SS-a / ro60 binding proteins, methods of making and uses

Abstract

The application provides an SS-A / Ro60 binding protein, a preparation method and application, and relates to the technical field of antibodies.The SS-A / Ro60 binding protein comprises a light chain variable region and a heavy chain variable region, wherein VH-CDR1 is shown as SEQ ID NO.1, VH-CDR2 is shown as SEQ ID NO.2, and VH-CDR3 is shown as SEQ ID NO.3; VL-CDR1 is shown as SEQ ID NO.4, the amino acid sequence of VL-CDR2 is AAS, and VL-CDR3 is shown as SEQ ID NO.5.The binding protein has good specific binding capacity with SS-A / Ro60, and can be used for identification of SS-A / Ro60 antibodies or SS-A / Ro60 antigens, and auxiliary diagnosis and detection of SS-A / Ro60 antibody positive diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to an SS-A / Ro60 binding protein, its preparation method, and its application. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Ro60 is an RNA-binding protein with a molecular weight of approximately 60 kDa, located in the cell nucleus. It binds to a small non-coding RNA molecule called "YRNA" and is one of the two target antigens of anti-SSA antibodies; the other is the Ro52 protein with a molecular weight of approximately 52 kDa. Mice lacking the gene encoding Ro60 exhibit autoimmune syndromes characterized by the presence of anti-ribosomal antibodies, anti-chromatin antibodies, and glomerulonephritis. Some researchers speculate that the Ro60 protein may play a role in preventing systemic autoimmune diseases.

[0004] Anti-SSA antibodies are associated with a variety of diseases, such as lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis. Although their diagnostic specificity is poor, they can assist in clinical diagnosis.

[0005] Currently, common diagnostic kits often use anti-SS-A / Ro60 antibodies derived from human positive serum. However, serum sources are difficult to obtain, and batch-to-batch variations are significant. Furthermore, methods using hybridoma technology to produce rabbit or mouse monoclonal antibodies, or purifying polyclonal antibodies from immunized animal positive serum and then conjugating them with human IgG, are complex, time-consuming, and costly. Moreover, they may not yield antibodies with ideal affinity suitable for diagnostic kit applications. Therefore, developing high-affinity recombinant monoclonal antibodies against SS-A / Ro60 that are easy to obtain, can be mass-produced, and can be widely used in clinical diagnostics, antigen protein purification, and identification is of great significance and value.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an SS-A / Ro60 binding protein that exhibits good specific binding ability to SS-A / Ro60. Based on the SS-A / Ro60 binding protein provided by this invention, another objective is to provide its applications. A further objective of this invention is to develop an SS-A / Ro60 binding protein with good binding activity using a simpler method, which can be used for the identification of SS-A / Ro60 antibodies to assist in the diagnostic detection of SS-A / Ro60 antibody-positive diseases.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] Definition of noun:

[0010] The SS-A / Ro protein consists of a 60kD protein and a 52kD protein. In this article, SS-A / Ro60 refers to the 60kD subunit of the SS-A / Ro protein. SS-A / Ro60 is an RNA-binding protein whose gene is located on chromosome 1, encoding 525 amino acid residues with a molecular weight of 80kDa. SS-A / Ro60 is present in both the nucleus and cytoplasm. In the cytoplasm, it can form complexes with small cytoplasmic RNAs called Y RNAs. SS-A / Ro60 is an autoantigen in various autoimmune diseases.

[0011] The SS-A / Ro60 binding protein provided by this invention can specifically bind to SS-A / Ro60. The SS-A / Ro60 bound by the SS-A / Ro60 binding protein includes SS-A / Ro60 expressed naturally in cells or expressed in cells transfected with the SS-A / Ro60 gene. The SS-A / Ro60 bound by the SS-A / Ro60 binding protein also includes artificially modified SS-A / Ro60, wherein the artificial modification includes, but is not limited to, mutated, truncated, or fused peptides or proteins with other structural domains, while retaining the necessary antigenic epitopes for antibody binding.

[0012] In this document, the technical term "binding protein" refers to a protein that binds to a specific antigen, broadly encompassing all proteins and protein fragments containing a complementarity-determining region (CDR). Proteins and protein fragments can be antibodies. The terms "antibody" and "full-length antibody" include both polyclonal and monoclonal antibodies. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. Non-naturally occurring antibodies are also referred to as "recombinant antibodies" in this document. The term "antibody" is used interchangeably with "immunoglobulin."

[0013] Proteins and protein fragments can also be antigen-binding fragments containing part or all of an antibody CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to antigens. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. These fragments are selected from, but are not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments, dsFv)), bispecific antibodies, nanobodies, and the smallest recognition unit of an antibody. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0014] The "variable region" or "variable domain" of a binding protein refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing the antigen. The heavy chain variable domain can be referred to as the "VH," and the light chain variable domain as the "VL." These domains are typically the most variable parts of the antibody and contain the antigen-binding site. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0015] The term "constant region" or "constant domain" of an antibody refers to the constant region of the antibody light chain, either alone or in combination, or the constant region of the antibody heavy chain. The antibody heavy chain has a variable domain (VH), followed by one or more constant domains or regions, such as hinges, CH1, CH2, CH3, and CH4. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the antibody heavy chain, and does not form a portion of the antibody's Fc region. The hinge region includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The N-terminus of CH2 is typically the CH3 domain, which usually forms the C-terminal portion of the antibody. In some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can originate from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms.

[0016] This invention does not limit the method of obtaining the SS-A / Ro60 binding protein. In some optional embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the SS-A / Ro60 binding protein to a vector and expressing it in cells. The vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a structure capable of expressing the SS-A / Ro60 binding protein. In other optional embodiments, the binding protein can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as automated peptide synthesizers (e.g., automated peptide synthesizers sold by Applied BioSystems, etc.); the antigen-binding fragment can also optionally be generated by enzymatic cleavage of antigen-binding molecules (including intact antibodies), such as pepsin or papain cleavage; or by chemical cleavage, such as by chemical reduction of disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0017] The terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of a binding protein to an epitope on a pre-defined antigen. Typically, binding proteins bind at a rate of approximately less than 10... -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K d Value binding. The K value of the antibody can be determined using methods well-established in the art. d Values. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry.

[0018] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides encode the aforementioned SS-A / Ro60 binding protein, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0019] In this article, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the cells.

[0020] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0021] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Transformation” and “transformed cell” include primary test cells and cultures derived from them.

[0022] In this document, the terms “purified” or “isolated” associated with peptides or nucleic acids mean that the peptide or nucleic acid is not in its native medium or in its native form. Therefore, the term “isolated” includes peptides or nucleic acids removed from their original environment, such as if they are naturally occurring. For example, isolated peptides typically do not contain at least some proteins or other cellular components that are normally bound to or mixed with or in solution with them. Isolated peptides include naturally produced peptides contained in cell lysates, peptides in purified or partially purified forms, recombinant peptides, peptides expressed or secreted by cells, and peptides in heterologous cells or cultures. As associated with nucleic acids, the terms “isolated” or “purified” indicate, for example, that the nucleic acid is not in its native genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into heterologous cells).

[0023] In this article, "SS-A / Ro60 antibody positive disease" refers to a disease that can cause a patient's SS-A / Ro60 antibody level to be higher than that before the disease. Exemplary SS-A / Ro60 antibody positive diseases include, but are not limited to, autoimmune diseases (such as lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis).

[0024] Firstly, an SS-A / Ro60 binding protein is provided, which includes a light chain variable region and a heavy chain variable region.

[0025] The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3.

[0026] The amino acid sequence of VH-CDR1 is GFTFNTNA (SEQ ID NO.1);

[0027] The amino acid sequence of VH-CDR2 is IRNKANGYTT (SEQ ID NO.2);

[0028] The amino acid sequence of VH-CDR3 is ARDNPTVGAY ((SEQ ID NO.3));

[0029] The amino acid sequence of VL-CDR1 is ESVDNYGISF (SEQ ID NO.4);

[0030] The amino acid sequence of VL-CDR2 is AAS;

[0031] The amino acid sequence of VL-CDR3 is QQNNEDPWT (SEQ ID NO.5).

[0032] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the SS-A / Ro60 binding protein is shown in SEQ ID NO. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7.

[0033] In an optional embodiment, the SS-A / Ro60 binding protein is one of the following: intact antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody, and antibody minimum recognition unit.

[0034] In an optional embodiment, the species from which the remaining portion of the antibody sequence originates include one or more of the following: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human. The remaining portion of the antibody sequence includes, but is not limited to, any one, several, or all of the backbone regions (FRs), and / or some or all of the constant regions.

[0035] In an optional implementation, the SS-A / Ro60 binding protein is an antibody or antigen-binding fragment including a constant region.

[0036] In an optional implementation, at least a portion of the constant region sequence of the SS-A / Ro60 binding protein is a human constant region sequence.

[0037] In an optional embodiment, the constant region sequence of the SS-A / Ro60 binding protein is selected from the sequence of part or all of the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, wherein IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD include their subclasses and mutant forms.

[0038] In an optional embodiment, the SS-A / Ro60 binding protein contains a heavy chain constant region.

[0039] In an optional embodiment, the heavy chain constant region sequence of the SS-A / Ro60 binding protein is selected from part or all of the constant region sequence of human IgG1, preferably including CH1, CH2 and CH3 of the constant region of human IgG1.

[0040] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.8.

[0041] In an optional embodiment, the SS-A / Ro60 binding protein contains a light chain constant region.

[0042] In an optional implementation, the light chain constant region sequence is selected from the light chain constant region of a mouse.

[0043] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.9.

[0044] In an optional embodiment, the SS-A / Ro60 binding protein is a human-mouse chimeric antibody, and the heavy chain amino acid sequence of the SS-A / Ro60 binding protein is shown in SEQ ID NO.10, and the light chain amino acid sequence is shown in SEQ ID NO.11.

[0045] In a second aspect, a biological material is also provided, comprising a polynucleotide, a carrier, or a cell; wherein the polynucleotide encodes the aforementioned SS-A / Ro60 binding protein; the carrier carries the polynucleotide; and the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the SS-A / Ro60 binding protein.

[0046] By linking the vector with a polynucleotide encoding the SS-A / Ro60 binding protein, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line that can express the SS-A / Ro60 binding protein, and the corresponding protein can be obtained through cell expression.

[0047] In an optional embodiment, the cells used to express SS-A / Ro60 binding protein are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0048] In an optional implementation, the cells used to express the SS-A / Ro60 binding protein are CHO cells (Chinese hamster ovary cells).

[0049] Thirdly, a method for preparing the SS-A / Ro60 binding protein of the first aspect is also provided, including culturing cells capable of expressing the SS-A / Ro60 binding protein.

[0050] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the SS-A / Ro60 binding protein into cells, and obtaining the SS-A / Ro60 binding protein by purification.

[0051] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the SS-A / Ro60 binding protein as needed, and / or preparing a suitable expression vector as needed, transforming the expression vector into the desired cells and expressing it, and obtaining the SS-A / Ro60 binding protein through purification.

[0052] In an optional embodiment, the cell is prepared by converting a polynucleotide encoding a SS-A / Ro60 binding protein as described in the first aspect into the cell, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the conversion comprising co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0053] In an optional implementation, the method includes fusing the C-terminus of the variable region of the heavy chain with a constant region fragment to construct a complete heavy chain expression plasmid, wherein the constant region fragment includes one or more of CH1, CH2, and CH3.

[0054] In an optional implementation, the constant region fragment includes a constant region fragment of human IgG1.

[0055] In an optional embodiment, the cell is a eukaryotic cell, preferably a mammalian cell.

[0056] In an optional embodiment, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

[0057] Fourthly, the application of the SS-A / Ro60 binding protein of the first aspect or the biological material of the second aspect in any of the following (I) to (VII) is also provided:

[0058] (I) Non-diagnostic and treatment-oriented detection of SS-A / Ro60 antibodies;

[0059] (II) Preparation of products for detecting SS-A / Ro60 antibodies;

[0060] (III) Prepare products for diagnosing diseases with positive SS-A / Ro60 antibodies;

[0061] (IV) Non-diagnostic and treatment-oriented testing of SS-A / Ro60;

[0062] (VII) Prepare products for the detection of SS-A / Ro60;

[0063] (VI) Used for purifying SS-A / Ro60;

[0064] (VII) Prepare products for purifying SS-A / Ro60;

[0065] For the detection of SS-A / Ro60 antibodies or in the diagnosis of SS-A / Ro60 antibody-positive diseases, SS-A / Ro60 binding protein can be used as a standard and / or quality control for the test to provide a reliable reference for the test results, and can also be used to construct a standard curve.

[0066] In an optional embodiment, the preparation of products for diagnosing SS-A / Ro60 antibody-positive diseases includes the preparation of products for diagnosing or assisting in the diagnosis of autoimmune diseases. These diseases include autoimmune diseases, including but not limited to one or more of the following: lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis.

[0067] The application described in aspect (IV) above can utilize the specific binding of the SS-A / Ro60 binding protein to SS-A / Ro60, enabling the detection of SS-A / Ro60 or cells expressing SS-A / Ro60 based on immunoassay techniques. For example, when the SS-A / Ro60 binding protein is an immunoconjugate, such as one linked to a fluorescent group, a fluorescence detection device can be used to locate or detect SS-A / Ro60 in real time. It can be used in techniques such as immunoblotting, immunoprecipitation, or flow cytometry that involve the specific binding properties of SS-A / Ro60 antigen and antibody to detect SS-A / Ro60. Based on the specific binding of the SS-A / Ro60 binding protein to SS-A / Ro60, it can also be used for the purification of SS-A / Ro60 in aspect (VI).

[0068] In optional embodiments, in aspects (II), (III), (V) or (VII) above, those skilled in the art can prepare corresponding products (such as the immunoconjugates mentioned above) according to actual uses, and select other reagent components in the product, including but not limited to one or more of the following: tracer markers, solid-phase carriers, buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, conjugates, negative controls, positive controls, standards, quality control products, and markers.

[0069] Fifthly, a reagent or kit is also provided, the reagent or kit comprising the SS-A / Ro60 binding protein of the first aspect, or the biological material of the second aspect.

[0070] In an optional embodiment, the kit is used to detect SS-A / Ro60 antibodies or to detect SS-A / Ro60 antibody-positive diseases; the kit includes standards and / or quality control products containing the SS-A / Ro60 binding protein.

[0071] In an optional embodiment, the kit further includes a detection reagent, which includes an SS-A / Ro60 antibody detection reagent.

[0072] In optional embodiments, the kit further includes detection reagents for one or more of the following: anti-Jo-1 antibody, anti-PL12 antibody, anti-PL7 antibody, anti-EJ antibody, anti-OJ antibody, anti-Ha antibody, anti-KS antibody, and anti-Zo antibody; and / or, the kit further includes detection reagents for antibodies against at least one of the following substances: dsDNA, Histone, Nucleosome, Ribosomal P, SS-A / Ro52, SS-A / Ro60, SSB / La, PM-scl, scl-70, U1-snRNP, Sm, CENP-B, AMAM2, Mi-2, PCNA, SRP-54, MDA-5, SAE-1, NXP-2, HMGCR, Ku, TIF1-γ, and SAE-2. This kit can be used to diagnose or assist in the diagnosis of autoimmune diseases using any one or more of the above-mentioned autoantibodies as markers. The autoantibody-positive diseases include, but are not limited to, one or more of the following: idiopathic inflammatory myopathy, antisynthetic enzyme syndrome, myositis, lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis, as well as other autoimmune diseases.

[0073] In an optional embodiment, the kit further includes a solid support.

[0074] In optional embodiments, the SS-A / Ro60 binding protein in the reagent or kit is coupled to a solid-phase support; or the SS-A / Ro60 binding protein in the reagent or kit and the solid-phase support are packaged separately. By coupling the SS-A / Ro60 binding protein to the solid-phase support, it can be used to capture SS-A / Ro60 protein in the sample to be tested. Alternatively, by coupling the SS-A / Ro60 binding protein to the solid-phase support, it can be used to purify SS-A / Ro60 protein.

[0075] In an optional embodiment, the SS-A / Ro60 antibody detection reagent includes a solid-phase carrier conjugated with the SS-A / Ro60 antigen.

[0076] In an optional implementation, the kit may further include a tracer marker.

[0077] In optional embodiments, the SS-A / Ro60 binding protein in the reagent or kit is coupled to a tracer label; or the SS-A / Ro60 binding protein and the tracer label in the reagent or kit are packaged separately. By coupling the SS-A / Ro60 binding protein to the tracer label, it can be used for localized detection of SS-A / Ro60 protein or for detecting SS-A / Ro60 protein in a sample via Western blotting.

[0078] The reagents or kits described above may also optionally include reagents and / or consumables well known to those skilled in the art for use in detecting reactions or purifying proteins, including but not limited to one or more of buffers, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality controls, and markers.

[0079] The above-described reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, or immunomagnetic beads. Those skilled in the art can formulate other reagents in the reagents or kits according to the corresponding detection methods, and the present invention does not limit this.

[0080] The tracer markers in any of the above embodiments include, but are not limited to, one or more of the following: enzymes, luminescent markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radioactive contrast agents, paramagnetic ions, metals, and photosensitizers.

[0081] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Luminescent labels include, but are not limited to, fluorescent proteins, synthetic small molecules, or polymer dyes. Specific examples include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C, Phycocyanin R, Thiamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red, one or more of these. Fluorescent microspheres, colored microspheres, and latex microspheres are each independently selected from products acceptable in the art, such as those derived from commercially available products. Radionuclides include, but are not limited to, those derived from... 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I,123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0082] The solid-phase support in any of the above embodiments includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or side-flow devices; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic beads. The chromatography matrix can be any known chromatography matrix acceptable in the art, including but not limited to polystyrene, polysaccharide polymers, or silica gel. In optional embodiments, the chromatography matrix includes gel particles.

[0083] This invention discloses an SS-A / Ro60-binding protein that specifically recognizes and binds to the SS-A / Ro60 antigen, which is then further used to prepare a human-mouse chimeric recombinant monoclonal antibody. This invention has the following beneficial effects:

[0084] (1) The preparation process of this method is simple and time-saving. The monoclonal antibody sequence is known and can be expressed by cells at any time. The expression level is high, the production process is controllable, and the batch-to-batch difference of the product is small.

[0085] (2) The SS-A / Ro60 binding protein disclosed in this invention can be used as a quality control in the detection kit, which can alleviate the problems of complicated operation of polyclonal antibodies and low subsequent conjugation efficiency, reduce production costs, stabilize product quality, and significantly improve reaction values; on the other hand, compared with the direct use of human serum, it can also avoid the problems of difficult sample sources and high costs.

[0086] (3) The SS-A / Ro60 binding protein conjugate disclosed in this invention is used for the detection of SS-A / Ro60 antigen.

[0087] (4) The SS-A / Ro60 binding protein disclosed in this invention can be coupled with gel particles for immunoaffinity chromatography purification of SS-A / Ro60 antigen, resulting in higher protein purity. Attached Figure Description

[0088] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0089] Figure 1 This is an electrophoresis diagram of the heavy chain (Fd) and light chains (κ, λ) used to construct the library in Embodiment 1 of the present invention;

[0090] Figure 2 This is an electrophoresis diagram of the Fab gene fragment used to construct the library in Example 1 of the present invention;

[0091] Figure 3 This is an SDS-PAGE protein electrophoresis image of the anti-SS-A / Ro60 recombinant monoclonal antibody in Example 2 of the present invention;

[0092] Figure 4 The results of the ELISA assay for the binding of recombinant monoclonal antibody to SS-A / Ro60 protein in Example 4 of this invention are shown. Ab-SS-A / Ro60 represents the binding activity of the recombinant monoclonal antibody to the SS-A / Ro60 antigen, and Ab-BSA represents the non-specific binding activity of the recombinant monoclonal antibody to the control protein BSA. Detailed Implementation

[0093] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0094] Example 1: Screening of SS-A / Ro60 binding proteins

[0095] (I) Preparation of phage display library

[0096] (1) Spleens were taken from mice immunized with SS-A / Ro60 antigen, and lymphocytes were separated using mouse lymphocyte separation solution.

[0097] (2) RNA extraction: Take 1×10 6Total RNA was extracted from lymphocytes using a cell extraction kit.

[0098] (3) Reverse transcription: The extracted total RNA is reverse transcribed to synthesize cDNA.

[0099] (4) Antibody gene fragment amplification: Using cDNA as a template, the κ and λ light chains and the VH-CH1(Fd) region of the heavy chain of the antibody are amplified by using specific amplification primers.

[0100] 20μL reaction system: 1μL cDNA, 0.8μL Prime F, 0.8μL Prime R, 10μL 2×phanta maxmaster mix, 7.4μL Nuclease-Free Water; Reaction program: Demothering at 95℃ for 30s, Annealing at 55℃ for 30s, Extension at 72℃ for 45s, 30 cycles.

[0101] After the reaction was complete, loading buffer was added to the system, and the mixture was identified by 1% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown, the target band is approximately 750 bp.

[0102] The target band was cut off, and the antibody heavy chain (Fd) gene fragment and light chain gene fragment (κ, λ) were recovered separately.

[0103] (5) Antibody light chain and heavy chain gene fragments are combined into complete Fab gene fragments by overlapping PCR.

[0104] 25 μL reaction system and procedure: 30 ng light chain, 4.3 ng linker, 30 ng heavy chain, 0.5 μL sfiⅠF (upstream primer), 0.5 μL sfiⅠR (downstream primer), 12.5 μL 2×phanta max master mix, and Nuclease-Free Water to bring the total volume to 25 μL.

[0105] Reaction procedure: Depolymerization at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, 30 cycles.

[0106] After the reaction was complete, loading buffer was added to the system, and the mixture was identified by 1% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 2 As shown, the target band is approximately 1500 bp. The target band was excised, and the antibody Fab fragment was recovered.

[0107] Upstream primer sfi IF: 5'>GAGCAGGAGCATAGGAGGATCGGGCGGCGGCC<3' (SEQ ID NO.12);

[0108] Downstream primer sfiⅠR: 5'>CCATGGCAATGGTGATTCTGCTGCGCGGCCTGGCC<3' (SEQ ID NO. 13).

[0109] (6) Plasmid construction: The antibody Fab fragment and pComb3xSS plasmid were digested with sfiI enzyme, mixed at a molar ratio of 3:1 between the digested fragment and the plasmid, and T4 DNA ligase was added. The mixture was then ligated overnight at 16°C.

[0110] (7) Library Construction: Recover the ligation product from the previous step. Take 1.5 μl of the recovered product, add TG1 competent cells, mix well, and transfer to an electroporation cuvette for electroporation. Parameter selection: Bac-Ec1. After electroporation, activate and incubate at 37℃ for 1 h. Transfer the activated bacterial solution to 200 mL of 2×YT medium, add 1 / 1000 ampicillin antibiotic and a final concentration of 2% glucose solution, and incubate at 37℃ and 220 rpm until OD600 = 0.6. Add 20 times the number of helper phage M13K07. Mix well and place in a shaker at 37℃ for static infection for 45 min. Centrifuge for 15 min to collect the bacteria, resuspend the precipitate in 200 mL of fresh 2×YT+Amp+Kana medium, and incubate at 30℃ and 220 rpm for 14 h for phage amplification. The next day, centrifuge to collect the supernatant, add 1 / 4 volume of 20% PEG6000 to precipitate the phage, and resuspend in PBS to obtain the phage display library.

[0111] (II) Screening for anti-SS-A / Ro60 antibodies using a phage display library

[0112] (1) Biotin-conjugated SS-A / Ro60 protein was used as the target antigen. After incubation with SA magnetic beads for 1 hour, 1×10⁻⁶ ppm was added to the reaction system. 12 The phages obtained in the previous step were incubated for 1 hour, and the specific phages were captured by the antigen. The cells were then washed with PBST (PBS + 0.05% Tween-20).

[0113] (2) Add the antigen-binding magnetic beads to the TG1 bacterial solution, incubate at 37°C for 45 min, and then activate and culture at 37°C and 220 rpm for 1 h. Take an appropriate amount of the incubated bacterial solution and dilute it serially to spread it on ampicillin plates. Add glucose (final concentration of 2%) and 1 / 1000 ampicillin antibiotic to the remaining bacterial solution, and shake at 37°C and 220 rpm for 3 h.

[0114] (3) Add 10 μL of M13K07 to the bacterial culture and incubate at 37°C for 45 min for infection.

[0115] (4) Centrifuge at 6000×g for 10 min, resuspend in 10 mL of 2×YT+Amp+Kana medium, and amplify the phage by shaking at 30℃ and 220 rpm for 14 h. On the second day, centrifuge to collect the supernatant, add 1 / 4 volume of 20% PEG6000 to precipitate the phage, and resuspend in PBS to obtain the phages selected in the first round of screening.

[0116] (5) Use the phages obtained in the first round of screening to repeat steps 1 to 4 for the second round of screening.

[0117] (6) Single clone identification: Single clones were selected from the second round of screening and plated in 96-well deep-well plates, and the phages were amplified by shaking overnight.

[0118] Plate coating: ELISA plates were coated with SS-A / Ro60 antigen, and BSA was used as a control protein for plate coating. The plates were incubated overnight at 4°C.

[0119] Blocking: On the second day, discard the coating solution, wash the plate with PBST, pat dry, add 3% milk, and block at 37°C for 2 hours.

[0120] Primary antibody preparation: Centrifuge 96-well deep-well plates, take the supernatant and dilute it in a final concentration of 1% milk, mix well and use it as the primary antibody for later use.

[0121] Primary antibody incubation: Discard the blocking solution, wash the plate with PBST, pat dry, add the primary antibody, and incubate at 37°C for 2 hours.

[0122] Secondary antibody incubation: Discard the primary antibody, wash the plate with PBST, blot dry, add 1:5000 diluted Anti-M13 Antibody (HRP) secondary antibody, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate with PBST, blot dry, add the chromogenic substrate for color development, and add stop solution after 15 minutes to stop the reaction. Read the value using a microplate reader.

[0123] (7) Select clones with high read values ​​(SS-A / Ro60 antigen well OD value > 1) and low non-specific binding (BSA control protein well OD value < 0.5) for sequencing.

[0124] Light chain sequencing primers: Bomp: 5'>GTGTGGAATTGTGAGCGG<3' (SEQ ID NO.14);

[0125] Heavy chain sequencing primers: PELB: 5'>ACCTATTGCCTACGGCAGCCG<3' (SEQ ID NO.15).

[0126] Twenty single clones were selected for sequencing. Sequencing results revealed three different SS-A / Ro60 binding protein sequences, each containing an antigen-binding domain, numbered SS-A / Ro60-Ab1 to 3.

[0127] Example 2: Expression and purification of recombinant anti-SS-A / Ro60 monoclonal antibody

[0128] After obtaining the Fab region sequence of the candidate antibody through sequencing, gene synthesis is performed.

[0129] The CH1 region of the heavy chain was replaced with the CH1 region of human IgG1, and the Fc region of human IgG1 was fused at the C-terminus. Using EcoRI+BamHI as the cloning site, the complete heavy chain fragment was inserted into the PTT5 plasmid. The heavy chain constant region sequence is shown in SEQ ID NO.8 and is a human-derived sequence.

[0130] The PTT5 plasmid was selected as the vector, and the EcoRI+BamHI cloning site was used to insert a light chain gene fragment. The light chain constant region sequence is shown in SEQ ID NO.9 and is a mouse-derived sequence.

[0131] Expression was performed using the mammalian cell 293F expression system.

[0132] (1) Plasmid extraction: The gene-synthesized plasmids were transformed into TOP10 competent cells, activated for 1 hour, and then transferred to LB medium. They were cultured overnight at 37°C. The plasmids were extracted the next day using an endotoxin-free plasmid extraction kit to obtain the corresponding heavy chain plasmids and light chain plasmids.

[0133] (2) One day before transfection, 293F cells were seeded into suspension cell culture flasks, and the cell density was controlled at 1×10⁻⁶. 6 per mL.

[0134] (3) On the second day, dilute 40 μg of heavy chain plasmid and 80 μg of light chain plasmid in 6 mL of transfection buffer and mix gently. Add 480 μL of PEI and mix gently. Incubate at room temperature for 20 minutes. Add the PEI dropwise to the cells and place the cells in an incubator for suspension culture at 98 rpm, 37 °C, and 5% CO2.

[0135] (4) After 6 days, the culture supernatant was collected, and IgG was purified using rProtein A packing material. Elution was performed with 0.1M Glycine (pH 3.0), followed by neutralization with 1M Tris (pH 8.0). After elution, the ultrafiltration centrifuge tubes were replaced with PBS buffer and concentrated. Protein concentration was determined, and purity was verified by SDS-PAGE. Results are as follows: Figure 3 As shown, the size of the non-reducing SDS-PAGE electrophoresis band is approximately 150 kDa, while the heavy chain and light chain of the reduced SDS-PAGE electrophoresis band are approximately 50 kDa and 25 kDa, respectively.

[0136] Example 3: Chemiluminescence assay to determine the binding activity of recombinant monoclonal antibody to SS-A / Ro60 antigen.

[0137] The detection performance of the three monoclonal antibodies was further tested using the following methods:

[0138] 1) Dilution: Dilute the antibody to 0.03 mg / mL, and further dilute the antibody according to Table 1.

[0139] 2) Incubation: Incubate the antibody solution to be tested with the magnetic barcode coated with the corresponding antigen at 37°C for 15 min, and wash to remove unbound substances; add phycoerythrin-labeled mouse anti-human IgG antibody, incubate at 37°C for 15 min, and wash to remove unbound substances.

[0140] 3) Result interpretation: The instrument automatically recognizes the magnetic barcode and detects the fluorescence intensity of the corresponding complex.

[0141] The test results are shown in Table 1. When the three recombinant antibodies were diluted to 0.001 mg / mL, only antibody No. 1 had an MFI value greater than 1000, which is qualified.

[0142] Table 1. Reactivity of three anti-SS-A / Ro60 recombinant monoclonal antibodies determined by chemiluminescence immunoassay.

[0143]

[0144] According to the sequencing results, the heavy chain variable region sequence of antibody 1 is shown in SEQ ID NO.6, and the light chain variable region sequence is shown in SEQ ID NO.7;

[0145] SEQ ID NO.6:

[0146] QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQPPGKALEWLGFIRNKANGYTTY YSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDNPTVGAYWGQGTLVTVSA.

[0147] SEQ ID NO.7:

[0148] DVVMTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVP ARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK.

[0149] Further analysis revealed that the antigen-binding domain comprises the following light and heavy chain CDRs:

[0150] Heavy chain CDR:

[0151] VH-CDR1 is GFTFNTNA (SEQ ID NO.1);

[0152] VH-CDR2 is IRNKANGYTT (SEQ ID NO.2);

[0153] VH-CDR3 is ARDNPTVGAY (SEQ ID NO.3);

[0154] Light chain CDR:

[0155] VL-CDR1 is ESVDNYGISF (SEQ ID NO.4);

[0156] VL-CDR2 is an AAS;

[0157] VL-CDR3 is QQNNEDPWT (SEQ ID NO.5).

[0158] The complete light chain sequence of the corresponding recombinant monoclonal antibody is shown in SEQ ID NO:11, and the complete heavy chain sequence is shown in SEQ ID NO:10.

[0159] Example 4: ELISA determination of the binding activity of recombinant monoclonal antibody to SS-A / Ro60 antigen.

[0160] Further testing was conducted to determine the binding activity of the selected recombinant antibody No. 1 to the SS-A / Ro60 protein:

[0161] (1) Coat SS-A / Ro60 protein (2 μg / mL) onto an ELISA plate, and simultaneously coat it with BSA as a non-specific binding control. Incubate overnight at 4°C.

[0162] (2) Discard the coating solution, wash the plate with PBST, pat dry, add 3% milk, and block at 37°C for 2 hours.

[0163] (3) Discard the blocking solution, wash the plate with PBST, pat dry, add 1000 nM of antibody initially diluted 3 times, and incubate at 37°C for 1.5 h.

[0164] (4) Discard the primary antibody, wash the plate with PBST, pat dry, add horseradish peroxidase (HRP) labeled mouse anti-human IgG secondary antibody, and incubate at 37°C for 1 h.

[0165] (5) Discard the secondary antibody, wash with PBST, pat dry, add chromogenic substrate for color development, add stop solution after 15 min to stop the reaction, and measure OD value with microplate reader.

[0166] (6) Plot a nonlinear fit graph with OD value on the ordinate and the logarithm of antibody molar concentration on the abscissa. For example... Figure 4 The EC50 value of the antibody is approximately 13 nM.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment against SS-A / Ro60, characterized in that, It includes variable regions for light chains and variable regions for heavy chains; The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3. The amino acid sequence of VH-CDR1 is shown in SEQ ID NO.1, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.3; The amino acid sequence of VL-CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL-CDR2 is AAS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

5.

2. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment against SS-A / Ro60 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment against SS-A / Ro60 is one of the following: intact antibody, F(ab')2, Fab', Fab, Fv, scFv, and dsFv.

4. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 1 or 2, characterized in that, The remaining sequence of the antibody is derived from one of the following species: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

5. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 1 or 2, characterized in that, It also includes constant regions.

6. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 5, characterized in that, At least a portion of the constant region sequence is a human constant region sequence.

7. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 5, characterized in that, The constant region sequence is selected from a portion or all of the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

8. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 5, characterized in that, The antibody or antigen-binding fragment against SS-A / Ro60 contains a heavy chain constant region, the sequence of which is selected from the constant region sequence of human IgG1.

9. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 8, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

8.

10. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 5, characterized in that, The antibody or antigen-binding fragment against SS-A / Ro60 contains a light chain constant region, the sequence of which is selected from the light chain constant region of mice.

11. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 10, characterized in that, The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.

9.

12. The antibody or antigen-binding fragment against SS-A / Ro60 according to claim 5, characterized in that, The heavy chain amino acid sequence of the antibody or antigen-binding fragment against SS-A / Ro60 is shown in SEQ ID NO.10, and the light chain amino acid sequence is shown in SEQ ID NO.

11.

13. A biomaterial, characterized in that, Including polynucleotides, carriers, or cells; The polynucleotide encodes the antibody or antigen-binding fragment against SS-A / Ro60 as described in any one of claims 1 to 12; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the antibody or antigen-binding fragment against SS-A / Ro60 as described in any one of claims 1 to 12.

14. A method for preparing the antibody or antigen-binding fragment against SS-A / Ro60 according to any one of claims 1 to 12, characterized in that, This includes culturing the cells as described in claim 13.

15. The preparation method according to claim 14, characterized in that, The cells are prepared by converting a polynucleotide encoding an antibody or antigen-binding fragment comprising any one of claims 1 to 12 against SS-A / Ro60 into the cells, wherein the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the conversion comprises co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

16. The preparation method according to claim 14, characterized in that, The cells in question are eukaryotic cells.

17. The preparation method according to claim 16, characterized in that, The cells in question are mammalian cells.

18. The preparation method according to claim 17, characterized in that, The mammalian cells include 293 cells or CHO cells.

19. The preparation method according to claim 18, characterized in that, The mammalian cells in question are 293F cells.

20. The use of the antibody or antigen-binding fragment against SS-A / Ro60 as described in any one of claims 1 to 12, or the biological material as described in claim 13, in any one of the following (I) to (VII): (I) Detection of SS-A / Ro60 antibodies for non-diagnostic and non-therapeutic purposes; (II) Preparation of products for detecting SS-A / Ro60 antibodies; (III) To prepare products for diagnosing diseases with positive SS-A / Ro60 antibodies; (IV) Non-diagnostic and treatment-oriented testing of SS-A / Ro60; (V) Prepare products for the detection of SS-A / Ro60; (VI) Used for purifying SS-A / Ro60; (VII) Prepare products for purifying SS-A / Ro60; The product described is for the diagnosis of SS-A / Ro60 antibody-positive diseases and is intended to be used to diagnose or assist in the diagnosis of autoimmune diseases. The autoimmune diseases mentioned are one or more of the following: lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis.

21. The application according to claim 20, characterized in that, The antibody or antigen-binding fragment against SS-A / Ro60 is used as a standard or quality control.

22. A reagent or kit, characterized in that, The reagent or kit contains an antibody or antigen-binding fragment against SS-A / Ro60 as described in any one of claims 1 to 12, or the biological material as described in claim 13.

23. The reagent or kit according to claim 22, characterized in that, The kit is used to detect SS-A / Ro60 antibodies or to detect SS-A / Ro60 antibody-positive diseases; the kit includes standards and / or quality control products, the standards and / or the quality control products containing the anti-SS-A / Ro60 antibody or antigen-binding fragment; The diseases that are positive for SS-A / Ro60 antibodies include one or more of the following: lupus erythematosus, Sjögren's syndrome, neonatal lupus erythematosus, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis.

24. The reagent or kit according to claim 22, characterized in that, The kit also includes an SS-A / Ro60 antibody detection reagent.

25. The reagent or kit according to claim 22, characterized in that, The kit further includes detection reagents for detecting one or more of the following: anti-Jo-1 antibody, anti-PL12 antibody, anti-PL7 antibody, anti-EJ antibody, anti-OJ antibody, anti-Ha antibody, anti-KS antibody, and anti-Zo antibody, and / or, the kit further includes detection reagents for antibodies against at least one of the following substances: dsDNA, Histone, Nucleosome, Ribosomal P, SS-A / Ro52, SSB / La, PM-scl, scl-70, U1-snRNP, Sm, CENP-B, AMA M2, Mi-2, PCNA, SRP-54, MDA-5, SAE-1, NXP-2, HMGCR, Ku, TIF1-γ, and SAE-2.

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