An anti-glycocholic acid antibody and use thereof

Abstract

The application provides an anti-glycocholic acid antibody and application thereof, and specifically, the application immunizes BALB / C mice with a glycocholic acid antigen, and more than ten positive monoclonal hybridoma cell strains are screened and obtained. Through performance determination, finally, 3 cell strains capable of specifically combining with glycocholic acid are obtained. Among them, the H15 antibody has high titer, good stability, and stable detection linearity, and can be used for clinical detection of glycocholic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to an anti-cholyglycine antibody and application thereof. BACKGROUND

[0002] Cholyglycine (CG) is a conjugated bile acid combined by cholic acid and glycine, and is one of main components of bile acids. Cholesterol is converted into primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA), in liver cells through extremely complex enzymatic reactions. The three hydroxyl groups (C3, C7, C12) on the steroid nucleus of cholic acid are combined with the hydroxyl group at the end of the side chain to form cholyglycine through a peptide bond, and the molecular weight of cholyglycine is 466.3. After being synthesized by liver cells, cholyglycine is discharged into the gallbladder through capillary bile ducts and bile ducts, and enters the duodenum with bile to help the digestion of fat in food. Under normal circumstances, the content of cholyglycine in peripheral blood is extremely low. When liver cells are damaged or bile stagnates, cholyglycine metabolism and circulation are disordered, the ability of liver cells to uptake cholyglycine is reduced, and the content of cholyglycine in blood is increased. The high and low values of cholyglycine are related to the severity of liver cell damage and bile acid metabolism disorder. The changes in the content of CG have important clinical significance in diseases such as liver cancer, liver cirrhosis, hepatitis, intrahepatic cholestasis of pregnancy (ICP), cholelithiasis, jaundice, biliary duct, gallbladder excretion dysfunction, obstructive liver disease, and intestinal-liver circulation disorder.

[0003] At present, the main method for determining cholyglycine is serological screening, in which the latex immunoturbidimetry is used to determine the content of cholyglycine in serum and has a wide application in clinic. The detection principle is that the cholyglycine antigen in the sample to be detected is specifically combined with the latex particle-enhanced anti-cholyglycine antibody to form an immune complex to produce turbidity, and the change in absorbance at 600 nm is detected, and the change degree is proportional to the concentration of cholyglycine in the sample. In the above method, the anti-cholyglycine antibody is undoubtedly the key to determining the content of CG, and therefore the effective development of the antibody becomes the primary task in the field.

[0004] Therefore, the technical personnel in the field are committed to developing an anti-cholyglycine antibody with more accurate detection effect, higher sensitivity and stable properties. SUMMARY

[0005] The present application aims to provide an anti-cholyglycine antibody and application thereof.

[0006] In a first aspect of the present application, an anti-cholyglycine monoclonal antibody is provided, and the antibody has:

[0007] (1) a heavy chain variable region; and / or

[0008] (2) a light chain variable region;

[0009] wherein the heavy chain variable region comprises the following three complementarity determining regions (CDRs):

[0010] CDR1 as shown in SEQ ID NO.: 1,

[0011] CDR2 as shown in SEQ ID NO.: 2, and

[0012] CDR3 as shown in SEQ ID NO.: 3;

[0013] the light chain variable region comprises the following three complementarity determining regions (CDRs):

[0014] CDR1' as shown in SEQ ID NO.: 5,

[0015] CDR2' as shown in SEQ ID NO.: 6, and

[0016] CDR3' as shown in SEQ ID NO.: 7.

[0017] In another preferred embodiment, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.: 4.

[0018] In another preferred embodiment, the light chain variable region has an amino acid sequence as shown in SEQ ID NO.: 8.

[0019] According to a second aspect of the present application, there is provided a polynucleotide encoding the antibody according to the first aspect of the present application.

[0020] According to a third aspect of the present application, there is provided a vector comprising the polynucleotide according to the second aspect of the present application.

[0021] In another preferred embodiment, the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0022] According to a fourth aspect of the present application, there is provided a genetically engineered host cell comprising the vector according to the third aspect of the present application or the polynucleotide according to the second aspect of the present application integrated into the genome.

[0023] According to a fifth aspect of the present application, there is provided an immunoconjugate comprising:

[0024] (a) the antibody according to the first aspect of the present application; and

[0025] (b) a conjugating moiety selected from the group consisting of a detectable label or an enzyme.

[0026] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label.

[0027] In a sixth aspect of the present application, there is provided the use of an antibody according to the first aspect of the present application, or an immunoconjugate according to the fifth aspect of the present application, for the manufacture of a reagent, assay plate or kit.

[0028] The reagent, assay plate or kit is used for detecting glycocholic acid in a sample.

[0029] In a seventh aspect of the present application, there is provided a method for detecting glycocholic acid in a sample, the method comprising the steps of:

[0030] (1) contacting the sample with an antibody according to the first aspect of the present application;

[0031] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of glycocholic acid in the sample.

[0032] In another preferred embodiment, the detection in step (2) is performed by ELISA or latex immunoturbidimetry.

[0033] In another preferred embodiment, the sample comprises a human or animal tissue sample, a sample of exfoliated cells.

[0034] In another preferred embodiment, the method is used for non-diagnostic purposes.

[0035] In an eighth aspect of the present application, there is provided a kit comprising an antibody according to the first aspect of the present application.

[0036] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Linear test results of H15 monoclonal antibody are shown. DETAILED DESCRIPTION

[0038] The present inventors, through in-depth research, after a large number of screening, successfully obtained a specific monoclonal antibody (H15 monoclonal antibody) against glycocholic acid. Experimental results show that the monoclonal antibody can specifically bind to glycocholic acid, has high titer and good stability. On this basis, the present application is completed.

[0039] Specifically, the present application uses glycocholic acid antigen to immunize BALB / C mice, and more than ten positive monoclonal hybridoma cell strains are screened. After performance determination, three cell strains capable of specifically binding to glycocholic acid are finally obtained. Among them, H15 has the best comprehensive performance.

[0040] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural core features. It is composed of two identical light (L) polypeptides and two identical heavy (H) polypeptides. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues are located at the interface between the variable regions of the light and heavy chains.

[0041] As used herein, the term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies, which is believed to be of significance in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of the heavy and light chains each comprise four FR regions, largely by β-sheet formation, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.

[0042] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chains of different classes of immunoglobulins have different constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0043] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the rabbit monoclonal antibodies herein are obtained by screening a phage library followed by construction of a full-length rabbit monoclonal antibody gene expression vector by molecular biology methods, and the vector is transformed into a eukaryotic expression system, and the supernatant is obtained after culture, and is not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0044] The present application also includes monoclonal antibodies having the amino acid sequences of the anti-glycocholic acid monoclonal antibody described, monoclonal antibodies having the variable region chains of the anti-glycocholic acid monoclonal antibody described, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing hypervariable regions (complementarity determining regions, CDRs) as long as the hypervariable regions are identical to or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and the heavy chain of the present application.

[0045] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by the combination of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the anti-glycocholic acid monoclonal antibody described or fragments thereof. The present application also includes cell surface markers or antigens bound to the anti-glycocholic acid monoclonal antibody described or fragments thereof.

[0046] The present application includes not only intact monoclonal antibodies, but also immunologically active fragments of antibodies, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains.

[0047] As used herein, the term "heavy chain variable region" is used interchangeably with "V H H".

[0048] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0049] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:

[0050] CDR1 having the amino acid sequence DYGVA (SEQ ID NO.: 1);

[0051] CDR2 having the amino acid sequence FISNLAYSIYYADTVTG (SEQ ID NO.: 2);

[0052] CDR3 having the amino acid sequence GGGYYPFFDY (SEQ ID NO.: 3).

[0053] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

[0054] EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGVAWFRQQPGKGPEW VAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEASRLRSEDTAMYYCARG GGYYPFFDYWGQGTTLTVSS (SEQ ID NO.: 4).

[0055] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region, which can be of murine, human or rabbit origin.

[0056] As used herein, the term "light chain variable region" is used interchangeably with "V L ".

[0057] In a preferred embodiment of the present application, the light chain variable region of the antibody according to the present application has a complementarity determining region (CDR) selected from the group consisting of:

[0058] CDR1' having the amino acid sequence RSSQSIVHSNGNTFLE (SEQ ID NO.: 5);

[0059] CDR2' having the amino acid sequence KVSGRFS (SEQ ID NO.: 6);

[0060] CDR3' having the amino acid sequence FQGSQIPYT (SEQ ID NO.: 7);

[0061] In another preferred embodiment, the amino acid sequence of the light chain variable region is:

[0062] DVLITQTPLSLPVSLGDQASISCRSSQSIVHSNGNTFLEWYLQKPGQSPK LLIYKVSGRFSGVPDRFSGSGSGTDFTLKISRVEMEDLGVYYCFQGSQIPYTF GAGTKLELK (SEQ ID NO.: 8).

[0063] In a preferred embodiment of the application, the light chain of the antibody comprises the above-mentioned variable region of light chain and a constant region of light chain, which can be of murine, human or rabbit origin.

[0064] In the present application, the terms "antibody of the application", "protein of the application" or "polypeptide of the application" are used interchangeably and refer to an antibody that specifically binds to glycocholic acid. They can or can not contain the initial methionine.

[0065] In another preferred embodiment, the antibody is a murine or human-murine chimeric monoclonal antibody against glycocholic acid, and the heavy chain constant region and / or the light chain constant region thereof can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgGl, IgG2, etc.

[0066] The present application also provides other proteins or fusion expression products having the antibody of the present application. Specifically, the present application includes any protein or protein conjugate and fusion expression product (i.e. immunoconjugate and fusion expression product) having a heavy chain and a light chain comprising a variable region, as long as the variable region is identical to or at least 90% homologous, preferably at least 95% homologous to the variable region of the heavy chain and the light chain of the antibody of the present application.

[0067] Generally, the antigen-binding properties of an antibody can be described by three specific regions in the variable regions of the heavy and light chains, referred to as complementarity-determining regions (CDRs), which are interspaced by four framework regions (FRs) whose amino acid sequences are relatively conserved and are not directly involved in the binding reaction. The CDRs form loop structures, and the beta sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

[0068] The variable regions of the heavy and / or light chains of the antibody of the present application are of particular interest, as at least part of them are involved in binding to the antigen. Therefore, the present application includes those molecules having the monoclonal antibody light chain and heavy chain variable regions with CDRs, as long as the CDRs thereof are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0069] This invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0070] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0071] The term "antibody" in this invention refers to a polypeptide having glycocholic acid binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of this invention.

[0072] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0073] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0074] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0075] Table A

[0076]

[0077] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0078] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0079] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0080] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0081] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0082] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0083] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0084] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0085] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0086] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0087] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0088] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0089] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0090] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. biotoxicants (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. gold nanoparticles / nanorobars (Lapotko et al., 2005, Cancer Communications). Letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Viral particles (Peng et al., 2004, Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Magnetic nanoparticles; 8. Prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapy agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0091] Preparation of monoclonal antibodies

[0092] The antibodies of the present invention can be prepared using various techniques known to those skilled in the art. For example, the antigens of the present invention can be administered to animals to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981), phage display technology, or recombinant DNA methods (US Patent No. 4,816,567).

[0093] Representative myeloma cells are those that are efficiently fused, produce stable, high levels of antibody-supporting cells through selected antibody production, and are sensitive to the culture medium (HAT medium matrix), including myeloma cell lines such as mouse myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0094] The culture medium in which hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with desired specificity, for example, by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of antibody-expressing cells can be detected using FACS. The hybridoma clone can then be converted into a subclone through a limiting dilution step and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Suitable culture media used for this purpose include, for example, DMEM or RPMI-1640. Furthermore, hybridoma cells can grow as ascites tumors in animals.

[0095] Monoclonal antibodies secreted by subclones are appropriately separated from culture media, ascites fluid, or serum using conventional immunoglobulin purification processes, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0096] Phage display technology is a screening technique that fuses exogenous peptides or proteins with the capsid protein of a phage. The fusion protein is displayed on the surface of the virus particle, while the DNA encoding the fusion protein is located inside the virus particle. This establishes a direct link between a large number of peptides and their DNA coding sequences, enabling the rapid identification of peptide ligands for various target molecules (antibodies, enzymes, cell surface receptors, etc.) through panning.

[0097] In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture is purified by affinity chromatography (Protein A / G-Sephrose).

[0098] In a preferred embodiment of the present invention, the monoclonal antibody is prepared using a method for producing monoclonal antibodies from Balb / C mouse ascites. Hybridoma cells are inoculated into the peritoneal cavity of sensitized mice, and the ascites fluid is extracted and purified using an affinity chromatography column (Protein A / G-Sephrose).

[0099] In a preferred embodiment of the present invention, the monoclonal antibody is constructed into a eukaryotic expression system using a recombinant DNA method, and the antibody is expressed by transiently transfecting HEK293 cells. The antibody secreted in the culture medium is then purified by affinity chromatography (Protein A / G-Sephrose).

[0100] Methods and Samples

[0101] This invention relates to a method for detecting glycocholic acid in tissue samples. The method generally involves the following steps: obtaining a tissue sample; and detecting the level of glycocholic acid in the sample. The sample used in this method is a serum sample.

[0102] Reagent test kit

[0103] The present invention also provides a kit containing only the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0104] The present invention further designs a detection kit for glycocholic acid, which includes an antibody that recognizes glycocholic acid, universal reagents and buffers required for detection, such as various buffers, enzyme-linked secondary antibodies, detection labels, detection substrates, etc. This detection kit can be an in vitro diagnostic device.

[0105] The main advantages of this invention are:

[0106] (1) The antibody against glycocholic acid provided by the present invention has high titer, good stability, and can be prepared in large quantities.

[0107] (2) The antibody detection for glycocholic acid provided by the present invention has extremely high sensitivity and linear stability, and can be used for the detection of serum samples.

[0108] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0109] Example 1: Preparation of Hybridoma Cells

[0110] (1) Preparation of feeder cells:

[0111] Healthy Balb / c mice were selected, and their peritoneal lavage fluid was collected and prepared into a cell suspension with a cell concentration of at least 5 × 10^5 cells / mL. 100 μL of the prepared cell suspension was added to each well of a 96-well culture plate and incubated in a CO2 incubator until day 2 at 37°C and 5% CO2 concentration.

[0112] (2) Preparation of myeloma cells:

[0113] Myeloma cells were resuscitated and cultured in advance. Myeloma cells in good growth condition and in the logarithmic growth phase (SP2 / 0 cells) were selected, and the cell concentration was adjusted to be no less than 0.2–1 × 10⁻⁶. 7 Cells / mL, incubated in a CO2 incubator.

[0114] (3) Preparation of positive control serum:

[0115] Synthetic small-molecule CG-conjugated carrier protein BSA was used as an antigen. Balb / c healthy mice were subcutaneously immunized twice, followed by a booster immunization intravenously. Positive control serum was obtained by collecting blood from the orbital vein.

[0116] (4) Preparation of mouse spleen cells:

[0117] After blood collection, mice were sacrificed to obtain spleen cell suspensions, and the cell count was adjusted to at least 5 × 10⁶ cells. 8 indivual.

[0118] (5) Cell fusion: Myeloma cells and spleen cells were mixed and centrifuged in 50 mL centrifuge tubes at a ratio of 1:2-5. The cells were resuspended twice in ECF buffer, and finally, 6.4 mL of ECF buffer was added to resuspend the cells again before electrofusion in an electrofusion tank. After fusion, DMEM basal culture medium was added for repair. After repair, 100 mL of HAT complete culture medium was added to resuspend the cells. 100 μL of the cell resuspension was added to each well of a 96-well culture plate containing feeder cells.

[0119] Example 2: Preparation of Monoclonal Cell Lines

[0120] Cell resuspension was cultured at 37°C and 7.5% CO2 for 7-10 days. Antibody detection was performed when visible clones appeared. Positive wells with only a single clone were marked under an inverted microscope and cloned again.

[0121] Repeat the above steps 3-5 times until the positive well rate is 100%. The obtained ten or so positive monoclonal cell lines were subjected to antibody testing, with their OD values ​​measured at 450 nm using a microplate reader. The optimal cell line test results are shown in Table 1. By comparing the OD values ​​with those of positive serum, it is confirmed that the obtained cell lines can produce positive antibodies. The obtained monoclonal cell lines were named H15, Q38, and V07.

[0122] Table 1

[0123]

[0124] Example 3 Antibody Performance Detection

[0125] The obtained cell lines were cultured in vitro and in mouse peritoneal cavity to obtain antibodies. The monoclonal antibodies on the cells and in the ascites fluid were then analyzed using ELISA to determine antibody titers (detection method referenced CN112391353A).

[0126] The ascites titer test results are shown in Table 2. The antibody secreted by the hybridoma cell line H15 of the present invention has a significantly better antibody titer than the control commercially available antibody titer.

[0127] Table 2

[0128]

[0129]

[0130] Among the three monoclonal cell lines tested, the H15 cell line had the best titer, which was significantly higher than that of commercially available antibodies.

[0131] The concentration of monoclonal antibodies produced by each cell line was uniformly diluted to 1 mg / mL with 10 mM pH 7.4 PBS. Starting from well 2, the antibodies were serially diluted 5-fold with 10 mM pH 7.4 PBS up to well 10. Positive control (serum from mice with the highest serum titer after four immunizations was diluted 500-fold) and PBS was used as negative control. The OD450 of each monoclonal antibody was measured by ELISA.

[0132] Antibody titer determination criteria: Plot a curve with Log (dilution) as the x-axis and antibody OD450 value as the y-axis. The curve equation is: y=min+(max-min) / (1+10^((LoGEC50-x)×Hillslope)). Fit the curve using Sigmaplot data processing software and obtain the median titer.

[0133] The test results are as follows:

[0134] Table 3

[0135] Antibody Median potency Commercially available antibody 105386.8 Q38 130642.4 H15 246952.6 V07 85247.3

[0136] Of the three monoclonal cell lines obtained, the H15 cell line had the best titer, more than twice that of commercially available antibodies.

[0137] Example 4 Biochemical Stability Determination

[0138] The thermostability of antibodies was detected using indirect immunofluorescence. The prepared anti-cholic acid antibody and commercially available control antibody were coupled to fluorescent tags and stored at 4℃, 25℃, 37℃, and 40℃ for 30 days, respectively. Then, 100 μL of the supernatant from each antibody was placed in a microplate reader to detect changes in fluorescence intensity, thus analyzing the thermostability of the antibodies. The fluorescence intensity of freshly prepared antibodies was considered 100%.

[0139] The test results are as follows:

[0140] Table 4

[0141] Relative fluorescence intensity 4℃ 25℃ 37℃ 40℃ Commercially available antibody 99.6％ 93.7％ 87.1％ 86.5％ Q38 99.3％ 86.2％ 80.4％ 72.6％ H15 99.8％ 98.5％ 98.3％ 97.9％ V07 99.6％ 96.4％ 95.7％ 92.7％

[0142] The test results showed that the H15 and V07 monoclonal antibodies had good stability, and the fluorescence intensity did not decrease significantly under storage conditions of 25℃-40℃.

[0143] Example 5 Biochemical Application Test

[0144] Standard concentrations of glycocholic acid samples were taken and tested using the H15 monoclonal antibody prepared according to this invention. The test results are shown in Table 5 below.

[0145] Table 5

[0146]

[0147]

[0148] Plot the average measurement results on the ordinate, perform linear regression, and calculate the slope, intercept, and correlation coefficient R. 2 The result is as follows Figure 1 As shown, R 2 =0.9981, indicating that the H15 antibody prepared in this invention has extremely high linear correlation.

[0149] Eight glycocholic acid analogues (cholic acid (CA), chenodeoxycholic acid, taurine, deoxycholic acid (DCA), lithocholic acid, ursodeoxycholic acid, and glycodeoxycholic acid) were prepared into test samples using negative human serum, and the specificity of antibody H15 was detected. The results showed that antibody H1 had an extremely low cross-reactivity with the glycocholic acid analogues, which met the requirements for specific detection.

[0150] Serum samples from 30 healthy individuals and 10 clinically diagnosed patients with liver disease exhibiting abnormal glycocholic acid levels were selected. The H15 monoclonal antibody prepared according to this invention was used to determine the glycocholic acid content in each serum sample using latex immunoturbidimetry. The results showed that the anti-glycocholic acid monoclonal antibody prepared according to this invention could clearly distinguish between the normal group and the case group samples, meeting the needs of clinical glycocholic acid detection.

[0151] Example 6 Antibody Sequencing

[0152] Hybridoma cells were preserved using lysis buffer, and total RNA from monoclonal antibody hybridoma cells was extracted, reverse transcribed, and then antibody gene cloned and sequenced. The obtained antibody sequences are as follows:

[0153] H15-VH:

[0154] EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGVAWFRQQPGKGPEW VAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEASRLRSEDTAMYYCARG GGYYPFFDYWGQGTTLTVSS(SEQ ID NO.:4)

[0155] H15-VL:

[0156] DVLITQTPLSLPVSLGDQASISCRSSQSIVHSNGNTFLEWYLQKPGQSPK LLIYKVSGRFSGVPDRFSGSGSGTDFTLKISRVEMEDLGVYYCFQGSQIPYTF GAGTKLELK(SEQ ID NO.:8)

[0157] The results of sequence analysis of the determined antibody heavy chain variable region and light chain variable region sequences are as follows:

[0158] Table 6

[0159]

[0160]

[0161] Table 7

[0162] Name Sequence SEQ ID NO. FR-L1 DVLITQTPLSLPVSLGDQASISC 13 CDR-L1 RSSQSIVHSNGNTFLE 5 FR-L2 WYLQKPGQSPKLLIY 14 CDR-L2 KVSGRFS 6 FR-L3 GVPDRFSGSGSGTDFTLKISRVEMEDLGVYYC 15 CDR-L3 FQGSQIPYT 7 FR-L4 FGAGTKLELK 16

[0163] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A monoclonal antibody against glycocholic acid, characterized in that, The antibody has a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the following three complementary determinant regions (CDRs): CDR1 shown in SEQ ID NO.1 CDR2 shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3; The light chain variable region includes the following three complementary determinant regions (CDRs): CDR1' shown in SEQ ID NO.5, CDR2' shown in SEQ ID NO.6, and CDR3' as shown in SEQ ID NO.

7.

2. The anti-glycocholic acid monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. The anti-glycocholic acid monoclonal antibody as described in claim 2, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

8.

4. A polynucleotide, characterized in that, It encodes the anticholic acid monoclonal antibody as described in claim 1.

5. A carrier, characterized in that, It contains the polynucleotide as described in claim 4.

6. A genetically engineered host cell, characterized in that, It contains the vector of claim 5 or the genome in which the polynucleotide of claim 4 is integrated.

7. An immunoconjugate, characterized in that, This immunoconjugate contains: (a) The antibody as described in claim 1; and (b) Selected from the following group of coupling parts: detectable markers or enzymes.

8. Use of the antibody as described in claim 1 and the immunoconjugate as described in claim 7 for the preparation of reagents, detection plates, or kits; wherein the reagents, detection plates, or kits are used to detect glycocholic acid in samples.

9. A reagent kit, characterized in that, The kit includes the antibody as described in claim 1.

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