Auto-polymerized immunoglobulin Fc region

Abstract

The invention relates to an immunoglobulin. The immunoglobulin has an Fc region as shown in SEQ ID NO: 1. The immunoglobulin can autopolymerize a monomer into a stable polymer structure, and has improved titer, detection sensitivity or correlation when being used for detection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application entitled “A Self-Aggregating Immunoglobulin Fc Region” filed with the State Intellectual Property Office on August 23, 2023, with application number 202311072501.6, the entire contents of which are incorporated by reference into this application. 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] Immunoglobulin (Ig) is composed of four polypeptide chains, each of which is connected by a varying number of interchain disulfide bonds. Ig can be in a "Y"-shaped structure, called an Ig monomer, which is the basic unit of antibodies. Natural Ig molecules contain 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 (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. IgM has a very large molecular weight, so it is very effective in antigen agglutination reactions. IgM is a multivalent antibody. Since a monomer antibody has 2 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 may 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-polymerizing Fc region 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 having an Fc region shown in SEQ ID NO:1.

[0008] The second aspect of the present application relates to an immunoglobulin having a constant region shown in SEQ ID NO:2.

[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, wherein the conjugate 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 antigen with the immunoglobulin, polymer, conjugate, kit or test strip as described above to form a conjugate, and detecting the conjugate.

[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 immunoglobulin provided in the present application and containing 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

[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 the present invention, the term "antibody" is used in the broadest sense, which may include monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies. It generally includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight. The amino acid sequences of the amino terminus (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 region 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.

[0022] In a first aspect, an embodiment of the present application provides an immunoglobulin having an Fc region shown in SEQ ID NO:1.

[0023] In a second aspect, an embodiment of the present application provides an immunoglobulin having a constant region shown in SEQ ID NO:2.

[0024] 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.

[0025] In a third aspect, an embodiment of the present application provides a nucleic acid encoding any of the immunoglobulins described above.

[0026] In a fourth aspect, an embodiment of the present application provides a vector comprising the above-mentioned nucleic acid.

[0027] 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.).

[0028] In a fifth aspect, an embodiment of the present application provides a cell containing the above-mentioned nucleic acid or vector.

[0029] 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.

[0030] In a sixth aspect, an embodiment of the present application provides a method for preparing an immunoglobulin, comprising culturing the above-mentioned cells.

[0031] 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.

[0032] In some embodiments, the polymer has a degree of polymerization of 2, 3, 4, 5 or 6.

[0033] 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.

[0034] In some embodiments, the conjugate further comprises biotin or a biotin derivative conjugated to the immunoglobulin or polymer.

[0035] In some embodiments, the conjugate further comprises a label coupled to the immunoglobulin or polymer.

[0036] In some embodiments, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents, and nanoparticle-based labels.

[0037] In some embodiments, the conjugate further comprises a solid phase carrier coupled to the immunoglobulin or polymer.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In a ninth aspect, embodiments of the present application provide a kit or test strip containing the above-mentioned immunoglobulin, polymer or conjugate.

[0044] 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.

[0045] The polymer is coated on the detection area and / or dropped on the conjugate pad.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In a tenth aspect, an embodiment of the present application provides a method for detecting an antigen, comprising:

[0050] 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.

[0051] 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).

[0052] 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).

[0053] 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.

[0054] 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.

[0055] In an eleventh aspect, embodiments of the present application provide use of the immunoglobulin, polymer, conjugate, kit or test strip as described above in immunoassays.

[0056] The implementation scheme of the present application will be described in detail below with reference to examples.

[0057] Example

[0058] 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.

[0059] Example 1 Preparation of polynucleotide encoding IgM heavy chain constant region

[0060] 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.

[0061] 1.1 Obtaining of polynucleotides encoding the CH region of murine IgM

[0062] 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:

[0063] Forward primer sequence (5′→3′): GAG AGT CAG TCC TTC CCA AAT GTC TTC CCC CTC GTCTC;

[0064] Reverse primer sequence (5'→3'): CCC GAATTC TTATCAATA GCA GGT GCC ACC TGT GTC AGACAT GAT CAG.

[0065] The EcoRI restriction site (GAATTC) was introduced by reverse primer immediately following the stop codon TGATAA (anticodon TTATCA). Double stop codons were used here. A polynucleotide of about 1.4 kb was obtained by PCR technology and introduced into the pMD-18T vector for sequencing confirmation.

[0066] The polynucleotide encodes the mouse IgM-CH region, and its amino acid sequence from N-terminus to C-terminus is as follows:

[0067] ESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPI PAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPK PITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASP STDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVED WNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQ LLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMS DTGGTCY (SEQ ID NO:3).

[0068] 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).

[0069] Example 2 Construction of expression plasmid for IgG / IgM hybrid recombinant antibody

[0070] The source of IgG antibody is not limited. In this embodiment, the IgG antibody used is anti-cTnI 2E7 antibody, and the heavy chain and light chain sequences of the antibody are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0071] The IgM sequence itself has the characteristics of a polymer. First, the polynucleotide encoding the IgG segment 1 of the 2E7 antibody was connected to the polynucleotide encoding the CH2-CH3-CH4 of mouse IgM by bridge PCR. The amino acid sequence of the IgG segment 1 of the 2E7 antibody is as follows:

[0072] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSDTHYNQMFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYFCAFITSVGDYWGQGTTL TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI(SEQ ID NO:4).

[0073] 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.

[0074] The polynucleotide encoding the IgG segment 3 of the 2E7 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 IgG segment 3 of the 2E7 antibody is as follows:

[0075] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSDTHYNQMFKDKATLTVDKSSNTAYMQLSSSLTSEDSAVYFCAFITSVGDYWGQ GTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI(SEQ IDNO:5). Connect to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-2E7Rmb3.

[0076] The polynucleotide encoding the IgG segment 4 of the 2E7 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 IgG segment 4 of the 2E7 antibody is as follows:

[0077] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSDSDTHYNQMFKDKATLTVDKSSNTAYMQLSSLTSEDSAVYFCAFITSVGDYWGQGTTLT VSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP(SEQ ID NO:6). Connect to pcDNA TM 3.4 vector, referred to as pcDNA3.4A-2E7Rmb4.

[0078] 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 the control antibody.

[0079] 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-cTnI2E7 antibody (shown in SEQ ID NO: 11).

[0080] Example 3 Expression and preparation of IgG / IgM hybrid recombinant antibody

[0081] 1. Linearization of recombinant antibody expression plasmid

[0082] 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.

[0083] 2. Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0084] 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.

[0085] 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 6 cells / 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.

[0086] 3. Cell expansion culture

[0087] 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.

[0088] 4. Shake flask production and purification

[0089] 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). 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.

[0090] Example 4 Application of IgG / IgM hybrid recombinant antibodies

[0091] 1) Preparation of fluorescence chromatography detection reagent card

[0092] 1.1) Preparation of nitrocellulose membrane

[0093] Preparation of nitrocellulose membrane: dilute 2E7Rmb-F1 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.

[0094] 1.2) Preparation of monoclonal antibodies labeled with fluorescence chromatography

[0095] 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.

[0096] 1.3) Preparation of fluorescent labeling pad

[0097] 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.

[0098] 1.4) Preparation of test strip sample pad

[0099] 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.

[0100] 1.5) Assembly of the test reagent card

[0101] 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-F1 fluorescence chromatography detection reagent card.

[0102] 2E7Rmb3, 2E7Rmb4 or control antibody (2E7) fluorescence chromatography detection reagent cards were prepared according to the method described in the above 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.

[0103] 2) IgG / IgM hybrid recombinant antibody fluorescence chromatography platform test results

[0104] 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 the test card is added 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 1.

[0105] Table 1

[0106]

[0107] 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.

[0108] 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.

[0109] Some of the amino acid sequences involved in this application are shown in Table 2:

[0110]

[0111]

Claims

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

1.

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

2.

3. A nucleic acid, a vector, and a cell, wherein the nucleic acid encodes the immunoglobulin according to any one of claims 1 to 2; the vector contains the above-mentioned nucleic acid; and the cell contains the above-mentioned nucleic acid or vector.

4. A method for preparing immunoglobulin, comprising culturing the cell according to claim 3.

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

6. A conjugate, comprising the immunoglobulin according to any one of claims 1 to 2 or the multimer according to claim 5; 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.

7. A kit or test strip containing the immunoglobulin according to any one of 1 to 2, the multimer according to claim 5, or the conjugate according to claim 6; 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.

8. A method for detecting an antigen, comprising: The immunoglobulin according to any one of claims 1 to 2, the multimer according to claim 5, the conjugate according to claim 6, or the kit or test strip according to claim 7 is contacted with the antigen to form a conjugate, and the conjugate is detected.

9. Use of the immunoglobulin according to any one of claims 1 to 2, the multimer according to claim 5, the conjugate according to claim 6, or the kit or test strip according to claim 7 in the preparation of an antigen immunoassay product.

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