Immunoglobulins and multimers thereof

Abstract

The invention relates to immunoglobulin which comprises a heavy chain, the heavy chain is obtained by fusing a first polypeptide and a second polypeptide, and the first polypeptide comprises a variable region located at the N end, an IgG CH1 region and a hinge region; the second polypeptide comprises an IgM CH2-CH3-CH4 region located at the C terminal; and the hinge region contains M prolines and N cysteines, M > = 2 and N > = 1, or M > = 1 and N > = 2. The polymer polymerized by the immunoglobulin has improved titer and excellent coating or labeling performance, so that the immunoglobulin can be widely applied to the field of detection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent applications with application number 202311072350.4 filed with the Chinese Patent Office on August 23, 2023, entitled “Immunoglobulins and their polymers” and with application number 202311703860.7 filed with the Chinese Patent Office on December 12, 2023, entitled “Immunoglobulins and their polymers”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of antibody engineering, and in particular, to an immunoglobulin and a polymer thereof. Background Art

[0004] The natural immunoglobulin (Ig) molecule contains four heterologous polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (L). The amino acid composition of the two heavy chains and the two light chains in the same Ig molecule is exactly the same. The molecular weight of the heavy chain is 50,000 to 75,000, and it is composed of 450 to 550 amino acid residues. Due to the different amino acid composition and arrangement order of the heavy chain constant region (CH region), its antigenicity is also different. Based on this, Ig can be divided into five classes, namely IgM, IgD, IgG, IgA and IgE. Among these five classes, IgG is a monomer structure. IgM is a basic antibody secreted by B cells. In the secretory form, it is mainly a pentamer composed of five Y-shaped monomers, which has extremely high affinity. The molecular weight of IgM is very large, so it is very effective in antigen agglutination reactions. IgM is a multivalent antibody. Since a monomeric antibody has two antigen-binding sites, IgM has 10 or 12 antigen-binding sites and is a multivalent antibody.

[0005] With the advancement of science and technology, in vitro diagnosis has become an important helper in clinical medical diagnosis, playing the role of the doctor's eyes. Immunodiagnostic reagents are a subdivision of in vitro diagnostic reagents. Immunodiagnosis is the application of immunological theories, techniques and methods to diagnose various diseases and determine immune status. Immunodiagnostic methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, fluorescence chromatography or colloidal gold chromatography. One of the main challenges in developing immunodiagnostic reagents is to obtain antibodies that strongly bind to antigens.

[0006] One of the most effective methods used in nature to achieve strong binding between antigens and their antibodies is through multivalent interactions. For example, naturally occurring IgM antibodies bind to antigens very tightly and efficiently, despite their relatively weak antigen-binding affinity in monomeric form. When more than one interaction occurs between two molecules, this functional affinity of multiple antibody-antigen interactions can be several orders of magnitude higher than the intrinsic affinity of a single antibody-antigen interaction. A variety of antibody multivalent engineering technologies have been developed, including domain swapping, linear fusion, chemical ligation, self-association, and heterodimerization. The development of an efficient self-aggregating immunoglobulin is of great significance for the preparation of high-performance multivalent antibodies. Summary of the invention

[0007] The first aspect of the present application relates to an immunoglobulin, comprising a heavy chain, wherein the heavy chain is obtained by fusion of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a variable region located at the N-terminus, an IgG CH1 region and a hinge region; and the second polypeptide comprises an IgM CH2-CH3-CH4 region located at the C-terminus; wherein the hinge region contains M proline and N cysteine, wherein M≥2 and N≥1, or wherein M≥1 and N≥2.

[0008] The second aspect of the present application relates to an immunoglobulin, comprising a heavy chain, wherein the amino acid sequence of the Fc region of the heavy chain is selected from any one of SEQ ID NO: 14 to SEQ ID NO: 20; or the constant region of the heavy chain is selected from any one of SEQ ID NO: 21 to SEQ ID NO: 27.

[0009] The third aspect of the present application relates to a nucleic acid encoding an immunoglobulin as described above.

[0010] The fourth aspect of the present application relates to a vector, which contains the nucleic acid as described above.

[0011] The fifth aspect of the present application relates to a cell containing the nucleic acid or vector as described above.

[0012] The sixth aspect of the present application relates to a method for preparing immunoglobulin, comprising culturing the cells as described above.

[0013] The seventh aspect of the present application relates to a multimer obtained by polymerizing the above-mentioned immunoglobulin as a monomer.

[0014] The eighth aspect of the present application relates to a conjugate, which comprises the above-mentioned immunoglobulin or polymer;

[0015] The ninth aspect of the present application relates to a kit or test strip containing the above-mentioned immunoglobulin, polymer or conjugate.

[0016] The tenth aspect of the present application relates to a method for detecting an antigen, comprising contacting the immunoglobulin, polymer, conjugate, kit or test strip as described above with the antigen to form a conjugate, and detecting the conjugate; wherein the antigen is an antigen that can specifically bind to the IgG variable region.

[0017] The eleventh aspect of the present application relates to the use of the immunoglobulin, polymer, conjugate, kit or test strip in the preparation of antigen immunoassay products.

[0018] The IgG / IgM chimeric immunoglobulin provided in the present application can self-polymerize monomers into a multimeric structure with desired valence, and has improved potency, sensitivity or correlation. DETAILED DESCRIPTION

[0019] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature described or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0021] In this document, the terms "include" or "have" are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0022] As used herein, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0023] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0024] The "at least 80% identity" of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0025] In the present invention, the term "antibody" is used in the broadest sense, and can be an antibody of natural structure or an antibody of various engineered structures. It can include monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies. Antibodies of natural structure generally include light chains with lighter molecular weight and heavy chains with heavier molecular weight. The amino acid sequences of the amino termini (N-terminus) of the heavy chain (H chain) and the light chain (L chain) vary greatly, which is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and varies little, which is called the constant region (C region). The C regions of the H chain and the L chain are respectively called the light chain constant region (CL region) and the heavy chain constant region (CH region). The CH region of IgG includes the CH1 region, hinge region, CH2 region and CH3 region from N-terminus to C-terminus, and the CH region of IgM includes the CH1 region, hinge region, CH2 region, CH3 region and CH4 region from N-terminus to C-terminus. "Fc region" refers to the part below the CH1 region of the CH region to the C-terminus, for example, containing the hinge region, CH2 region, CH3 region and CH4 region. The terms "antibody" and "immunoglobulin" are used interchangeably.

[0026] The inventors of the present application unexpectedly discovered during the experiment that the difference in the hinge region of IgG / IgM chimeric antibodies would seriously affect the self-aggregation of the antibodies. Further research found that the flexibility of the hinge region has a very large impact on the self-aggregation of IgG / IgM chimeric antibodies.

[0027] Thus, IgG / IgM chimeric antibodies with different hinge regions are constructed and applied to the coating end or labeling end of in vitro detection reagents. The IgG / IgM chimeric antibodies of the present application have improved titer, sensitivity or correlation compared to existing IgG antibodies or multivalent antibodies.

[0028] In a first aspect, the present application provides an immunoglobulin obtained by fusing a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a variable region located at the N-terminus, an IgG CH1 region and a hinge region. And the second polypeptide comprises an IgMCH2-CH3-CH4 region located at the C-terminus; wherein the hinge region contains M prolines and N cysteines, M≥2 and N≥1, or M≥1 and N≥2.

[0029] On the other hand, an embodiment of the present application provides an immunoglobulin, comprising a heavy chain, wherein the heavy chain is obtained by fusion of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a variable region located at the N-terminus, an IgG CH1 region and a hinge region; and the second polypeptide comprises an IgM CH2-CH3-CH4 region located at the C-terminus; wherein the hinge region contains M proline and N cysteine, M≥2 and N≥1, or M≥1 and N≥2.

[0030] In this application, when a single amino acid is located in a polypeptide chain, the single amino acid and the amino acid residue can be used interchangeably. Therefore, as described above, "proline" can be used interchangeably with "proline residue", and the same applies to other single amino acids.

[0031] As used herein, the term "IgG variable region" refers to the variable region of an IgG antibody, and is synonymous with "variable region".

[0032] In some embodiments, the immunoglobulin further comprises a light chain or a variable region thereof.

[0033] In some embodiments, 3≤M+N≤8.

[0034] In some embodiments, M+N=3, 4, 5, 6, 7, or 8.

[0035] In some embodiments, 1≤M≤6.

[0036] In some embodiments, M=1, 2, 3, 4, 5, or 6.

[0037] In some embodiments, 1≤N≤4.

[0038] In some embodiments, N=1, 2, 3, or 4.

[0039] In some embodiments, the species origin of the variable region, CH1 region, hinge region, CH2 region, CH3 region or CH4 region is independently selected from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans.

[0040] In some embodiments, the hinge region type is any one of IgG, IgA, and IgD.

[0041] In some embodiments, the IgG is selected from IgGl, IgG2a, IgG2b, IgG2c, IgG3, or IgG4.

[0042] In some embodiments, the IgG and IgM are selected from the same species origin.

[0043] In some embodiments, the hinge region is a full-length or truncated native hinge region.

[0044] When the hinge region is truncated from the natural hinge region, rather than artificially synthesizing a non-natural hinge, the hinge is closer to the flexibility of the corresponding segment of the natural hinge.

[0045] The full-length native hinge regions are exemplified below, and the hinge regions of various other immunoglobulin heavy chains not listed in Table 1 can be easily identified through appropriate sequence alignment.

[0046] Table 1

[0047] Isotype Full length natural hinge Mouse IgG2b EPSGPISTINPCPPCKECHKCPAPNLEGGP Mouse IgG2a EPRGPTIKPCPPCKCPAPNLLGGP Mouse-IgG1 VPRDCGCKPCICTVPEVS Rabbit IgG APSTCSKPTCPPPELLGGP Rabbit IgG APSTCSKPMCPPPELPGGP

[0048] It should be understood that due to allotypes and allelic variations in the population, the natural amino acid residues at these positions may be different from the listed amino acids, so there may be individual amino acid differences between the presented full-length native hinge region sequence and the sequence in Table 1.

[0049] The definition of the full-length native hinge region may be different for those skilled in the art. Therefore, the N-terminus or C-terminus of the hinge region in Table 1 may be extended or shortened by 1, 2, 3, 4 or 5 amino acids.

[0050] The term "truncated" refers to truncations from the N-terminus and / or C-terminus of the native hinge region.

[0051] In some embodiments, the hinge region is a C-terminally truncated native hinge region.

[0052] In some embodiments, M+N=3, 4, 7, 8.

[0053] In some embodiments, M=3 and N=1.

[0054] In some embodiments, M=2 and N=1.

[0055] In some embodiments, M=1 and N=2, or M=3 and N=4.

[0056] In some embodiments, M=2 and N=2, M=5 and N=2, or M=6 and N=2.

[0057] In some embodiments, M+N=4.

[0058] In some embodiments, the hinge region is the native hinge region of mouse IgG2b that is C-terminally truncated.

[0059] In some embodiments, M+N=3.

[0060] In some embodiments, the hinge region is the native hinge region of mouse IgG2a that is C-terminally truncated.

[0061] In some embodiments, M+N=3 or 7.

[0062] In some embodiments, the hinge region is the native hinge region of mouse IgG1 that is C-terminally truncated.

[0063] In some embodiments, M+N=4, 7, or 8.

[0064] In some embodiments, the hinge region is the native hinge region of a C-terminally truncated rabbit IgG.

[0065] In some embodiments, M=3 and N=1; the hinge region is the native hinge region of C-terminally truncated murine IgG2b.

[0066] In some embodiments, M=2 and N=1; the hinge region is the native hinge region of C-terminally truncated murine IgG2a.

[0067] In some embodiments, M=1 and N=2, or M=3 and N=4; the hinge region is a native hinge region of C-terminally truncated murine IgG1.

[0068] In some embodiments, M=2 and N=2, M=5 and N=2, or M=6 and N=2; the hinge region is a native hinge region of a C-terminally truncated rabbit IgG.

[0069] In some embodiments, the amino acid sequence of the hinge region is selected from any one of SEQ ID NO: 1 to SEQ ID NO:7.

[0070] In some embodiments, the amino acid sequence of IgG CH1 is selected from any one of SEQ ID NO: 8 to SEQ ID NO: 11 or has at least 80% identity with any one;

[0071] In some embodiments, the amino acid sequence of the IgM CH2-CH3-CH4 region is selected from any one of SEQ ID NOs: 12 to 13 or has at least 80% identity with any one of them.

[0072] In a second aspect, an embodiment of the present application provides an immunoglobulin, wherein the amino acid sequence of the Fc region of the immunoglobulin is selected from any one of SEQ ID NO: 14 to SEQ ID NO: 20.

[0073] On the other hand, an embodiment of the present application provides an immunoglobulin, comprising a heavy chain, wherein the amino acid sequence of the Fc region of the heavy chain is selected from any one of SEQ ID NO: 14 to SEQ ID NO: 20.

[0074] The Fc region includes the hinge region at the N-terminus and the IgM CH2-CH3-CH4 region at the C-terminus.

[0075] In some embodiments, the immunoglobulin further comprises a light chain or a variable region thereof.

[0076] On the other hand, an embodiment of the present application provides an immunoglobulin, wherein the constant region of the immunoglobulin is selected from any one of SEQ ID NO: 21 to SEQ ID NO: 27.

[0077] On the other hand, an embodiment of the present application provides an immunoglobulin, comprising a heavy chain, wherein the constant region of the heavy chain is selected from any one of SEQ ID NO: 21 to SEQ ID NO: 27. The constant region comprises the CH1 region and hinge region of IgG at the N-terminus, and the IgM CH2-CH3-CH4 region at the C-terminus.

[0078] In some embodiments, the immunoglobulin further comprises a light chain or a variable region thereof.

[0079] The immunoglobulin provided in the present application can self-polymerize monomers into a stable polymer structure with desired valence, and has higher polymerization stability or potency.

[0080] In a third aspect, an embodiment of the present application provides a nucleic acid encoding any of the immunoglobulins described above.

[0081] In a fourth aspect, an embodiment of the present application provides a vector comprising the above-mentioned nucleic acid.

[0082] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40). In some embodiments, the vector described in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0083] In a fifth aspect, an embodiment of the present application provides a cell containing the above-mentioned nucleic acid or vector.

[0084] The term "cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The cell is preferably a eukaryotic cell, more preferably a mammalian cell.

[0085] In a sixth aspect, an embodiment of the present application provides a method for preparing an immunoglobulin, comprising culturing the above-mentioned cells.

[0086] In a seventh aspect, an embodiment of the present application provides a multimer obtained by polymerizing the immunoglobulin as described in any one of the above items as monomers.

[0087] In some embodiments, the polymer has a degree of polymerization of 2, 3, 4, 5 or 6.

[0088] In an eighth aspect, an embodiment of the present application provides a conjugate, wherein the conjugate includes any one of the immunoglobulins or polymers described above.

[0089] In some embodiments, the conjugate further comprises biotin or a biotin derivative conjugated to the immunoglobulin or polymer.

[0090] In some embodiments, the conjugate further comprises a label coupled to the immunoglobulin or polymer.

[0091] In some embodiments, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents, and nanoparticle-based labels.

[0092] In some embodiments, the conjugate further comprises a solid phase carrier coupled to the immunoglobulin or polymer.

[0093] In some embodiments, the solid phase carrier is a plastic carrier, a microparticle or a membrane carrier; the plastic carrier can be a polystyrene carrier; the microparticle can be a magnetic microparticle; the membrane carrier can be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane.

[0094] In some embodiments, the solid phase carrier is selected from the wells of a test tube, an EP tube, a multi-well plate, a chromatography column, and a microplate.

[0095] In the present application, the term "microparticle" may be a sphere, a nearly sphere, a cube, a polyhedron or an irregular shape. The diameter of the microparticle may be 10 nm to 1 mm, such as 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm.

[0096] The particles are preferably magnetic particles, and the composition thereof contains magnetic substances. The magnetic substances may be metals (metallic elements or alloys), non-metals, or composites formed by metals and non-metals. Metals include iron, aluminum nickel cobalt, etc.; non-metals include ferrite non-metals (preferably Fe2O3 or Fe3O4 magnetic nanoparticles); composites formed by metals and non-metals include NdFeB rubber magnetic composite materials.

[0097] The multi-well plate is preferably an ELISA plate, which may have 8 wells, 16 wells, 32 wells, 48 ​​wells, 64 wells, 96 wells or more wells.

[0098] In a ninth aspect, embodiments of the present application provide a kit or test strip containing the above-mentioned immunoglobulin, polymer or conjugate.

[0099] In some embodiments, the test strip includes a sample pad, a conjugate pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area.

[0100] The polymer is coated on the detection area and / or dropped on the conjugate pad.

[0101] It is easy to understand that when the detection area and the binding pad both contain polymers, the antigen epitopes bound by the IgG variable regions of the polymers are different, and the second polypeptide terminal domains may be the same or different.

[0102] In some embodiments, the kit may also include sample pretreatment reagents (such as sample purification and enrichment reagents, lysis solutions, etc.), cleaning solutions (such as water, etc.), buffer solutions (such as PBS or Tris, etc.), colorimetric reagents for signal substances (such as ECL or DAB for horseradish peroxidase as a signal substance), etc.

[0103] The immunoglobulin provided in the present application can self-polymerize monomers into a stable polymer structure with a desired valence, and has higher stability or titer. Therefore, when used for labeling or coating, it has higher detection sensitivity or correlation.

[0104] In a tenth aspect, an embodiment of the present application provides a method for detecting an antigen, comprising:

[0105] The immunoglobulin, polymer, conjugate as described above or the kit or test strip as described above is contacted with the antigen to form a conjugate, and the conjugate is detected, wherein the antigen is an antigen that can specifically bind to the IgG variable region.

[0106] The conjugate can be detected by any method known to those skilled in the art, such as the double antibody sandwich method, that is, after the antigen is contacted with the solid phase carrier, the conjugate is detected with an additional antibody against the antigen (which is usually labeled with a signal substance, or detected with a secondary antibody labeled with a signal substance).

[0107] Alternatively, conventional antibodies are used as antibodies coated on a solid phase carrier and incubated with the antigen to be detected, and the polymer is detected using a free detection antibody (which is usually labeled with a signal substance, or detected with a secondary antibody labeled with a signal substance).

[0108] Alternatively, the antigen is detected by using a polymer-coated solid phase carrier in combination with another polymer, in which case the polymer coated on the solid phase carrier and the other polymer are paired antibodies.

[0109] The signal substance can be any one of a fluorescent substance, quantum dots, digoxigenin-labeled probe, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold or enzymes. In some embodiments, the signal substance is colloidal gold, fluorescein, fluorescent microspheres, acridinium esters, horseradish peroxidase, alkaline phosphatase or β-galactosidase.

[0110] In an eleventh aspect, embodiments of the present application provide the use of the immunoglobulin, polymer, conjugate, kit or test strip as described above in immunoassay.

[0111] The implementation scheme of the present application will be described in detail below with reference to examples.

[0112] Example

[0113] In the following examples, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara, pMD-18T vector was purchased from Takara, plasmid extraction kit was purchased from Tiangen, primer synthesis and gene sequencing were completed by Invitrogen.

[0114] Example 1 Preparation of polynucleotide encoding IgM heavy chain constant region

[0115] In the present application, the polynucleotide encoding the antibody constant region can be obtained from peripheral blood cell DNA by PCR, or can be obtained from a hybridoma cell line secreting a corresponding type of monoclonal antibody by PCR.

[0116] 1.1 Obtaining polynucleotides encoding mouse IgM CH region

[0117] The polynucleotide encoding the mouse IgM-CH region was obtained from the genome of the hybridoma cell line 6M9 (available from Feipeng Bio) that secretes IgM type mouse monoclonal antibodies by PCR. The primers used are as follows:

[0118] Forward primer sequence (5′→3′): GAG AGT CAG TCC TTC CCA AAT GTC TTC CCC CTC GTCTC;

[0119] Reverse primer sequence (5'→3'): CCC GAA TTC TTA TCA ATA GCA GGT GCC ACC TGT GTCAGA CAT GAT CAG.

[0120] The EcoRI restriction site (GAATTC) was introduced by reverse primer immediately following the stop codon TGA TAA (anticodon TTA TCA). Double stop codons were used here. A polynucleotide of about 1.4 kb was obtained by PCR technology and introduced into pMD-18T vector for sequencing confirmation.

[0121] The polynucleotide encodes the mouse IgM-CH region, and its amino acid sequence from N-terminus to C-terminus is as follows:

[0122] ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPI PAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPK PITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASP STDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVED WNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQ LLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMS DTGGTCY (SEQ ID NO:28).

[0123] A polynucleotide encoding the CH2-CH3-CH4 domain of the mouse IgM CH region was prepared in a similar manner (the underlined portion of SEQ ID NO: 28 is CH2-CH3-CH4).

[0124] 1.2 Obtaining polynucleotide encoding rabbit IgM CH region

[0125] The nucleotide encoding the rabbit IgM-CH region was obtained by referring to the method in 1.1 above, and its amino acid sequence from N-terminus to C-terminus is as follows:

[0126] VSLSSPTLYPLVSCEGALTDGNLVAMGCLARDFLPSSVTFSWSFKNNSEISSRTVRTFPVVKRGDKYMATSQVLVPSKDVLQGTEEYLVCKVQHSNSNRDLRVSFP VDSELPPNVSVFIPPRDSFSGSGTRKSRLICQATGFSP KQISVSWLRDGQKVESGVLTKPVEAETKGAGPATFSISSMLTITESDWLSQSLYTCRVDHRGIFFDKNVSMSSECS TTPSPGIQVFPIAPSFADTFLSKSARLICLVTDLTTYGSLNISWASHNGKALDTHMNITESHPNATFSAMGEASVC AEDWESGEQFTCTVTHADLPFPLKHTISKSREVAKHPPAVYVLPPAREQLVLRESATVTCLVKGFSPADVFVQWQQ RGQPLSSDKYVTSAPAPEPQAPGLYFTHSTLTVTEEDWNSGETFTCVVGHEALPHMVTERTVDKSTGKPTLYNVSL IMSDTASTCY (SEQ ID NO:29).

[0127] A polynucleotide encoding the CH2-CH3-CH4 domain of the rabbit IgM CH region was prepared in a similar manner (the underlined portion of SEQ ID NO: 29 is CH2-CH3-CH4).

[0128] Example 2 Construction of expression plasmid for mouse IgG / IgM hybrid recombinant antibody

[0129] 2.1 Construction of expression plasmid for mouse IgG2b / IgM hybrid recombinant antibody

[0130] In this embodiment, the mouse IgG2b antibody used is anti-Pan-PLDH 9G7 monoclonal antibody (hereinafter referred to as MA9G7), and the heavy chain and light chain sequences of the antibody are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.

[0131] First, the polynucleotide encoding the IgG2b segment 1 (VH+CH1+hinge region 1, the underlined amino acids are hinge region 1) of the MA 9G7 antibody was connected to the polynucleotide encoding the CH2-CH3-CH4 of mouse IgM by bridge PCR. The amino acid sequence of the IgG2b segment 1 of the MA9G7 antibody is as follows:

[0132] QVQLQQPGAELVRPGASVKLSCKASGYSFTNYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSLTAEDSAVYYCARSGDFYTYYFD YWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKL EPS GPIST (SEQ ID NO:30)

[0133] The polynucleotide obtained by PCR was double-digested with HindIII / EcoRI and ligated to pcDNA cut with the corresponding restriction enzymes. TM 3.4 The resulting vector was named pcDNA3.4A-9G7Rmb1.

[0134] The polynucleotide encoding the IgG2b segment 2 (VH+CH1+hinge region 2, the underlined amino acids are hinge region 2 (SEQ ID NO: 1)) of the MA 9G7 antibody was selected and connected to the polynucleotide encoding the CH2-CH3-CH4 of mouse IgM by bridge PCR. The amino acid sequence of the IgG2b segment 2 of the MA 9G7 antibody is as follows:

[0135] QVQLQQPGAELVRPGASVKLSCKASGYSFTNYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSLTAEDSAVYYCARSGDFYTYYFD YWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKL EPS GPISTINPC (SEQ ID NO:31), ligated to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-9G7Rmb2.

[0136] The polynucleotide encoding the IgG2b heavy chain of MA 9G7 was ligated to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-9G7 control, as the control antibody.

[0137] The IgM sequence itself has the polymeric characteristics. Since the polymeric characteristics of IgM depend on the heavy chain, the light chain sequence of the antibody in this example is the same as the light chain of MA 9G7.

[0138] 2.2 Construction of expression plasmid for mouse IgG2a / IgM hybrid recombinant antibody

[0139] In this example, the mouse IgG2a antibody used is an anti-cTnI 2E7 antibody, and the heavy chain and light chain sequences of the antibody are shown in SEQ ID NO: 34 and SEQ ID NO: 35, respectively.

[0140] Referring to the construction method in Example 2.1, the polynucleotide encoding the VH+CH1+hinge region of IgG2a was connected to the polynucleotide encoding the CH2-CH3-CH4 of mouse IgM by bridge PCR. Expression plasmids of 2E7Rmb-H1, 2E7Rmb3, and 2E7Rmb4 with different hinge region lengths were constructed respectively. Among them, the hinge region of 2E7Rmb-H1 is EPRGPTIKPC (SEQ ID NO: 2), 2E7Rmb3 has no hinge region, and the hinge region of 2E7Rmb4 is EPRGPTIKP.

[0141] 2.3 Construction of expression plasmid for mouse IgG1 / IgM hybrid recombinant antibody

[0142] In this embodiment, the mouse IgG1 antibody uses the 3E50 antibody, which is recorded in Chinese Patent Publication No. CN112239501A, which is incorporated herein by reference. The antibody can be selected from any antibody having any group of mutations in the mutation combination 1 to mutation combination 68 in the section

[0049] to

[0052] of CN112239501A, such as mutation combination 58, which have similar properties. In this embodiment, the mouse IgG1 antibody uses the 5F27 antibody, which is recorded in Chinese Patent CN112239500A, which is incorporated herein by reference. The antibody can be selected from any antibody having any group of mutations in the mutation combination 1 to mutation combination 42 in the section

[0048] to

[0049] of CN112239500A, such as mutation combination 19, which have similar properties.

[0143] Referring to the construction method in Example 2.1, the polynucleotide encoding the VH+CH1+hinge region of IgG1 was connected to the polynucleotide encoding the CH2-CH3-CH4 of mouse IgM by bridge PCR. Expression plasmids containing Rmbs of different hinge region lengths were constructed respectively. Among them, the hinge region of 3E50Rmb1 is VPRDC, the hinge region of 3E50Rmb2 is VPRDCGC ​​(SEQ ID NO: 3), the hinge region of 3E50Rmb3 is VPRDCGCKPCICTVPEVS (SEQ ID NO: 4), and the hinge region of 5F27Rmb1 is VPRDC.

[0144] 2.4 Construction of expression plasmid of rabbit IgG / IgM hybrid recombinant antibody

[0145] In this embodiment, the rabbit IgG antibody used is the anti-COVID-7A12 antibody, the heavy chain and light chain sequences of which are shown in SEQ ID NO:36 and SEQ ID NO:37, respectively.

[0146] Referring to the construction method in Example 2.1, the polynucleotide encoding the VH+CH1+hinge region of IgG was connected to the polynucleotide encoding the CH2-CH3-CH4 of rabbit IgM by bridge PCR. Expression plasmids containing Rmbs of different hinge region lengths were constructed respectively. Among them, the hinge region of 7A12Rmb1 is APSTCSKPTC (SEQ ID NO: 5), the hinge region of 7A12Rmb2 is APSTCSKPTCPPPELLGG (SEQ ID NO: 6), and the hinge region of 7A12Rmb3 is APSTCSKPMCPPPELPGG (SEQID NO: 7).

[0147] Example 3 Expression and preparation of IgG / IgM hybrid recombinant antibody

[0148] 1. Linearization of recombinant antibody expression plasmid

[0149] Prepare the following enzyme digestion system: restriction enzyme buffer 50ul, recombinant plasmid 100ug / tube, PvuⅠ enzyme 10ul, sterile water to 500ul, and digest in a 37℃ water bath overnight; first use phenol / chloroform / isoamyl alcohol (25:24:1) (lower layer) with an equal volume to the enzyme digestion system, and then use chloroform (aqueous phase) to extract in sequence; use 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volumes of ethanol to precipitate DNA on ice, rinse the DNA precipitate with 70% ethanol, remove the organic solvent, and re-dissolve with an appropriate amount of sterile water after the ethanol is completely evaporated, and finally measure the concentration.

[0150] 2. Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0151] The heavy chain plasmid and light chain plasmid constructed in Example 2 were diluted to 40ug / 100ul with ultrapure water at a ratio of 1:1. A new centrifuge tube was taken and the CHO cell concentration was adjusted to 1.43×10 7 cells / ml, 100ul plasmid was mixed with 700ul cells, and transferred into an electroporation cup for electrotransfection. Cells were cultured after transfection and counted the next day; 25umol / L MSX 96-well pressurized culture was performed for about 25 days.

[0152] Observe the clone wells with cells under a microscope and record the confluence; take the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration and transfer them to 24-well plates, and transfer them to 6-well plates after about 3 days; keep the seeds for batch culture after 3 days and adjust the cell density to 0.5×10 6 cells / ml, with a volume of 2.2 ml for batch culture and a cell density of 0.3×10 6cells / ml, 2ml for seed preservation; after 7 days, the batch culture supernatant in the 6-well plate was sent for sample detection, and the cell lines with smaller antibody concentration and cell diameter were selected and transferred to TPP tubes for seed preservation and subculture to obtain cell lines expressing IgG / IgM hybrid recombinant antibodies.

[0153] 3. Cell expansion culture

[0154] After cell recovery, the cells were first inoculated in a 125 ml shake flask containing 100% Dynamis medium, with an inoculation volume of 30 ml, and cultured in a shaker at 120 r / min, 37°C, and 8% carbon dioxide. After 72 h of culture, 5 × 10 5 The inoculation density was 5×10 cells / ml, and the expanded culture volume was calculated according to production requirements. The culture medium used was 100% Dynamis medium. After that, the culture was expanded every 72 hours. When the cell volume met the production requirements, the inoculation density was strictly controlled to 5×10 5 Produced at around cells / ml.

[0155] 4. Shake flask production and purification

[0156] Shake flask production parameters: shaker speed 120r / min, temperature 37℃, carbon dioxide content 8%. Feeding: Start feeding every day when the culture reaches 72h in the shake flask, feed 3% of the initial culture volume of HyClone every day. TM CellBoost TM Feed 7a, daily feed amount is 1 / 1000 of the initial culture volume of HyClone TM Cell Boost TM Feed7b until the 12th day (feeding on the 12th day). Add 3g / L glucose on the 6th day. Collect samples on the 13th day. Using captoL (cytiva) and CHT (Bio-Rad) as fillers, the multimeric antibodies of IgG / IgM hybrid recombinant antibodies were purified in two steps by affinity chromatography. Take 10ul of purified antibody (concentration of 1mg / ml) for reducing SDS-PAGE. Two bands are shown in each lane, one with a relative molecular mass (Mr) of 70-80kD (heavy chain) and the other with Mr of 20-35kD (light chain).

[0157] The constant region (CH) sequence of the above-constructed antibody was determined as follows:

[0158] Antibody ID Constant region (CH) sequence 9G7Rmb2 SEQ ID NO:21 2E7Rmb-H1 SEQ ID NO:22 3E50Rmb2 SEQ ID NO:23 3E50Rmb3 SEQ ID NO:24 7A12Rmb1 SEQ ID NO:25 7A12Rmb2 SEQ ID NO:26 7A12Rmb3 SEQ ID NO:27

[0159] Example 4 Application of IgG / IgM hybrid recombinant antibodies

[0160] 4.1 Application of mouse IgG2b / IgM hybrid recombinant antibody on colloidal gold platform

[0161] Preparation of MA-9G7Rmb1 Colloidal Gold Test Paper

[0162] 1) Preparation of nitrocellulose membrane

[0163] Preparation of nitrocellulose membrane: Dilute MA-9G7Rmb1 recombinant antibody to 1-5 mg / ml with coating buffer, draw a line as T, which is the detection line, and T line is close to the colloidal gold end; dilute goat anti-mouse IgG antibody (produced by Feipeng Biotechnology Co., Ltd., product number BA-PAB-MU0001) to 1-5 mg / ml with coating buffer, draw a line as C, which is the control line, and C line is close to the absorption pad. Dry at 37℃, package and set aside.

[0164] 2) Preparation of colloidal gold and gold-labeled monoclonal antibodies

[0165] 2.1) Preparation of colloidal gold

[0166] Dilute 1% chloroauric acid to 0.01% with double distilled deionized water, boil on an electric stove, add 2ml 1% trisodium citrate for every 100ml 0.01% chloroauric acid, continue boiling until the liquid turns bright red, then stop heating, and make up for the water loss after cooling to room temperature. The prepared colloidal gold is pure, transparent, without precipitation and floating matter, and is valid for one week.

[0167] 2.2) Preparation of colloidal gold labeled antibodies

[0168] Adjust the pH value of colloidal gold to 8.2 with 0.1M potassium carbonate, add another MA-labeled antibody (available from Feipeng Bio) at 8-10μg antibody / ml colloidal gold, mix with a magnetic stirrer for 30 minutes, add BSA to a final concentration of 1% under stirring and let stand for 1 hour. Centrifuge the liquid at 13000rpm and 4℃ for 30 minutes, discard the supernatant, wash the precipitate twice with the labeling washing and preservation solution, resuspend the precipitate with one-tenth of the initial colloidal gold volume of the labeling washing and preservation solution, and place at 4℃ for use. The validity period is one week.

[0169] 3) Preparation of gold label pad

[0170] Soak the gold-labeled pad in the blocking solution for 30 minutes and dry it at 37°C. Then spread the prepared colloidal gold-labeled antibody evenly on the gold-labeled pad, with 20 square centimeters per milliliter of solution, freeze-dry, package, and place at 4°C for later use.

[0171] 4) Preparation of test strip sample pad

[0172] The sample pad was immersed in the blocking solution (containing BSA) for 30 min, dried at 37°C, packaged, and placed at 4°C for later use.

[0173] 5) Assembly of test strips

[0174] The absorption pad (purchased from Millipore), nitrocellulose membrane, gold label pad, and sample pad were placed on a non-absorbent support sheet and cut into 3 mm wide strips. Ten strips were packed into one pack, desiccant was added, and vacuum packaged to obtain MA-9G7Rmb1 colloidal gold test paper.

[0175] The colloidal gold test strips of MA-9G7Rmb2 recombinant antibody or control antibody (original MA-9G7 IgG2b) were prepared by referring to the method described for the above-mentioned MA-9G7Rmb1 colloidal gold test strips, wherein the mass concentration of the T-line hybrid recombinant antibody was the same as that of the control antibody.

[0176] 6) Application of colloid test strips

[0177] The assembled test strips are used to detect whether the material under test contains Pan-PLDH protein (hereinafter referred to as MA protein), so as to determine the effect of MA antibody on MA protein detection. During the detection, MA protein first combines with colloidal gold-labeled MA antibody to form MA-colloidal gold-labeled-MA antibody complex. Due to capillary action, MA-colloidal gold-labeled-MA antibody complex swims forward along the nitrocellulose membrane. When it reaches the detection line, MA-colloidal gold-labeled-MA antibody complex will combine with the MA-coated antibody on the line to form MA antibody-MA-colloidal gold-labeled-MA antibody complex, thereby enriching on the detection line to form a red precipitation line. Table 2 is the corresponding detection data. The depth of the red precipitation line indicates the strength of the reaction. The darker the red, the stronger the reactivity, and vice versa. At the same time, the strength of the reaction is represented by a combination of letters C and numbers. The smaller the number after C, the stronger the reactivity, and vice versa, the weaker the reactivity. No red precipitation line is formed, which is represented by B.

[0178] Table 2

[0179]

[0180] 4.2 Application of mouse IgG1 / IgM hybrid recombinant antibody on colloidal gold platform

[0181] The colloidal gold platform application of SARS-CoV-2 3E50 and 5F27 IgG1 / IgM hybrid recombinant antibodies was used to perform performance evaluation according to the method of Example 4.1. The specific performance evaluation results are shown in the following table:

[0182] Table 3 Detection data of hybrid recombinant antibodies used as coating antibodies and labeled antibodies

[0183]

[0184]

[0185] Note: Vaccine refers to the inactivated sample of the new coronavirus.

[0186] 4.3 Application of mouse IgG2a / IgM hybrid recombinant antibody on fluorescence chromatography platform

[0187] 1) Preparation of fluorescence chromatography detection reagent card

[0188] 1.1) Preparation of nitrocellulose membrane

[0189] Preparation of nitrocellulose membrane: dilute 2E7Rmb-H1 recombinant antibody to 1-2 mg / ml with coating buffer, draw T line (detection line), T line is close to the end of gold pad, about 5 mm away from the end of gold pad; dilute goat anti-mouse IgG antibody (obtained from Feipeng Bio) to 1-2 mg / ml with coating buffer, draw C line (control line), C line is close to the absorption pad, about 3 mm away from the absorption pad. Dry at 37℃, package and set aside.

[0190] 1.2) Preparation of monoclonal antibodies labeled with fluorescence chromatography

[0191] Take 1 ml of fluorescent microspheres with 1% solid content, centrifuge and remove the supernatant, add an equal volume of labeled activation solution as the microspheres and mix by ultrasonic, then add activators EDC (final concentration 5 mg / ml) and NHS (final concentration 5 mg / ml), shake and mix in the dark for 20 minutes, centrifuge and remove the supernatant, add an equal volume of labeled coupling solution as the microspheres and mix by ultrasonic, then add 0.2-1 mg of another paired cTnI labeled antibody, shake and mix in the dark for 3 hours, finally add labeled blocking solution for blocking, shake and mix in the dark for 45 minutes, terminate the labeling, centrifuge and remove the supernatant, re-dissolve the microspheres with microsphere labeling preservation solution, mix by ultrasonic, and place at 4°C for use. The shelf life is 1 month.

[0192] 1.3) Preparation of fluorescent labeling pad

[0193] After diluting the marker 5 to 10 times with marker diluent, use a spray pad to spray the diluted marker on the glass fiber pad. Seal the marker pad and place it at 4°C for later use.

[0194] 1.4) Preparation of test strip sample pad

[0195] The glass fiber pad was soaked in blocking solution (containing BSA) for 30 min, dried at 37°C, and the sample pad was sealed and placed at 4°C for later use.

[0196] 1.5) Assembly of the test reagent card

[0197] The absorption pad (purchased from Millipore), nitrocellulose membrane, labeling pad and sample pad were placed on a non-absorbent support sheet, cut into 3 mm wide strips, and loaded into a card shell, one pack per card, added with a desiccant, and vacuum-sealed in an aluminum foil bag to obtain a 2E7Rmb-H1 fluorescence chromatography detection reagent card.

[0198] 2E7Rmb3, 2E7Rmb4 or control antibody (original 2E7-IgG2a) fluorescence chromatography detection reagent cards were prepared according to the method described for the above-mentioned 2E7Rmb-H1 fluorescence chromatography detection reagent card, wherein the mass concentration of the T-line hybrid recombinant antibody was the same as that of the control antibody.

[0199] 2) cTnI 2E7 IgG2a / IgM hybrid recombinant antibody fluorescence chromatography platform test results

[0200] The purchased cTnI antigen was used as the test sample. Tear open the aluminum foil bag and take out the test card. For each test sample, aspirate 50 μL of the sample, add the sample to the sample diluent, mix thoroughly and then aspirate 50 μL to add to the sample well of the test card; the reaction time after adding the sample to the test card is 15 minutes; the fluorescence detector will automatically test and read the results. At the same time, 30 clinical fixed-value specimens collected from the hospital were used as test samples to detect the specimen correlation r of the antibody. The specific test results are shown in Table 4.

[0201] Table 4

[0202] Clinical value specimen (ng / ml) Control Antibodies 2E7Rmb4 2E7Rmb-H1 2E7Rmb3 50 2.103 4.089 7.382 2.629 18 0.765 1.558 2.742 0.956 10.5 0.452 0.965 1.655 0.563 3 0.138 0.371 0.567 0.171 0.7 0.042 0.189 0.234 0.051 0.15 0.019 0.146 0.154 0.022 0.08 0.016 0.140 0.144 0.018 0.04 0.015 0.137 0.138 0.016 Detection of correlation of 30 samples 0.990 0.994 0.998 0.991

[0203] 4.4 Application of rabbit IgG / IgM hybrid recombinant antibody in ELISA platform

[0204] The 7A12 IgG / IgM hybrid recombinant antibody or the control antibody (7A12 IgG) was used as a labeled antibody and matched with another paired SARS-CoV-2 N protein antibody (purchased from Feipeng). The performance evaluation was performed according to the method of Example 4.1. The specific performance evaluation results are shown in the following table:

[0205] Table 5

[0206]

[0207] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0208] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

[0209] Some of the amino acid sequences involved in this application are shown in Table 6:

[0210]

[0211]

[0212]

[0213]

Claims

1. An immunoglobulin, comprising a heavy chain, wherein the heavy chain is obtained by fusion of a first polypeptide and a second polypeptide: The first polypeptide includes a variable region, an IgG CH1 region and a hinge region at the N-terminus; and the second polypeptide includes an IgM CH2-CH3-CH4 region at the C-terminus; wherein, The hinge region contains M proline residues and N cysteine ​​residues, wherein M≥2 and N≥1, or wherein M≥1 and N≥2.

2. The immunoglobulin according to claim 1, wherein 3≤M+N≤8; Optionally, 1≤M≤6; Optionally, 1≤N≤4; Optionally, the species origin of the variable region, CH1 region, hinge region, CH2 region, CH3 region or CH4 region is independently selected from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese or humans; Optionally, the hinge region type is any one of IgG, IgA, and IgD; Optionally, the IgG is selected from IgG1, IgG2a, IgG2b, IgG2c, IgG3 or IgG4; Optionally, the hinge region is a full-length or truncated native hinge region; Optionally, the hinge region is a natural hinge region truncated at the C-terminus; Optionally, M+N=3, 4, 7, 8; Optionally, M=3 and N=1; Optionally, M=2 and N=1; Optionally, M=1 and N=2, or M=3 and N=4; Optionally, M=2 and N=2, M=5 and N=2, or M=6 and N=2.

3. The immunoglobulin according to any one of claims 1 to 2, wherein the amino acid sequence of the hinge region is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 7; Optionally, the amino acid sequence of the IgG CH1 is selected from any one of SEQ ID NO: 8 to SEQ ID NO: 11; Optionally, the amino acid sequence of the IgM CH2-CH3-CH4 region is selected from any one of SEQ ID NOs: 12 to 13.

4. An immunoglobulin comprising a heavy chain, wherein the amino acid sequence of the Fc region of the heavy chain is selected from any one of SEQ ID NO: 14 to SEQ ID NO: 20; or The constant region of the heavy chain is selected from any one of SEQ ID NO:21 to SEQ ID NO:

27.

5. A method for preparing a nucleic acid, a vector, a cell or an immunoglobulin, wherein the nucleic acid encodes the immunoglobulin described in any one of claims 1 to 4; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the preparation method includes culturing the above-mentioned cell.

6. A multimer obtained by polymerizing the immunoglobulin according to any one of claims 1 to 4 as a monomer; Optionally, the degree of polymerization of the polymer is 2, 3, 4, 5 or 6.

7. A conjugate, comprising the immunoglobulin according to any one of claims 1 to 4 or the multimer according to claim 6; Optionally, the conjugate further comprises biotin or a biotin derivative coupled to the immunoglobulin or polymer; Optionally, the conjugate further comprises a label coupled to the immunoglobulin or polymer; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the conjugate further comprises a solid phase carrier coupled to the immunoglobulin or polymer.

8. A kit or test strip containing the immunoglobulin according to any one of 1 to 4, the multimer according to claim 6, or the conjugate according to claim 7; Optionally, the test strip comprises a sample pad, a conjugation pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area; The polymer is coated on the detection area and / or dropped on the conjugate pad.

9. A method for detecting an antigen, comprising: The immunoglobulin according to any one of claims 1 to 4, the multimer according to claim 6, the conjugate according to claim 7, or the kit or test strip according to claim 8 is contacted with the antigen to form a conjugate, and the conjugate is detected, wherein the antigen is an antigen that can specifically bind to the IgG variable region.

10. Use of the immunoglobulin according to any one of claims 1 to 4, the multimer according to claim 6, the conjugate according to claim 7, or the kit or test strip according to claim 8 in the preparation of an antigen immunoassay product.

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