Self-polymerized immunoglobulin fc region

Abstract

The present application relates to an immunoglobulin which has an Fc region as shown in SEQ ID NO: 1. The immunoglobulin can self-polymerize monomers into a stable polymer structure, and has improved titer, detection sensitivity or correlation when being used for detection.

Description

A self-aggregating immunoglobulin Fc region

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent applications with application number 202311072501.6 filed with the Chinese Patent Office on August 23, 2023, entitled “A self-aggregating immunoglobulin Fc region” and Chinese patent application number 202311703507.9 filed with the Chinese Patent Office on December 12, 2023, entitled “A self-aggregating immunoglobulin Fc region”, 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 a self-aggregating immunoglobulin Fc region. Background Art

[0004] Natural immunoglobulin (Ig) molecules contain four heterologous polypeptide chains. The two chains with larger molecular weights are called heavy chains (H), while the two chains with smaller molecular weights 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 identical. The heavy chain has a molecular weight of 50,000 to 75,000 and is composed of 450 to 550 amino acid residues. The antigenicity varies due to the amino acid composition and arrangement sequence of the heavy chain constant region (CH region). Therefore, Ig can be divided into five classes: IgM, IgD, IgG, IgA, and IgE. Of these five classes, IgG is a monomeric structure. IgM is a basic antibody secreted by B cells. In its secreted form, it is primarily a pentamer composed of five Y-shaped monomers and has extremely high affinity. Due to its extremely large molecular weight, IgM 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 advances in science and technology, in vitro diagnostics (IVDs) have become a crucial aid in clinical diagnosis, serving as the doctor's eyes. Immunodiagnostic reagents are a sub-segment of IVD. Immunodiagnostics apply immunological theories, techniques, and methods to diagnose various diseases and measure immune status. Immunodiagnostic methods include radioimmunoassays, enzyme-linked immunosorbent assays, chemiluminescence, fluorescence chromatography, or colloidal gold chromatography. One of the major challenges in developing immunodiagnostic reagents is obtaining 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 very tightly and efficiently to antigens, despite their relatively weak antigen-binding affinity in monomeric form. When more than one interaction occurs between two molecules, the 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. Developing an efficient self-aggregating Fc region is of great significance for the preparation of high-performance multivalent antibodies.

[0007] Summary of the Invention

[0008] The first aspect of the present application relates to an immunoglobulin having an Fc region shown in SEQ ID NO: 1.

[0009] The second aspect of the present application relates to an immunoglobulin having a constant region shown in SEQ ID NO: 2.

[0010] The third aspect of the present application relates to a nucleic acid encoding an immunoglobulin as described above.

[0011] The fourth aspect of the present application relates to a vector comprising the nucleic acid as described above.

[0012] The fifth aspect of the present application relates to a cell containing the nucleic acid or vector as described above.

[0013] The sixth aspect of the present application relates to a method for preparing immunoglobulin, comprising culturing the cells as described above.

[0014] The seventh aspect of the present application relates to a multimer obtained by polymerizing the above-mentioned immunoglobulin as a monomer.

[0015] The eighth aspect of the present application relates to a conjugate, comprising the immunoglobulin or polymer described above;

[0016] The ninth aspect of the present application relates to a kit or test strip containing the above-mentioned immunoglobulin, polymer or conjugate.

[0017] 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.

[0018] 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.

[0019] The immunoglobulin provided in the present application, which contains the Fc region shown in SEQ ID NO: 1, can self-polymerize monomers into a multimeric structure with desired valence, and has improved potency, sensitivity or correlation. DETAILED DESCRIPTION

[0020] 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 to illustrate, not to limit, the present application. Indeed, 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, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] 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.

[0023] As used herein, the terms "optionally," "optional," "optionally," "optional," or "optionally" 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.

[0024] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or 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 Foundation, Washington, DC). 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. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .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.

[0025] The "at least 80% identity" of the present invention can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly 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.

[0026] In the present invention, the term "antibody" is used in the broadest sense and can refer to antibodies with naturally occurring structures or antibodies with various engineered structures. It can include monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies. Naturally occurring antibodies typically include a light chain with a relatively low molecular weight and a heavy chain with a relatively high molecular weight. The amino acid sequences at the amino terminus (N-terminus) of the heavy chain (H chain) and light chain (L chain) vary greatly and are called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and varies very little and is called the constant region (C region). The C regions of the H chain and L chain are called the light chain constant region (CL region) and the heavy chain constant region (CH region), respectively. The CH region of IgG includes, from N-terminus to C-terminus, the CH1 region, hinge region, CH2 region, and CH3 region, while the CH region of IgM includes, from N-terminus to C-terminus, the CH1 region, hinge region, CH2 region, CH3 region, and CH4 region, in order. The "Fc region" refers to the portion of the CH region from the CH1 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.

[0027] In a first aspect, an embodiment of the present application provides an immunoglobulin having an Fc region shown in SEQ ID NO: 1.

[0028] In some embodiments, the immunoglobulin comprises a heavy chain having an Fc region set forth in SEQ ID NO:1.

[0029] In some embodiments, the heavy chain has a constant region set forth in SEQ ID NO: 2, or at least 80% identical thereto.

[0030] In some embodiments, the heavy chain includes a variable region at the N-terminus and a constant region at the C-terminus.

[0031] In some embodiments, the immunoglobulin further comprises a light chain or a variable region thereof.

[0032] In a second aspect, an embodiment of the present application provides an immunoglobulin having a constant region shown in SEQ ID NO:2.

[0033] In some embodiments, the immunoglobulin comprises a heavy chain having a constant region set forth in SEQ ID NO:2.

[0034] In some embodiments, the heavy chain includes a variable region at the N-terminus and a constant region at the C-terminus.

[0035] In some embodiments, the immunoglobulin further comprises a light chain or a variable region thereof.

[0036] The immunoglobulin provided in the present application can self-polymerize monomers into a stable multimeric structure with desired valence, and has higher polymerization stability or potency.

[0037] In a third aspect, an embodiment of the present application provides a nucleic acid encoding any one of the immunoglobulins described above.

[0038] In a fourth aspect, an embodiment of the present application provides a vector comprising the above-mentioned nucleic acid.

[0039] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); 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, and papovaviruses (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.).

[0040] In a fifth aspect, an embodiment of the present application provides a cell containing the above-mentioned nucleic acid or vector.

[0041] The term "cell" refers to cells 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.

[0042] In a sixth aspect, an embodiment of the present application provides a method for preparing immunoglobulin, comprising culturing the above-mentioned cells.

[0043] 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.

[0044] In some embodiments, the degree of polymerization of the multimer is 2, 3, 4, 5, or 6.

[0045] 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.

[0046] In some embodiments, the conjugate further comprises biotin or a biotin derivative conjugated to the immunoglobulin or polymer.

[0047] In some embodiments, the conjugate further comprises a label coupled to the immunoglobulin or polymer.

[0048] In some embodiments, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0049] In some embodiments, the conjugate further comprises a solid phase carrier coupled to the immunoglobulin or polymer.

[0050] 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.

[0051] In some embodiments, the solid phase carrier is selected from the group consisting of a test tube, an EP tube, a multi-well plate, a chromatography column, and a well of a micro reaction plate.

[0052] In this application, the term "microparticle" may be a sphere, a nearly spherical shape, a cube, a polyhedron, or an irregular shape. The diameter of the microparticle may be 10 nm to 1 mm, for example, 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm.

[0053] The particles are preferably magnetic particles containing a magnetic substance. The magnetic substance can be a metal (elemental metal or alloy), a non-metal, or a metal-non-metal composite. Examples of metals include iron and aluminum nickel cobalt; non-metals include ferrite non-metals (preferably Fe2O3 or Fe3O4 magnetic nanoparticles); and metal-non-metal composites include neodymium iron boron rubber magnetic composites.

[0054] 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.

[0055] In a ninth aspect, embodiments of the present application provide a kit or test strip containing the above-mentioned immunoglobulin, polymer or conjugate.

[0056] 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.

[0057] The polymer is coated on the detection area and / or dropped on the conjugate pad.

[0058] It is easy to understand that when the detection area and the binding pad both contain multimers, the IgG variable regions of the multimers bind to different antigenic epitopes, and the second polypeptide terminal domains may be the same or different.

[0059] In some embodiments, the kit may also include sample pretreatment reagents (such as sample purification and enrichment reagents, lysis buffer, etc.), cleaning solutions (such as water, etc.), buffer solutions (such as PBS or Tris, etc.), color development reagents for signal substances (such as ECL or DAB for horseradish peroxidase as a signal substance), etc.

[0060] The immunoglobulin provided in this application can self-polymerize monomers into a stable multimeric structure with a desired valence, and thus has higher stability or potency. Therefore, when used for labeling or coating, it has higher detection sensitivity or correlation.

[0061] In a tenth aspect, an embodiment of the present application provides a method for detecting an antigen, comprising:

[0062] The immunoglobulin, polymer, conjugate, or the kit or test strip described above is contacted with the antigen to form a conjugate, and the conjugate is detected.

[0063] In some embodiments, the antigen is an antigen that can specifically bind to the variable region of the immunoglobulin.

[0064] 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).

[0065] 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).

[0066] Alternatively, the antigen is detected using a polymer-coated solid support in combination with another polymer, in which case the polymer coated on the solid support and the other polymer are paired antibodies.

[0067] The signal substance can be any one of a fluorescent substance, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radiocontrast 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.

[0068] 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 immunoassays.

[0069] The embodiments of the present application will be described in detail below with reference to examples.

[0070] Example

[0071] 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, and primer synthesis and gene sequencing were performed by Invitrogen.

[0072] Example 1 Preparation of polynucleotide encoding IgM heavy chain constant region

[0073] 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 the corresponding type of monoclonal antibody by PCR.

[0074] 1.1 Obtaining polynucleotides encoding mouse IgM CH regions

[0075] Polynucleotides encoding the mouse IgM-CH region were obtained by PCR from the genome of the hybridoma cell line 6M9 (available from Feipeng Bio) that secretes IgM type mouse monoclonal antibodies. The primers used are as follows:

[0076] Forward primer sequence (5′→3′): GAG AGT CAG TCC TTC CCA AAT GTC TTC CCC CTC GTC TC;

[0077] Reverse primer sequence (5'→3'): CCC GAA TTC TTA TCA ATA GCA GGT GCC ACC TGT GTC AGA CAT GAT CAG.

[0078] An EcoRI restriction site (GAATTC) was introduced immediately after the stop codon TGA TAA (anticodon TTA TCA) using a reverse primer. Double stop codons were used. A polynucleotide of approximately 1.4 kb was obtained by PCR and introduced into the pMD-18T vector for sequencing confirmation.

[0079] The polynucleotide encodes the mouse IgM-CH region, and its amino acid sequence from N-terminus to C-terminus is as follows:

[0080] 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: 3 is CH2-CH3-CH4).

[0081] Example 2 Construction of expression plasmid for IgG / IgM hybrid recombinant antibody

[0082] The source of IgG antibodies is not limited. In this example, the IgG antibody used is an anti-cTnI 2E7 antibody, the heavy chain and light chain sequences of which are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0083] The IgM sequence itself exhibits multimeric properties. First, the polynucleotide encoding the IgG segment 1 of the 2E7 antibody was ligated to the polynucleotide encoding the CH2-CH3-CH4 region of mouse IgM using bridge PCR. The amino acid sequence of the IgG segment 1 of the 2E7 antibody is as follows:

[0084] 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-2E7Rmb-F1.

[0085] A polynucleotide encoding the IgG segment 3 of the 2E7 antibody was selected and ligated to a polynucleotide encoding the CH2-CH3-CH4 region of mouse IgM using bridge PCR. The amino acid sequence of the IgG segment 3 of the 2E7 antibody is as follows:

[0086] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSDTHYNQMFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYFCAFITSVGDYWGQ GTTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI(SEQ ID NO:5). Connect to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-2E7Rmb3.

[0087] A polynucleotide encoding IgG segment 4 of the 2E7 antibody was selected and ligated to a polynucleotide encoding the CH2-CH3-CH4 region of mouse IgM using bridge PCR. The amino acid sequence of IgG segment 4 of the 2E7 antibody is as follows:

[0088] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSDTHYNQMFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYFCAFITSVGDYWGQGTTLT VSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP(SEQ ID NO:6). Connect to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-2E7Rmb4.

[0089] Ligation of the polynucleotide encoding the IgG heavy chain of 2E7 into pcDNA TM 3.4 vector, referred to as pcDNA3.4A-2E7 control, as a control antibody.

[0090] Since the multimerization property of IgM depends on the heavy chain, the light chain sequence of the antibody in this example is the same as the light chain of the above-mentioned anti-cTnI 2E7 antibody (shown in SEQ ID NO: 11).

[0091] Example 3 Expression and Preparation of IgG / IgM Hybrid Recombinant Antibodies

[0092] 1. Linearization of recombinant antibody expression plasmid

[0093] Prepare the following enzyme digestion system: 50ul restriction enzyme buffer, 100ug / tube recombinant plasmid, 10ul PvuⅠ enzyme, and make up to 500ul with sterile water, and digest in a 37°C water bath overnight; first use an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) (lower layer) as the enzyme digestion system, and then use chloroform (aqueous phase) for extraction; use 0.1 times the volume (aqueous phase) of 3M sodium acetate and 2 times the volume of ethanol to precipitate DNA on ice, rinse the DNA precipitate with 70% ethanol, remove the organic solvent, and after the ethanol is completely evaporated, re-dissolve it with an appropriate amount of sterile water, and finally measure the concentration.

[0094] 2. Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0095] The heavy chain plasmid and light chain plasmid constructed in Example 2 were diluted with ultrapure water at a ratio of 1:1 to 40 μg / 100 μl. A new centrifuge tube was taken and the CHO cell concentration was adjusted to 1.43×10 7 cells / ml, mixed 100ul plasmid with 700ul cells, transferred to an electroporation cup for electrotransfection, continued to culture cells after transfection, and counted the next day; cultured under pressure in 25umol / L MSX 96-well plates for about 25 days.

[0096] 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; after 3 days, preserve the seeds for batch culture and adjust the cell density to 0.5×10 6 cells / ml, and cultured in batches in a volume of 2.2 ml at a cell density of 0.3 × 10 6 cells / ml, 2ml was used 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 passage, and the cell lines expressing IgG / IgM hybrid recombinant antibodies were obtained.

[0097] 3. Cell Expansion Culture

[0098] After cell recovery, the cells were inoculated into 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 Cells / ml were inoculated and expanded. The expanded culture volume was calculated based on production requirements. The culture medium used was 100% Dynamis medium. The culture was expanded every 72 hours. When the cell quantity met the production requirements, the inoculation density was strictly controlled to 5×10 5 Produced at a rate of approximately cells / ml.

[0099] 4. Shake flask production and purification

[0100] Shake flask production parameters: shaker speed 120r / min, temperature 37℃, carbon dioxide content 8%. Feeding: Start feeding every day after 72h of culture in shake flask, and feed 3% of the initial culture volume of HyClone every day. TM Cell Boost TM Feed 7a, daily feed rate is 1 / 1000 of the initial culture volume of HyClone TM Cell Boost TM Feed 7b until day 12 (feeding on day 12). 3g / L glucose was added on day 6. Samples were collected on day 13. Multimeric antibodies of IgG / IgM hybrid recombinant antibodies were purified in two steps by affinity chromatography using captoL (cytiva) and CHT (Bio-Rad) as fillers. 10ul of purified antibody (at a concentration of 1mg / ml) was subjected to reducing SDS-PAGE. Two bands were shown in each lane, one with a relative molecular mass (Mr) of 70-80kD (heavy chain) and the other with a Mr of 20-35kD (light chain). After sequencing, the Fc sequence of the 2E7Rmb-F1 antibody constructed above is shown in SEQ ID NO: 1, the constant region sequence of the 2E7Rmb-F1 antibody is shown in SEQ ID NO: 2, the constant region sequence of the 2E7Rmb3 antibody is shown in SEQ ID NO: 7, the constant region sequence of the 2E7Rmb4 antibody is shown in SEQ ID NO: 8, and the constant region sequence of the control antibody is shown in SEQ ID NO: 9.

[0101] Example 4 Application of IgG / IgM hybrid recombinant antibodies

[0102] 1) Preparation of fluorescence chromatography detection reagent card

[0103] 1.1) Preparation of nitrocellulose membrane

[0104] Prepare nitrocellulose membrane: Dilute 2E7Rmb-F1 recombinant antibody to 1-2 mg / ml in coating buffer. Draw a T-line (test line) approximately 5 mm from the gold-labeled pad. Dilute goat anti-mouse IgG antibody (obtained from Feipeng Bio) to 1-2 mg / ml in coating buffer. Draw a C-line (control line) approximately 3 mm from the absorbent pad. Dry at 37°C and seal for later use.

[0105] 1.2) Preparation of fluorescence chromatography-labeled monoclonal antibodies

[0106] Take 1 ml of 1% solid content fluorescent microspheres, centrifuge and remove the supernatant, add an equal volume of labeling activation solution to the microspheres and mix by ultrasonication, 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 labeling coupling solution to the microspheres and mix by ultrasonication, then add 0.2-1 mg of another paired cTnI labeled antibody, shake and mix in the dark for 3 hours, finally add labeling blocking solution for blocking, shake and mix in the dark for 45 minutes, terminate labeling, centrifuge and remove the supernatant, re-dissolve the microspheres with microsphere labeling preservation solution, mix by ultrasonication, and store at 4°C for use. The shelf life is 1 month.

[0107] 1.3) Preparation of fluorescent labeling pad

[0108] Dilute the marker 5-10 times with marker diluent and spray the diluted marker onto the glass fiber mat using a spray pad device. Seal the marker mat and store at 4°C until ready for use.

[0109] 1.4) Preparation of test strip sample pad

[0110] Soak the glass fiber pad in blocking solution (containing BSA) for 30 minutes, dry it at 37°C, seal the sample pad, and store it at 4°C for later use.

[0111] 1.5) Assembly of the test reagent card

[0112] 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 placed in a card shell, one pack per card. Desiccant was added and the cards were vacuum-sealed in an aluminum foil bag to obtain the 2E7Rmb-F1 fluorescence chromatography detection reagent card.

[0113] 2E7Rmb3, 2E7Rmb4 or control antibody (2E7) fluorescence chromatography detection reagent cards were prepared according to the method described for the above-mentioned 2E7Rmb-F1 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.

[0114] 2) IgG / IgM hybrid recombinant antibody fluorescence chromatography platform test results

[0115] Purchased cTnI antigen was used as the test sample. The aluminum foil pouch was opened and the test card removed. For each test sample, 50 μL of the sample was pipetted into the sample diluent, mixed thoroughly, and then 50 μL was pipetted into the sample well of the test card. The reaction time after adding the sample to the test card was 15 minutes. The fluorescence detector automatically performed the test and read the results. Thirty clinically confirmed specimens collected from the hospital were also used as test samples to test the antibody's specimen correlation r. The specific test results are shown in Table 1.

[0116] Table 1

[0117] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0118] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

[0119] Some of the amino acid sequences involved in this application are shown in Table 2:

Claims

1. An immunoglobulin having an Fc region shown in SEQ ID NO:

1.

2. The immunoglobulin according to claim 1, wherein The immunoglobulin comprises a heavy chain having an Fc region as shown in SEQ ID NO: 1; Optionally, the heavy chain has a constant region shown in SEQ ID NO:

2.

3. An immunoglobulin having a constant region shown in SEQ ID NO:

2.

4. A nucleic acid, a vector, and a cell, wherein the nucleic acid encodes the immunoglobulin according to any one of claims 1 to 3; the vector contains the above-mentioned nucleic acid; and the cell contains the above-mentioned nucleic acid or vector.

5. A method for preparing immunoglobulin, comprising culturing the cell according to claim 4.

6. A multimer obtained by polymerizing the immunoglobulin according to any one of claims 1 to 3 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 3 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 claims 1 to 3, 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 3, 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.

10. Use of the immunoglobulin according to any one of claims 1 to 3, 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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