Specific antibody for resisting glycocholic acid-antibody compound and application thereof

Abstract

The invention discloses a specific antibody for resisting a glycocholic acid-antibody compound and application of the specific antibody, and belongs to the technical field of glycocholic acid-antibody detection. The antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region sequentially comprise the 24 to 34 sites, the 50 to 56 sites and the 89 to 97 sites in SEQ ID NO. 1; the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region sequentially comprise the 31st site to the 35th site, the 50th site to the 65th site and the 98th site to the 106th site in SEQ ID NO.2. The antibody prepared by the invention can efficiently recognize a glycocholic acid-antibody compound, and provides a theoretical basis for screening and diagnosis of liver and gall diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cholyglycine-antibody detection, and more particularly to a specific antibody of an anti-cholyglycine-antibody complex and an application thereof. BACKGROUND

[0002] There is poor specificity in diagnosing liver diseases by using conventional liver function indicators, which is easily affected by drugs or patient living habits, and it is difficult to accurately reflect the damage degree of liver cells. Cholyglycine (CG) is a metabolic product of bile acid combined with glycine, which is mainly synthesized by the liver and excreted through the biliary tract. Abnormal elevation of serum CG level is closely related to various hepatobiliary diseases, such as intrahepatic cholestasis of pregnancy (ICP), liver and bile duct obstruction, chronic hepatitis, cirrhosis, etc. Cholyglycine is an important clinical indicator of hepatobiliary diseases, which provides an important basis for the diagnosis, treatment and prognosis analysis of hepatobiliary diseases.

[0003] Cholyglycine is a small molecule compound with a relative molecular mass of only 465.6. The existing detection methods are high performance liquid chromatography, enzyme cycle method, radioimmunoassay, ELISA immunization method, etc. At present, the cholyglycine detection kit on the market is mainly based on competitive inhibition method. The competitive inhibition method has the advantages of high specificity, high sensitivity, low cost, etc., but also has the limitations of poor repeatability, negative correlation between signal concentration and intensity, etc. The ELISA immunization method has the defects of poor specificity of antibody and antigen combination and high probability of false positive. SUMMARY

[0004] Therefore, the application provides a specific antibody for detecting cholyglycine-antibody complex and an application thereof, which overcomes the defects of poor repeatability and specificity in detecting serum CG level in the prior art.

[0005] In order to achieve the above purpose, the application adopts the following technical solutions: The application provides a specific antibody or antigen binding fragment thereof for anti-cholyglycine-antibody complex, which includes a heavy chain variable region and a light chain variable region. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region include SEQ ID NO. 1 24-34, 50-56 and 89-97, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region include SEQ ID NO. 2 31-35, 50-65 and 98-106, respectively.

[0006] Further, the light chain variable region further comprises framework regions FR1, FR2, FR3, FR4, and the amino acid sequences of FR1, FR2, FR3, FR4 comprise SEQ ID NO. 1 1-23, 35-49, 57-88, 98-107, respectively; The heavy chain variable region further comprises framework regions FR1, FR2, FR3, FR4, and the amino acid sequences of FR1, FR2, FR3, FR4 comprise SEQ ID NO. 2 1-30, 36-49, 66-97, 107-117, respectively.

[0007] Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2.

[0008] As used herein, the term "glycocholic acid-antibody complex" refers to a complex of glycocholic acid with an antibody capable of specifically recognizing and binding it, in which all of the CG antigen binding sites of the CG antibody are saturatedly occupied by CG molecules. The term "antibody" is used in the broadest sense, and includes polyclonal antibodies and monoclonal antibodies, including intact antibodies and functional (antigen binding) antibody fragments, including antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobodies) fragments. The term encompasses immunoglobulins that are genetically engineered and / or otherwise modified, e.g., intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies such as bispecific antibodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and subclasses thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgGl constant region. The antibody can comprise a human IgG4 constant region.

[0009] The term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as "antigen-binding portion", "antigen-binding domain". In some cases, the antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen-binding ability to the polypeptide.

[0010] The antibody or antigen-binding fragment thereof described above includes a heavy chain variable region and a light chain variable region, both of which are composed of a complementarity determining region and a framework region; the complementarity determining region of the heavy chain variable region and the light chain variable region are each composed of CDR1, CDR2 and CDR3; the CDR of the present application is "complementarity determining region", which is a region in the variable domain of an antibody that is highly variable in sequence and forms a structurally determined "hypervariable loop" and / or contains "antigen contact residues" "antigen contact points". CDR is mainly responsible for binding to the epitope of the antigen. One variable region usually contains 3 CDR regions, in order from the N-terminal, CDR1, CDR2 and CDR3.

[0011] Further, the antigen-binding fragment includes any one of Fab, Fv, scFv, F(ab')2, linear antibody, single domain antibody.

[0012] Further, in the specific antibody or antigen-binding fragment thereof described above, the specific antibody or antigen-binding fragment thereof can further include a constant region.

[0013] Further, the constant region can be a heavy chain constant region and a light chain constant region.

[0014] Further, the heavy chain constant region can be IgG, IgM, IgA.

[0015] Further, the IgG can be IgG1, IgG2, IgG3 or IgG4.

[0016] Further, the light chain constant region can be a lamda (λ) constant region or a Kappa (κ) constant region.

[0017] Further, the specific antibody or antigen-binding fragment thereof can be murine, human, chimeric or humanized.

[0018] As used herein, the term "monoclonal antibody" or "antibody," unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of a "light" (L) chain and a "heavy" (H) chain. In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. The constant region mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding sites. The allocation of amino acids to the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of this invention, the heavy chain may be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0019] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0020] A second aspect of the invention provides biological materials associated with the said antibody or its antigen-binding fragment, said biological materials comprising at least one of A1) to A12): A1) A nucleic acid molecule encoding the specific antibody or its antigen-binding fragment described herein; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganisms containing the nucleic acid molecules described in A1); A6) Recombinant microorganisms containing the expression cassette described in A2); A7) Recombinant microorganisms containing the recombinant vector described in A3); A8) Recombinant microorganisms containing the recombinant vector described in A4); A9) Recombinant cells containing the nucleic acid molecules described in A1); A10) Recombinant cells containing the expression cassette described in A2); A11) Recombinant cells containing the recombinant vector described in A3); A12) Recombinant cells containing the recombinant vector described in A4).

[0021] In the aforementioned biological materials, the expression cassette described in A2) refers to DNA capable of expressing the specific antibody or its antigen-binding fragment in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all regulatory sequences necessary for the expression of the aforementioned protein. The regulatory sequences, under compatible conditions, guide the coding sequence to express the aforementioned protein in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0022] In the aforementioned biological materials, the recombinant vector described in A3) can be a cloning vector or an expression vector. As used herein, 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, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors 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, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0023] When preparing an expression vector, the nucleic acid molecule encoding the aforementioned protein can be located within the vector so that it can be operatively linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, integrates into the chromosome and replicates along with the integrated chromosome. The vector contains one or more selection markers that facilitate the selection of transformed cells. A selection marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers protrophic anatrophy, etc. Examples of bacterial selection markers include the dal gene of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable stable integration of the vector into the host cell genome or ensure autonomous replication of the vector independently of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling autonomous replication in the target host cell. The origin of replication may carry a mutation that makes it temperature-sensitive in the host cell (see, for example, f. Ehrlich, 1978, Proceedings of the National Academy of Sciences 75: 1433). The yield of the gene product can be increased by inserting more than one copy of the nucleic acid molecule encoding the aforementioned protein into the host cell. This copy number increase can be achieved by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selection marker along with the nucleic acid molecule, and by culturing cells in the presence of a suitable selection reagent to select cells containing the amplified copy of the selective marker gene, thereby containing the additional copy of the nucleic acid molecule. The operations for connecting the above elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0024] In the aforementioned biological materials, the recombinant microorganisms described in A5) can specifically be bacteria, yeast, algae, and fungi. The bacteria can be any of the following: 1) Prokaryotic microorganisms; 2) Gram-negative bacteria; 3) Bacteria of the genus Escherichia; 4) Escherichia coli.

[0025] In the aforementioned biological materials, the recombinant cells described in A9)-A12) can be animal cells, and the animal cell lines can be non-reproductive materials. The animal cells can be isolated mammalian cells. The mammals include humans or mice. The mammalian cells may not include animal germ cells, animal fertilized eggs, and animal embryonic stem cells; they may be somatic cells or cell lines. The animal cells may be cell lines or somatic cells derived from mice.

[0026] A third aspect of the present invention provides a method for preparing the specific antibody or its antigen-binding fragment thereof, comprising: immunizing a mammal with the glycocholic acid-antibody complex under suitable conditions, separating spleen cells and fusing them with myeloma cells, then screening hybridoma cells that secrete the target antibody, and purifying the antibody.

[0027] A fourth aspect of the present invention provides an antibody conjugate comprising an antibody portion and a conjugation portion, wherein the antibody portion comprises the specific antibody or an antigen-binding fragment thereof.

[0028] The fifth aspect of the present invention provides the use of the specific antibody or its antigen-binding fragment, or the biomaterial or the antibody conjugate, in at least one of the following: B1) Preparation of products for detecting or assisting in the detection or diagnosis of glycocholic acid-antibody complexes; B2) Prepare products for screening or assisting in screening, diagnosis or assisting in the diagnosis of hepatobiliary diseases; B3) Preparation of products containing glycocholic acid-antibody complexes; The detection methods include direct immunoassay and indirect immunoassay. The product includes reagent kits; the reagent kits include latex-enhanced immunoturbidimetric kits, chemiluminescent immunoassay kits, and ELISA kits.

[0029] The sixth aspect of the present invention provides a product for detecting or assisting in the detection of glycocholic acid-antibody complexes, comprising the specific antibody or its antigen-binding fragment, the biological material, or the antibody conjugate.

[0030] Furthermore, the product for detecting or assisting in the detection of glycocholic acid-antibody complexes has at least one of the following functions: C1) Detection or auxiliary detection of glycocholic acid-antibody complex; C2) Screening or auxiliary screening for hepatobiliary diseases; C3) binds to the glycocholic acid-antibody complex. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Appendix Figure 1 This is a graph showing the results of the CG-1 antibody specificity test. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0034] The experimental equipment used in the examples is as follows: Experimental equipment: Micro-electronic balance, Mettler Toledo GmbH, Germany PCR instrument Hangzhou Borui Technology Co., Ltd. Benchtop centrifuges from Thermo Fisher Scientific, USA. Electrophoresis apparatus and electrophoresis tanks are manufactured by Bio-Rad, a US company. Gel Imaging System Invitrogen (Shanghai) Trading Co., Ltd. Shanghai Boxun Industrial Co., Ltd. Ultrasonic pulverizer, Ningbo Xinzhi Biotechnology Co., Ltd. Pipettes, manufactured by Sorenson Biosciences, USA Multichannel pipettes Shanghai Dalong Medical Equipment Co., Ltd. Multifunctional microplate reader from Molecular Devices, Austria Biosafety cabinets Shanghai Likang Scientific Instruments Co., Ltd. Shanghai Likang Scientific Instruments Co., Ltd. (CO2 Incubator) Main reagents and consumables PCR amplification kit from Takara Corporation, Japan. PCR Product Purification Kit (Shanghai Sangon Biotech Co., Ltd.) DNA Gel Recovery Kit Shanghai Sangon Biotech Co., Ltd. Plasmid DNA Mini-Extraction Kit (Shanghai Sangon Biotech Co., Ltd.) FastDigest BamHI endonuclease, Thermo Fisher Scientific, USA FastDigest Xho I endonuclease, Thermo Fisher Scientific, USA DEPC Water Shanghai Sangon Biotech Co., Ltd. Gel Red Nucleic Acid Dye, Shanghai Sangon Biotech Co., Ltd. DNA Loading Buffer, Shanghai Sangon Biotech Co., Ltd. DNA Molecular Weight Marker, Shanghai Sangon Biotech Co., Ltd. MICA / pCMV-XL6 plasmid, Origene Corporation, USA pET-32a carrier, manufactured by Novagen, USA. BL21 (DE3) Escherichia coli, produced by Novagen, USA. E. coli competent cell preparation kit (Shanghai Sangon Biotech Co., Ltd.) Recombinant protein refolding kit, Novagen (USA) Dithiothreitol, Sigma-Aldrich (USA) HRP-labeled goat anti-mouse IgG antibody, Jackson Immuno Research, USA Laboratories IMEM culture medium, Corning Incorporated (USA) 100× HAT, Invitrogen (USA) Protein G Agarose Resin Jiangsu Changzhou Tiandi Renhe Biotechnology Co., Ltd. Ultrafiltration tube (MW30000) Sartorius, Germany Bradford reagents, Bio-Rad Laboratories, USA Disposable low-protein adsorption filter, Sartorius GmbH, Germany 5× Sample Loading Buffer Shanghai Sangon Biotech Co., Ltd. Protein molecular weight marker, Thermo Fisher Scientific, USA Highly Sensitive Rapid Coomassie Brilliant Blue Staining Kit (Shanghai Sangon Biotech Co., Ltd.) T4 Ligase, Takara Corporation, Japan Isopropyl-β-D-thiogalactoside (IPTG) Shanghai Sangon Biotech Co., Ltd. Freund's Complete Adjuvants, Sigma-Aldrich, USA Freund's incomplete adjuvant, Sigma-Aldrich (USA) Newborn calf serum Hangzhou Tianhang Biotechnology Co., Ltd. DMEM high-glucose culture medium, Corning Incorporated (USA) Fetal bovine serum (FBS) from Corning Incorporated, USA 100× Penicillin-Streptomycin Mixture Hangzhou Gino Biomedical Technology Co., Ltd. 0.01M PBS, pH 7.4 Wuhan Sewell Biotechnology Co., Ltd. 0.25% pancreatic enzyme solution (containing 0.02% EDTA) Hangzhou Gino Biomedical Technology Co., Ltd. CG, inspired by Aladdin CG antibody, sourced from Hangzhou Bopu Pharmaceutical Co., Ltd. CG-BSA protein, derived from laboratory preparation. CG-F(ab)2 antibody, derived from laboratory preparation. The CG-F(ab)2-CG complex was derived from a laboratory-made product. The glycocholic acid test kit was prepared in-house. The experimental animals are as follows: The standard Balb / c mice were obtained from Shanghai Slack Laboratory Animal Supply Company.

[0035] Example 1: Animal Immunization 1) Preparation of glycocholic acid-antibody complex: Excess glycocholic acid reacts fully with antibody at room temperature, and then excess glycocholic acid molecules are removed by dialysis, which is the glycocholic acid-antibody complex.

[0036] 2) Immunization of animals: Balb / c mice were injected with the prepared glycocholic acid-antibody complex; each mouse was injected with 100 μL of 100 μg / mL glycocholic acid-antibody complex, for a total of 5 mice. Each mouse was immunized 3-5 times, with an interval of 2 weeks between each immunization.

[0037] 3) ELISA titer test: Blood was collected from the tail vein of mice and centrifuged to obtain plasma. The titer of the glycocholic acid-antibody complex in mouse plasma was tested by ELISA, and mice with the highest titer were selected.

[0038] Example 2: Hybridoma cell production of specific antibodies against glycocholic acid-antibody complex Mice were sacrificed by cervical dislocation to obtain their spleens. The spleens were washed with antibiotics and then with PBS until they turned grayish-white. After centrifugation and washing, a spleen cell suspension was obtained. SP2 / 0 cells and spleen cells were mixed at a ratio of 1:10 and then seeded into 96-well plates. The cell supernatant was collected after 8-10 days.

[0039] Positive clones of glycocholic acid-antibody complexes were screened by ELISA, and negative clones of CG-F(ab)2 antibody and CG-BSA protein were then screened from them; the cells were then expanded to hybridoma cell lines containing monoclonal antibodies.

[0040] Hybridoma cell lines secreting monoclonal antibodies, obtained through the above screening, were cultured, and total RNA was extracted from the cells using conventional biological methods. Using the total RNA as a template, cDNA was synthesized via reverse transcription using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TAKARA). The cDNA was then amplified using primers for the constant region of the antibody. After separation by agarose gel electrophoresis, the DNA fragments were purified and recovered, including the nucleotide sequence encoding the variable region of the light chain: pcDNA3.4-ZSH014-53-34-WT-LC( Kappa ) GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCAACTGCAAGGCCAGTCAAAATGTGGATACTTCTCTGGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAATTATTGATTTATTGGGCATCCACCCGG CACACTGGAGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTACCAATGTGCAGTCTGAAGACTTGGCACATTATTTCTGTCAACAATATAGTACCTATCCTCTCACATTCGGAGGGGGGACCAGGCTGGAAGTAAAA, SEQ ID NO.3.

[0041] Nucleotide sequence encoding the variable region of the heavy chain: pcDNA3.4-ZSH014-53-34-WT-HC GAGGTAAAGCTTCAGGAGTCGGGACCTGACCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAAGCAGATATGGTGTACACTGGGTTCGCCAGTTTCCAGGGGAGGGTCTGGAGTGGCTGGTCCTGATATGGAGTGATGGAAGCACAACCTAT AATTCAGCTCTCAGATCCAGAGTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAACATTGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGTGTCAGACATGTGCTGGGCTATGCTATGGACAATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA, SEQ ID NO.4.

[0042] The amino acid sequence deduced from this is: Finally, the light chain variable region of the monoclonal antibody is obtained: pcDNA3.4-ZSH014-53-34-WT-LC( Kappa ) DIVMTQSHKFMSTSVGDRVSINCKASQNVDTSLAWYQQKPGQSPKLLIYWASTRHTGVPDRFIGSGSGTDFTLTITNVQSEDLAHYFCQQYSTYPLTFGGGTRLEVK, SEQ ID NO.1; Heavy chain variable region: pcDNA3.4-ZSH014-53-34-WT-HC EVKLQESGPDLVAPSQSLSITCTVSGFSLSRYGVHWVRQFPGEGLEWLVLIWSDGSTTYNSALRSRVSISKDNSKSQVFLTLNSLQTDDTAMYYCVRHVLGYAMDNWGQGTSVTVSS, SEQ ID NO.2.

[0043] Based on the above amino acid sequence, the CDR and FR of the antibody variable region are divided using the Kappa numbering rule. The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region include positions 24-34, 50-56, and 89-97 of SEQ ID NO. 1, respectively. The light chain variable region also includes the framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 include positions 1-23, 35-49, 57-88, and 98-107 of SEQ ID NO. 1, respectively.

[0044] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region include positions 31-35, 50-65, and 98-106 of SEQ ID NO. 2, respectively. The heavy chain variable region also includes framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 include positions 1-30, 36-49, 66-97, and 107-117 of SEQ ID NO. 2, respectively.

[0045] Example 3: Preparation of monoclonal antibodies

[0046] The sequenced monoclonal antibody variable region was spliced ​​with the human IgG1 constant region to generate the target gene fragment, which was then cloned into the pcDNA3.4 expression vector to prepare a transfection-grade expression plasmid. Cells were cultured in serum-free medium (ThermoFisher Scientific), seeded in shake flasks, and incubated on a shaker at 37°C and 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and ExpiFectamine™ 293 transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 16-18 hours of transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 were added. After 6 days of cell culture, the supernatant was collected for purification. The final purified monoclonal antibody CG-1 was analyzed for purity by SDS-PAGE and A280 concentration was determined.

[0047] Human IgG1 constant region sequence: ASTKGPSVFPLAPSSKSGSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO.5.

[0048] Example 4: Antibody Specificity Detection

[0049] Dilute CG-BSA protein, CG-F(ab)2 antibody, and CG-F(ab)2-CG complex to a final concentration of 2.5 μg / mL using CBS at pH 9.6. Add 100 μL to each well and coat overnight at 4°C. After drying, pre-wash once with TBST washing buffer, then wash four times. Block the enzyme-labeled wells with 10% newborn calf serum TBST and incubate at 37°C for 1 h. Dilute CG-1 antibody to 1 μg / mL, repeat 3 times per group, adding 100 μL to each well and incubating at 37°C for 1 h. After drying, pre-wash once with TBST washing buffer, then wash four times. Dilute anti-mouse Fc with 2% newborn calf serum TBST at a ratio of 1:100000, adding 100 μL to each well and incubating at 37°C for 30 min. After drying, pre-wash once with TBST washing buffer, then wash four times. Add 50 μL of chromogenic solution A and 50 μL of chromogenic solution B to each well and incubate at room temperature for 5 minutes. After min, add 50 μL of stop solution, set the microplate reader to main wavelength 450 nm, secondary wavelength 630 nm, optical path 1.0, and detect OD value.

[0050] Test results as follows Figure 1 As shown, CG-1 can hardly bind to CG-BSA and CG-F(ab)2 antibodies, but it can specifically bind to the CG-F(ab)2-CG complex.

[0051] Example 5: CG-1 Affinity Detection

[0052] The CG-F(ab)2-CG complex was diluted 200-fold and 400-fold with CBS at pH 9.6, 100 μL per well, and coated overnight at 4°C. After drying, the wells were pre-washed once with TBST washing buffer and washed four times. The wells were then blocked with 10% newborn calf serum (NBS) TBST and incubated at 37°C for 1 hour. The CG-1 antibody was diluted 1000-fold and serially diluted 15 times, 100 μL per well, and incubated at 37°C for 1 hour. After drying, the wells were pre-washed once with TBST washing buffer and washed four times. GAM and 10% NBS TBST were diluted 1:4000, 100 μL per well, and incubated at 37°C for 30 minutes. After drying, the wells were pre-washed once with TBST washing buffer and washed four times. 50 μL of chromogenic solution A and 50 μL of chromogenic solution B were added to each well, and the reaction was allowed to stand at room temperature for 5 minutes. Then, 50 μL of stop solution was added, and the microplate reader was set to a main wavelength of 450 nm. nm, secondary wavelength 630nm, optical path 1.0, OD value detected.

[0053] Table 1 Affinity of CG-1

[0054] As shown in Table 1, the affinity between the CG-1 antibody and the CG-F(ab)2-CG complex is approximately 1.56 × 10⁻⁶. 11 .

[0055] Example 6: Repeatability test of glycocholic acid reagent kit The reagents were placed into the AU5800 biochemical analyzer, calibrated, and passed the quality control test.

[0056] Prepare high, medium, and low CG concentration samples, and repeat the test 20 times for each sample. Calculate the CV.

[0057] Table 2. Repeatability of the Glycocholic Acid Reagent Kit

[0058] As shown in Table 2, the CVs of high, medium, and low samples are all ≤10%, indicating good repeatability.

[0059] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A specific antibody or antigen-binding fragment thereof against an anticholic acid-antibody complex, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region include positions 24-34, 50-56 and 89-97 of SEQ ID NO.1, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region include positions 31-35, 50-65 and 98-106 of SEQ ID NO.2, respectively.

2. The specific antibody or antigen-binding fragment of the anti-cholic acid-antibody complex according to claim 1, characterized in that, The light chain variable region further includes framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 sequentially include positions 1-23, 35-49, 57-88, and 98-107 of SEQ ID NO.1; The heavy chain variable region further includes framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 include positions 1-30, 36-49, 66-97, and 107-117 of SEQ ID NO.2, respectively.

3. The specific antibody or antigen-binding fragment thereof of the anticholic acid-antibody complex according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

4. The specific antibody or antigen-binding fragment thereof of the anticholic acid-antibody complex according to claim 1 or 2, characterized in that, The antigen-binding fragment includes any one of Fab, Fv, scFv, F(ab')2, linear antibody, and single-domain antibody.

5. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, characterized in that, The biomaterial includes at least one of A1) to A12): A1) A nucleic acid molecule encoding the specific antibody or antigen-binding fragment thereof as described in any one of claims 1-4; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganisms containing the nucleic acid molecules described in A1); A6) Recombinant microorganisms containing the expression cassette described in A2); A7) Recombinant microorganisms containing the recombinant vector described in A3); A8) Recombinant microorganisms containing the recombinant vector described in A4); A9) Recombinant cells containing the nucleic acid molecules described in A1); A10) Recombinant cells containing the expression cassette described in A2); A11) Recombinant cells containing the recombinant vector described in A3); A12) Recombinant cells containing the recombinant vector described in A4).

6. A method for preparing the specific antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, include: Under suitable conditions, mammals are immunized with the glycocholic acid antibody complex, spleen cells are isolated and fused with myeloma cells, hybridoma cells that secrete the target antibody are then screened, and the antibody is purified.

7. An antibody conjugate, comprising an antibody portion and a conjugation portion, characterized in that: The antibody portion comprises the specific antibody or its antigen-binding fragment as described in any one of claims 1-4.

8. The use of the specific antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the biomaterial as described in claim 5, or the antibody conjugate as described in claim 7 in at least one of the following: B1) Preparation of products for detecting or assisting in the detection or diagnosis of glycocholic acid-antibody complexes; B2) Prepare products for screening or assisting in screening, diagnosis or assisting in the diagnosis of hepatobiliary diseases; B3) Preparation of products containing glycocholic acid-antibody complexes; in, The detection methods include direct immunoassay and indirect immunoassay. The product includes reagent kits; the reagent kits include latex-enhanced immunoturbidimetric kits, chemiluminescent immunoassay kits, and ELISA kits.

9. A product for detecting or assisting in the detection of glycocholic acid-antibody complexes, characterized in that, Includes the specific antibody or its antigen-binding fragment as described in any one of claims 1-4, the biological material as described in claim 5, or the antibody conjugate as described in claim 7.

10. The product for detecting or assisting in the detection of glycocholic acid-antibody complexes according to claim 9, characterized in that, The product has at least one of the following functions: C1) Detection or auxiliary detection of glycocholic acid-antibody complex; C2) Screening or auxiliary screening for hepatobiliary diseases; C3) binds to the glycocholic acid-antibody complex.

Images