown in SEQ ID NO:9 or has at least 80% homology therewith;

The HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or has at least 80% homology therewith;

The amino acid sequence of LFR1 is as shown in any of SEQ ID NO: 11, 22, or has at least 80% homology therewith;

The amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;

The amino acid sequence of LFR3 is as shown in any of SEQ ID NO: 13, 23, or has at least 80% homology therewith;

The LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.

Herein, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs according to The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

It should be noted that, in other embodiments, the amino acid sequence of each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the above-mentioned corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12 , 13 or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% homology. In an optional embodiment, the antibody or its functional fragment has a KD≤10 ^-7 M, KD≤10 ^-8 M, KD≤10 ^-9 M, KD≤10 ^-10 M or KD≤10 ^-11 binding affinity to human IgM.

In an alternative embodiment, the antibody or functional fragment thereof binds to human IgM with an affinity of KD≤1.06×10 ⁻¹⁰ or KD≤6.14×10 ⁻¹¹ or KD≤8.02×10 ⁻¹² . The detection of KD is carried out with reference to the method in the examples of the present disclosure.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 24;

In an optional embodiment, the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 16, 25, 26, or 27.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26.

In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.

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

In an alternative embodiment, the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region" can be represented as CH; a "light chain constant region" can be represented as CL .

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

In an optional embodiment, the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, gamecock or man.

In an optional embodiment, the species source of the constant region is mouse.

In an optional embodiment, the heavy chain constant region sequence (CH) is such as SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17; the light chain constant region is SEQ ID NO: 17 ID NO: 18 or an amino acid sequence having more than 80% homology with SEQ ID NO: 18.

It should be noted that, in other embodiments, the constant region sequence provided by the present disclosure 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% homology.

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

Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived. Those skilled in the art can easily understand from the content of the present disclosure that the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or methods of splitting disulfide bonds by chemical reduction. On the basis of the structure of the intact antibody disclosed in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.

Functional fragments of the above-mentioned antibodies can also be synthesized by recombinant genetics techniques also known to those skilled in the art or by, for example, automatic peptide synthesizers such as those sold by Applied BioSystems.

In an optional embodiment, the amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 28; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 29, 30, 31 Show.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:29.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 30.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 31.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:28; the amino acid sequence of the light chain is shown in SEQ ID NO:20.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:28; the amino acid sequence of the light chain is shown in SEQ ID NO:29.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:28; the amino acid sequence of the light chain is shown in SEQ ID NO:30.

In an optional embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO:28; the amino acid sequence of the light chain is shown in SEQ ID NO:31.

Some embodiments of the present disclosure also provide an anti-human IgM antibody or a functional fragment thereof, the antibody or its functionality comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region The above-mentioned HCDR1-HCDR3 and the above-mentioned HFR1-HFR4 are included, and the light chain variable region includes the above-mentioned LCDR1-LCDR3 and the above-mentioned LFR1-LFR4.

In an optional embodiment, the species source of the constant region is mouse.

Some embodiments of the present disclosure also provide an anti-human IgM antibody or a functional fragment thereof, including a heavy chain and/or a light chain, and the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; The light chain includes the above-mentioned light chain variable region and the above-mentioned light chain constant region.

Some embodiments of the present disclosure also provide an antibody conjugate comprising the above-mentioned antibody or a functional fragment thereof and a coupling moiety coupled thereto.

In an optional embodiment, the coupling moiety is selected from a purification tag (such as a His tag); a detectable label, such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromogenic Group labels, electron-dense labels such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase One or more of enzymes, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling.

In an alternative embodiment, the coupling moiety is selected from a solid phase carrier.

In an optional embodiment, the solid support is selected from microspheres, plates or membranes.

In an optional embodiment, the solid phase support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

In an optional embodiment, the solid phase carrier is magnetic microspheres.

Some embodiments of the present disclosure also provide a nucleic acid encoding the above antibody or a functional fragment thereof.

Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence. DNA nucleic acid is used when it is ligated into a vector.

Some embodiments of the present disclosure also provide vectors comprising the nucleic acid molecules described above.

Some embodiments of the present disclosure also provide cells containing the vectors described above.

Some embodiments of the present disclosure also provide a method for preparing an antibody or a functional fragment thereof, which includes: culturing the above-mentioned cells, and separating and purifying the antibody or a functional fragment thereof from the culture product.

On the basis of the amino acid sequence of the antibody or its functional fragment in the present disclosure, those skilled in the art can easily think of using genetic engineering technology or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, from the It is easy for those skilled in the art to separate and purify the antibody or its functional fragment from the culture product of recombinant cells expressing the antibody or its functional fragment as described in any one of the above. Any technique used to prepare the antibody or its functional fragments of the present disclosure falls within the protection scope of the present disclosure.

Some embodiments of the present disclosure also provide the application of the above antibodies or functional fragments thereof in immunoassay or in the preparation of immunoblockers.

Some embodiments of the present disclosure also provide a blocking agent, which includes the above-mentioned antibody or a functional fragment thereof.

A blocking agent is a biological agent added to an immunoassay system that reacts with endogenous antibodies to effectively prevent non-analyte-mediated antibody bridging.

The antibody of the present invention or its functional fragment is specific to human IgM immunoglobulin, and can specifically, actively and efficiently neutralize interfering IgM components, thereby blocking the generation of unintended combination. The antibody or its functional fragments of the present invention can effectively block only a low concentration, and minimize the impact.

In an optional embodiment, the concentration of the antibody in the blocking agent is 5-100 μg/ml.

In an optional embodiment, the concentration of the antibody in the blocking agent is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/ml.

Some embodiments of the present disclosure also provide a detection reagent or kit, which includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate or the above-mentioned blocking agent.

In an alternative embodiment, the blocking agent contained in the kit may be in the form of a liquid solution, attached to a solid support, or a dry powder. When the blocking agent is a liquid solution, the liquid solution may be an aqueous solution. When the immunoblocking agent is in the form attached to a solid support, the preferred solid support may be a chromatographic medium such as a film, test strip, plastic bead or plate, or a microscope slide. When the blocking agent is a dry powder, the powder can be reconstituted by adding an appropriate solvent.

Some embodiments of the present disclosure also provide a method for reducing/eliminating endogenous interference, adding the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate, or the above-mentioned blocking agent to the immune detection system.

In an optional embodiment, the endogenous interference is rheumatoid factor interference or heterophile antibody interference.

Some embodiments of the present disclosure also provide an immunoassay method, comprising: adding the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate, or the above-mentioned blocking agent to an immunoassay system.

Some embodiments of the present disclosure also provide a method for detecting IgM, comprising: A) using any of the above-mentioned antibodies or functional fragments thereof, or the above-mentioned antibody conjugates under conditions sufficient for a binding reaction to occur; The conjugate, or the above-mentioned reagent or kit is contacted with the sample from the subject to carry out the binding reaction; and B) detecting the immune complex generated by the binding reaction.

In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. For those that do not indicate specific conditions in the examples or implementations, proceed according to conventional conditions or conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit dosages herein, some methods and materials are now described. Unless otherwise stated, techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. This technique is fully explained in the literature, e.g., Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( M.J.Gait edited, 1984); "Animal Cell Culture (Animal Cell Culture)" (R.I.Freshney edited, 1987); "Methods in Enzymology" (Academic Press, Inc.); " "Handbook of Experimental Immunology" (D.M.Weir and C.C.Blackwell edited); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller and M.P.Calos edited, 1987); "Contemporary Methods in Molecular Biology (Current Protocols in Molecular Biology)" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., eds., 1994); and Contemporary Immunology Methods (Current Protocols in Immunology)" (J.E. Coligan et al. eds., 2011), each of which is expressly incorporated herein by reference.

In the following examples, restriction enzymes and rTaq DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.

Example

The features and performances in the present disclosure will be further described in detail in conjunction with the examples below.

Embodiment 1: Preparation of 8G5MRAb 1 antibody

1. Expression plasmid construction

(1) 8G5MRAb1 antibody gene preparation

In the early stage, the inventor obtained hybridoma cell lines secreting anti-human IgM monoclonal antibody (8G5MRAb1 antibody) through hybridoma preparation technology, extracted mRNA from the hybridoma cell line secreting anti-human IgM monoclonal antibody, and obtained DNA by RT-PCR method The product, which was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, was transformed into DH5α competent cells, and the Heavy Chain (heavy chain) and Light Chain (light chain) genes were respectively obtained after the colonies grew out. Each of the 4 clones was sent to a gene sequencing company for sequencing.

(2) Sequence analysis of 8G5MRab1 antibody variable region gene

Put the gene sequence obtained by sequencing in the above step 1-(1) into the Kabat antibody database for analysis, and use VNTI11.5 software to analyze and confirm that the genes amplified by the heavy chain and light chain primer pairs are correct, where Light In the gene fragment amplified by Chain, the light chain variable region (VL) gene sequence is 324bp, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the reconnected variable region (VH ) gene sequence is 348bp, belongs to the VH1 gene family, and has a 57bp leader peptide sequence in front of it.

(3) Construction of recombinant antibody expression plasmid

pcDNA ^™ 3.4

Vector is a recombinant antibody eukaryotic expression vector constructed. The expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, EcoRI, etc., and named as pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector subsequently; according to the above steps 1- (2) The obtained antibody variable region gene sequencing results in the pMD-18T vector, designed VL and VH gene-specific primers of the 8G5MRAb1 antibody, with HindIII and EcoRI restriction sites and protective bases at both ends, and amplified by PCR. The 0.73KB Light Chain gene fragment and the 1.4kb Heavy Chain gene fragment were amplified by the amplification method. The Heavy Chain and Light Chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. 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 Recombinant expression plasmids for Heavy Chain and Light Chain.

2. Screening of stable cell lines

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

Dilute the plasmid prepared in the above step 1-(3) to 40 μg/100 μL with ultrapure water, adjust the cell concentration of CHO cells to 1.43×10 ⁷ cells/mL in a centrifuge tube, mix 100 μL of the above plasmid with 700 μL of cells , transferred to the electroporation cup for electroporation, samples were counted on the 3rd, 5th, and 7th days after electroporation, and samples were collected on the 7th day for testing.

Dilute human IgM (purchased from Fapon) to 1 μg/mL with coating solution (main component NaHCO ₃ ), 100 μL per well, overnight at 4°C; the next day, wash with washing solution (main component Na ₂ HPO ₄ +NaCl) for 2 Once, pat dry; add blocking solution (20% BSA+80% PBS), 120 μL per well, incubate at 37°C for 1 hour, pat dry; add diluted CHO cell supernatant, 100 μL/well, incubate at 37°C Incubate under low temperature for 30 min (partial supernatant for 1 h); wash with washing solution 5 times, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, and incubate at 37°C for 30 min; wash with washing solution 5 times, pat dry; Color solution A (50 μL/well, main components citric acid + sodium acetate + acetanilide + carbamide peroxide), add color solution B (50 μL/well, main components citric acid + EDTA·2Na + TMB + concentrated HCl), Incubate for 10 min; add stop solution (50 μL/well, EDTA·2Na+concentrated H ₂ SO ₄ ); read OD value at 450 nm (reference 630 nm) on a microplate reader.

The results showed that the reaction OD was still greater than 1.0 after adding CHO cell supernatant and diluting 1000 times, and the reaction OD of wells without CHO cell supernatant was less than 0.1, indicating that the 8G5MRAb1 antibody produced after plasmid transient transfer was active against human IgM.

(2) Linearization of recombinant antibody expression plasmid

Prepare the following reagents: 50 μL of Buffer, 100 μg/tube of the plasmid prepared in step 1-(3), 10 μL of PvuⅠ enzyme, make up to 500 μL with sterile water, digest overnight in a 37°C water bath; first use an equal volume of phenol/chloroform/iso Amyl alcohol (lower layer) (the volume ratio of phenol: chloroform: isoamyl alcohol is 25:24:1), and then sequentially extract with chloroform (water phase); 0.1 volume (water phase) of 3M sodium acetate and 2 volumes Precipitate with ethanol on ice, rinse the precipitate with 70% ethanol, remove the organic solvent, rethaw with an appropriate amount of sterilized water after the ethanol has evaporated completely, and finally measure the concentration.

(3) Stable transfection of recombinant antibody expression plasmids, pressurized selection of stable cell lines

Dilute the plasmid obtained in step 2-(2) to 40 μg/100 μL with ultrapure water, adjust the cell concentration of CHO cells to 1.43×10 ⁷ cells/mL in a centrifuge tube, mix 100 μL of the above plasmid with 700 μL CHO cells, and transfect Put into the electroporation cup for electroporation, and count the next day; 25μmol/L MSX 96-well pressurized culture for about 25 days.

Observe and mark the clone wells with cells under a microscope, and record the confluence; take the culture supernatant and send samples for detection; select cell lines with high antibody concentration and relative concentration and transfer to 24 wells, and transfer to 6 wells in about 3 days; preserve the species after 3 days Batch culture, adjust the cell density of CHO cells to 0.5×10 ⁶ cells/mL, take 2.2 mL for batch culture, adjust the cell density of CHO cells to 0.3×10 ⁶ cells/mL, take 2 mL for seed preservation; 7 days 6 After the wells were cultured in batches, the supernatant was sent for testing, and the cell lines with smaller antibody concentration and cell diameter were selected to be transferred to TPP for preservation and passage.

3. Production of recombinant antibodies

(1) Cell expansion

After recovery, the cells obtained in step 2-(3) were first cultured in a 125mL shake flask, the inoculation volume was 30mL, the medium was 100% Dynamis medium, placed at a speed of 120r/min, the temperature was 37°C, and the carbon dioxide was 8% in the shaker. After culturing for 72 hours, inoculate the expanded culture at an inoculation density of 500,000 cells/mL. The expanded culture volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.

(2) Shake flask production and purification

Shake flask parameters: rotation speed 120r/min, temperature 37°C, carbon dioxide concentration 8%. Fed-batch feeding: Feed feeding starts every day when the shake flask is cultured for 72 hours. HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day). Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day. The protein A affinity chromatography column was used for affinity purification, and the purification steps were carried out by conventional methods in the art. Take 6.6 μg of the purified antibody for reducing SDS-PAGE, and the electropherogram is shown in Figure 1. Two bands were shown after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain). The results showed that the purified antibody was 8G5MRAb1.

8G5MRAb1 obtained through the above steps, after sequencing and Kabat analysis, the heavy chain CDR1, CDR2, and CDR3 of 8G5MRAb1 are shown in the amino acid sequence of SEQ ID NO: 1-3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: As shown in the amino acid sequence of 4 to 6, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain has the amino acid sequence shown in SEQ ID NO: 16. The amino acid sequence shown in ID NO:19, the light chain has the amino acid sequence shown in SEQ ID NO:20.

4. The inventor analyzed the structure of 8G5MRAb1, designed mutation primers, repeated steps 1-(3) to 3-(2), and obtained mutant antibodies after activity identification, which were named 8G5MRAb2, 8G5MRAb3, 8G5MRAb4, 8G5MRAb5, 8G5MRAb6, 8G5MRAb7, 8G5MRAb8. Among them, the amino acid sequences of the heavy chains and light chains of the eight antibodies 8G5MRAb1 to 8G5MRAb8 (abbreviated as 8G5MRAb1-8) are shown in Table 2.

Table 2: Amino acid sequences of the heavy and light chains of 8G5MRab 1 to 8

Example 2: Affinity analysis, activity identification and performance evaluation of different antibodies

1. Affinity analysis

Using the AMC sensor, the purified antibody (8G5MRAb1-8) obtained in Example 1 and the control antibody were diluted to 20 μg/mL with PBST, and human IgM (purchased from Fapon) was serially diluted with PBST.

Operation process: Equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH value of 1.69 The GLY solution and buffer solution 3 (PBST) were used to regenerate the sensor, and output data, the specific data are shown in Table 3.

Table 3: Affinity results for different antibodies

Note: KD means the equilibrium dissociation constant, that is, affinity; Kon means the association rate constant; Kdis means the dissociation rate constant.

2. Activity identification

Dilute human IgM (purchased from Fapon) to 3 μg/mL with coating solution (main component NaHCO ₃ ), 100 μL per well, overnight at 4°C; the next day, wash with washing solution (main component Na ₂ HPO ₄ +NaCl) for 2 Once, pat dry; add blocking solution (20% BSA+80% PBS), 120 μL per well, incubate at 37°C for 1 h, pat dry; add diluted 8G5MRAb1-8 antibodies and control antibodies obtained in Example 1, 100 μL/well, incubate at 37°C for 30 min; wash with washing solution 5 times, pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37°C for 30 min; wash with washing solution 5 times, pat dry; Add chromogenic solution A (50 μL/well, main components citric acid + sodium acetate + acetanilide + carbamide peroxide), add chromogenic solution B (50 μL/well, main components citric acid + EDTA 2Na + TMB + concentrated HCl ), incubate for 10 min; add stop solution (EDTA·2Na+ concentrated H ₂ SO ₄ ), 50 μL/well; read the OD value at 450 nm (refer to 630 nm) on a microplate reader, and the detection results are shown in Table 4. 8G5MRAb1～8 antibodies The binding activity of the antibody was higher than that of the control antibody.

Table 4: Binding activity results of different antibodies

3. Antibody performance evaluation

3.1 Verify the blocking effect on the CTNI fluorescence platform

In the paired detection of the CTNI fluorescence platform, the experimental group used the 8G5 series of blocking agent raw materials (8G5MRAb1-8 obtained in Example 1 as the blocking agent, namely the experimental group) and the market mainstream blocking agent raw materials to treat the sample pads respectively. The sample pads of the group were not treated (ie no blocking agent (blank) group); the false positive samples were detected respectively, and the experimental results are shown in Table 5. The results showed that the experimental group had an obvious elimination effect on false positive samples, indicating that the 8G5 series blocker raw materials had a blocking effect, and it was also slightly better than the mainstream blocker raw materials in the market.

Table 5: Blocking effects of different antibodies

In Table 4, the T/C value description: the sample to be tested is added to the sample port of the detection reagent card, under the action of the lateral capillary, the sample to be tested first passes through the binding pad, and has a specific interaction with the fluorescent group-labeled antibody on the binding pad. Immunobinding, each combined to form an antigen-antibody fluorescent complex, which is immobilized in the T-line. The C line is coated with a substance that reacts with the free fluorescent group-labeled antibody. When the free fluorescent group-labeled antibody passes through the C line, it can specifically immunocombined with the substance on the C line, thereby being fixed on the C line. in line. The fluorescence intensity of the two bands detected by the fluorescence immunoassay analyzer is reflected by the peak area, and the T/C value is calculated by the calculation software of the instrument itself. The instrument reading T/C indicates the ratio of T peak area to C peak area. In quality control samples and positive samples, the higher the T/C, the higher the activity; the lower the T/C in the false positive samples, it represents the blocking effect The better; when the T/C value <0.1, it is judged as a negative sample.

3.2 Verify the blocking effect of different antibodies on the GRP chemiluminescence platform

In the paired detection of the GRP chemiluminescence platform, the experimental group added 8G5 series blocker raw materials (8G5MRAb1-8 obtained in Example 1 as blockers, namely the experimental group) and market mainstream blocker raw materials were added to the coating system Among them, the control group was not added to the coating system (that is, no blocking agent (blank) group); RF samples were tested respectively, and the experimental results are shown in Table 6. The results showed that the experimental group had an obvious elimination effect on RF specimens, indicating that the 8G5 series blocking agent raw materials had a blocking effect, and it was also slightly better than the mainstream blocking agent raw materials in the market.

Table 6: Blocking effects of different antibodies

The values in Table 5 are the OD values read by the chemiluminescence immunoassay analyzer, the lower the OD value, the weaker the detection signal, indicating the better blocking effect.

Example 3: Detection of antibody stability

The 8G5MRAb1-8 antibodies obtained in Example 1 were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the antibody samples were taken at 7 days, 14 days, and 21 days for state observation. The activity of the 21-day antibody sample was detected, and the OD result was detected by enzyme immunoassay. For the specific operation steps, refer to step 2 of Example 2. For the detection results, see Table 7. The results showed that under the three assessment conditions, the antibody was left for 21 days without any obvious change in the protein state, and the activity did not decrease with the increase of the assessment temperature. Therefore, it is further demonstrated that the 8G5MRAb1-8 antibodies obtained in Example 1 are stable.

Table 7: Antibody stability test results

样品浓度(ng/mL)Sample concentration (ng/mL)	125125	15.62515.625	00
4℃，21天样品4°C, 21 days sample	2.2622.262	1.9531.953	0.0380.038
-80℃，21天样品-80℃, 21 days sample	2.1922.192	1.9621.962	0.0210.021
37℃，21天样品37°C, 21 days sample	2.2422.242	1.9881.988	0.0360.036

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Industrial Applicability

The anti-IgM antibody provided by the present disclosure has high affinity, high reactivity, high sensitivity or specificity for human IgM. It has a good effect of blocking and eliminating immune interference when used in immune detection, and provides an important source of blocking agent raw materials for immunodiagnosis. Therefore, the anti-IgM antibody, immunoassay reagent and kit provided by the present disclosure all have excellent practical performance and broad market application prospects.

Claims

An anti-human IgM antibody or a functional fragment thereof, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that the HCDR1-HCDR3 comprises any of SEQ ID NO: 15 and SEQ ID NO: 24 The amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region; the LCDR1-LCDR3 includes LCDR1-LCDR3 of the light chain variable region shown in any one of SEQ ID NO:16 and SEQ ID NO:25-27 consensus amino acid sequence.
The antibody or its functional fragment according to claim 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, Chothia, IMGT, AbM or Contact system.
The antibody or its functional fragment according to claim 1, wherein the antibody or its functional fragment comprises the following complementarity determining regions:

HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;

HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;

HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of it;

LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of it;

LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of it;

LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
The antibody or its functional fragment according to any one of claims 1 to 3, wherein the antibody or its functional fragment further comprises framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region;

Wherein, HFR1 includes an amino acid sequence as shown in any one of SEQ ID NO:7, 21, or an amino acid sequence having at least 80% homology therewith;

HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto;

HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto;

HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;

LFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 22, or an amino acid sequence having at least 80% homology therewith;

LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto;

LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 13, 23, or an amino acid sequence having at least 80% homology therewith;

LFR4 comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.
An anti-human IgM antibody or a functional fragment thereof, characterized in that the antibody or its functionality comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region comprises claim 1 HCDR1-HCDR3 described in any one of claims 1-3 and HFR1-HFR4 described in claim 4, the light chain variable region comprising LCDR1-LCDR3 described in any one of claims 1-3 and claim 4 LFR1~LFR4;

Optionally, the amino acid sequence of the heavy chain variable region is shown in any of SEQ ID NO: 15, 24;

The amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 16, 25, 26, or 27.
The antibody or functional fragment thereof according to any one of claims 1-5, further comprising a constant region;

Optionally, said constant region comprises a heavy chain constant region and/or a light chain constant region;

Optionally, 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 ;

Optionally, the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , turkeys, fighting cocks or people;

Optionally, the species source of the constant region is mouse;

Optionally, the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;

The light chain constant region is SEQ ID NO: 18 or an amino acid sequence having more than 80% homology with SEQ ID NO: 18.
The antibody or its functional fragment according to any one of claims 1-6, wherein the functional fragment is selected from F(ab') 2 , Fab', Fab, Fv and scFv of the antibody any of the
An anti-human IgM antibody or a functional fragment thereof, comprising a heavy chain and/or a light chain, wherein the heavy chain comprises the heavy chain variable region according to claim 5 and the heavy chain variable region according to claim 6 chain constant region;

The light chain comprises the light chain variable region of claim 5 and the light chain constant region of claim 6;

Optionally, the amino acid sequence of the heavy chain is shown in any of SEQ ID NO: 19, 28;

The amino acid sequence of the light chain is shown in any one of SEQ ID NO: 20, 29, 30, 31.
An antibody conjugate, characterized in that the antibody conjugate comprises the antibody or its functional fragment according to any one of claims 1-8 and a coupling moiety coupled thereto;

Optionally, the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioactive isotope , fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, one or more of glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling;

Optionally, the coupling moiety is selected from solid phase supports, such as magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
A nucleic acid, characterized in that the nucleic acid encodes the antibody or a functional fragment thereof according to any one of claims 1-8.
A cell, characterized in that the cell comprises the nucleic acid according to claim 10.
A method for preparing the antibody or functional fragment thereof according to any one of claims 1-8, characterized in that the method comprises culturing the cell according to claim 11.
The use of the antibody or its functional fragment according to any one of claims 1 to 8 in immunoassay or in the preparation of immunoblocking agent.
A blocking agent, characterized in that the blocking agent comprises the antibody or functional fragment thereof according to any one of claims 1-8;

Optionally, the concentration of the antibody in the blocking agent is 5-100 μg/ml.
A detection reagent or kit, characterized in that the reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 8, the antibody conjugate of claim 9 or the antibody conjugate of claim 9 14 said blocking agent.
A method for reducing/eliminating endogenous interference, characterized in that the antibody or its functional fragment according to any one of claims 1 to 8, or the antibody conjugate of claim 9 is added to the immune detection system , or the blocking agent described in claim 14.
A method for immunodetection, characterized in that the method comprises:

The antibody or functional fragment thereof according to any one of claims 1 to 8, the antibody conjugate according to claim 9, or the blocking agent according to claim 14 is added to the immunoassay system.
A method for detecting IgM, characterized in that the method comprises:

A) Using the antibody or functional fragment thereof according to any one of claims 1 to 8, the antibody conjugate according to claim 9, or the blocking agent according to claim 14 under conditions sufficient for a binding reaction to occur or the reagent or kit of claim 15 is contacted with a sample from said subject for a binding reaction; and

B) Detection of immune complexes generated by the binding reaction.