Anti-glycocholic acid antibody and application thereof

Abstract

The invention provides an anti-glycocholic acid antibody and application thereof, and particularly relates to more than ten positive monoclonal hybridoma cell strains obtained by adopting glycocholic acid antigen immune BALB / C mice and screening. Through performance determination, three cell strains capable of being specifically combined with glycocholic acid are finally obtained. Wherein the H15 antibody is high in titer, good in stability and stable in detection linearity, and can be used for clinical detection of glycocholic acid.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to an anti-glycocholic acid antibody and an application thereof. Background Art

[0002] Cholyglycine (CG) is a conjugated bile acid formed by the combination of bile acid and glycine, and is one of the main components of bile acid. Cholesterol undergoes extremely complex enzymatic reactions in liver cells and is converted into primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA). There are three hydroxyl groups (C3, C7, C12) on the steroid nucleus of bile acid. The hydroxyl group at the end of the side chain is combined with glycine by a peptide bond to form cholyglycine, with a molecular weight of 466.3. After being synthesized by liver cells, cholyglycine is discharged into the gallbladder through the bile capillaries and bile ducts, and enters the duodenum along with bile to help digest fat in food. Under normal circumstances, the content of cholyglycine in peripheral blood is extremely low. When human liver cells are damaged or bile is stagnant, it will cause cholyglycine metabolism and circulation disorders, reduce the ability of liver cells to absorb cholyglycine, and lead to an increase in the content of cholyglycine in the blood. The level of cholyglycine is related to the severity of liver cell damage and bile acid metabolism disorders. Changes in CG content values ​​have important clinical significance in diseases such as liver cancer, cirrhosis, hepatitis, intrahepatic cholestasis of pregnancy (ICP), cholelithiasis, jaundice, bile duct and gallbladder excretion dysfunction, obstructive liver disease, and enterohepatic circulation disorder.

[0003] At present, the main method for determining glycocholic acid is serological screening, among which the latex immunoturbidimetry method for determining the glycocholic acid content in serum is widely used in clinical practice. The detection principle is that the glycocholic acid antigen in the sample to be tested specifically binds to the anti-glycocholic acid antibody enhanced by latex particles to form an immune complex and produce turbidity, and the change in its absorbance can be detected at 600nm, and the degree of change is proportional to the concentration of glycocholic acid in the sample. In the above method, the anti-glycocholic acid antibody is undoubtedly the key to determining the CG content, so the effective development of this antibody has become a top priority in this field.

[0004] Therefore, those skilled in the art are committed to developing anti-glycocholic acid antibodies with more accurate detection effects, higher sensitivity and stable properties. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-glycocholic acid antibody and application thereof.

[0006] In a first aspect of the present invention, there is provided an anti-glycocholic acid monoclonal antibody, wherein the antibody has:

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

[0008] (2) light chain variable region;

[0009] Wherein, the heavy chain variable region includes the following three complementarity determining regions CDR:

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

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

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

[0013] The light chain variable region includes the following three complementarity determining regions CDR:

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

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

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

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

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

[0019] The second aspect of the present invention provides a polynucleotide encoding the antibody as described in the first aspect of the present invention.

[0020] The third aspect of the present invention provides a vector comprising the polynucleotide described in the second aspect of the present invention.

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

[0022] The fourth aspect of the present invention provides a genetically engineered host cell, which contains the vector described in the third aspect of the present invention or the polynucleotide described in the second aspect of the present invention integrated into its genome.

[0023] The fifth aspect of the present invention provides an immunoconjugate, which comprises:

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

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

[0026] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, etc.

[0027] The sixth aspect of the present invention provides the use of the antibody according to the first aspect of the present invention or the immunoconjugate according to the fifth aspect of the present invention for preparing a reagent, a detection plate or a kit;

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

[0029] The seventh aspect of the present invention provides a method for detecting glycocholic acid in a sample, the method comprising the steps of:

[0030] (1) contacting a sample with the antibody described in the first aspect of the present invention;

[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 includes: human or animal tissue samples, exfoliated cell samples.

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

[0035] The eighth aspect of the present invention provides a kit, which comprises the antibody described in the first aspect of the present invention.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] The inventors have successfully obtained a monoclonal antibody (H15 monoclonal antibody) specific to glycocholic acid through in-depth research and extensive screening. The experimental results show that the monoclonal antibody can specifically bind to glycocholic acid, has high titer and good stability. On this basis, the present invention was completed.

[0039] Specifically, the present invention uses glycocholic acid antigen to immunize BALB / C mice, and screens and obtains more than ten positive monoclonal hybridoma cell lines. After performance testing, three cell lines that can specifically bind 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 with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). 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. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0041] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0042] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0043] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of substantially uniform populations, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually different antibodies for 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 constructing a full-length rabbit monoclonal antibody gene expression vector by molecular biological methods after phage library screening, transferring the vector into a eukaryotic expression system, and collecting the cell supernatant after cultivation, without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially uniform antibody population, and this should not be interpreted as requiring any special method to produce the antibody.

[0044] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-glycocholic acid monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-glycocholic acid monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention 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 a hypervariable region (complementarity determining region, CDR), as long as the hypervariable region is identical to the hypervariable region of the light chain and heavy chain of the present invention or has at least 90% homology, preferably at least 95% homology.

[0045] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the anti-glycocholic acid monoclonal antibody or its fragment to form a conjugate. The present invention also includes a cell surface marker or antigen combined with the anti-glycocholic acid monoclonal antibody or its fragment.

[0046] The present invention includes not only complete monoclonal antibodies, but also antibody fragments having immunological activity, such as Fab or (Fab') 2 Fragment; Antibody heavy chain; Antibody light chain.

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

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

[0049] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three complementarity determining regions CDR:

[0050] CDR1, whose amino acid sequence is DYGVA (SEQ ID NO.: 1);

[0051] CDR2, whose amino acid sequence is FISNLAYSIYYADTVTG (SEQ ID NO.: 2);

[0052] CDR3, the amino acid sequence of which is 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 invention, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region, and the heavy chain constant region may be of mouse, human or rabbit origin.

[0056] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

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

[0058] CDR1', whose amino acid sequence is RSSQSIVHSNGNTFLE (SEQ ID NO.: 5);

[0059] CDR2', whose amino acid sequence is KVSGRFS (SEQ ID NO.: 6);

[0060] CDR3', whose amino acid sequence is FQGSQIPYT (SEQ ID NO.: 7);

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

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

[0063] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and a light chain constant region, and the light chain constant region may be of mouse, human or rabbit origin.

[0064] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to antibodies that specifically bind to glycocholic acid. They may or may not contain an initial methionine.

[0065] In another preferred embodiment, the antibody is a mouse or human-mouse chimeric monoclonal antibody against glycocholic acid, and its heavy chain constant region and / or light chain constant region 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 IgG1, IgG2, etc.

[0066] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains containing variable regions, as long as the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibodies of the present invention.

[0067] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0068] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0069] The present invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0070] As used herein, the terms "fragment", "derivative" and "analog" 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 conservative or non-conservative amino acid residues (preferably conservative 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 a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0071] The antibody of the present invention refers to a polypeptide having glycocholic acid binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0072] Variant forms of the polypeptide include: homologous sequences, conservative 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 stringency 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 almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, 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.

[0074] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0075] Table A

[0076]

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

[0078] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0079] The present invention also relates to polynucleotides that hybridize to the above-mentioned 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 can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.

[0080] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the 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 by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0082] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0083] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

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

[0085] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

[0087] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0088] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

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

[0090] The therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2, etc. (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 / nanorods (Lapotko et al., 2005, Cancer Communications (Cancer Communications) letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic 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 by 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. For monoclonal antibodies, hybridoma technology can be used to prepare (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 method (U.S. Pat. No. 4,816,567) can be used.

[0093] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium (HAT medium medium), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma 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 the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with the desired specificity, such as, by in vitro binding analysis, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. Then, the hybridoma clones can be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 pages). Suitable culture media used to achieve this purpose include, for example, DMEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0095] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0096] Phage display technology is a screening technology that fuses exogenous polypeptides or proteins with the capsid protein of 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, thereby establishing a direct connection between a large number of polypeptides and their DNA coding sequences, allowing the polypeptide ligands of various target molecules (antibodies, enzymes, cell surface receptors, etc.) to be quickly identified through screening.

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

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

[0099] In a preferred embodiment of the present invention, a eukaryotic expression system is constructed by recombinant DNA method for monoclonal antibody, HEK293 cells are transiently transfected to express the antibody, and the antibody secreted in the culture medium is purified by affinity chromatography column (Protein A / G-Sephrose).

[0100] Methods and samples

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

[0102] Reagent test kit

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

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

[0105] The main advantages of the present 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 sensitivity against glycocholic acid provided by the present invention is extremely high and linearly stable, and can be used for the detection of serum samples.

[0108] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0109] Example 1 Hybridoma Cell Preparation

[0110] (1) Preparation of feeder cells:

[0111] Select healthy Balb / c mice, take the peritoneal lavage fluid, and prepare a cell suspension with a cell concentration of at least 5×10^5 cells / mL. Add 100 μL / well of the prepared cell suspension to a 96-well culture plate and place it in a carbon dioxide incubator for culture until the second day at a temperature of 37°C and CO 2 Concentration 5%.

[0112] (2) Preparation of myeloma cells:

[0113] Resuscitate and culture myeloma cells in advance, take myeloma cells (SP2 / 0 cells) in logarithmic growth phase with good growth status, and adjust the cell concentration to no less than 0.2-1×10 7 / mL, place CO 2 Culture in incubator.

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

[0115] The synthetic small molecule CG coupled carrier protein BSA was used as an antigen, and Balb / c healthy mice were subcutaneously immunized twice and intravenously boosted once to obtain boosted immunized mice. Blood was collected from the orbital vein to obtain positive control serum.

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

[0117] After blood collection, the mice were killed to obtain spleen cell suspension, and the cell number was adjusted to at least 5×10 8 indivual.

[0118] (5) Cell fusion: Myeloma cells and spleen cells were added to a 50 mL centrifuge tube at a ratio of 1:2-5 and mixed and centrifuged. The cells were resuspended twice with ECF Buffer, and finally 6.4 mL of ECF Buffer was added to resuspend the cells. Then, the cells were added to the electrofusion tank for electrofusion. 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 above cell resuspension was added to the 96-well culture plate with feeder cells.

[0119] Example 2 Preparation of monoclonal cell lines

[0120] The cells were resuspended in 37°C and 7.5% CO 2 After 7-10 days of wet culture, antibodies can be detected when clones visible to the naked eye appear; observe under an inverted microscope, mark the positive wells where only a single clone grows, and clone again.

[0121] Repeat the above steps 3 to 5 times until the positive pore rate is 100%. The obtained more than ten positive monoclonal cell strains were subjected to antibody detection, and their OD values ​​were detected on a microplate reader at 450nm. The results of the better cell strain detection are shown in Table 1. By comparing with the positive serum OD value, it is shown that the obtained cell strain can produce positive antibodies. The obtained monoclonal cell strains were named H15, Q38, and V07.

[0122] Table 1

[0123]

[0124] Example 3 Antibody Performance Testing

[0125] The cell lines were cultured in vitro and in the peritoneal cavity of mice to obtain antibodies. The monoclonal antibodies in the cells and the ascites were tested for antibody titer by Elisa (the detection method refers to CN112391353A).

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

[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 the titer of commercially available antibodies.

[0131] The concentration of the monoclonal antibody produced by each cell line was uniformly diluted to 1 mg / mL with 10 mM PBS, pH 7.4. Starting from the second well, it was diluted 5-fold to the tenth well with 10 mM PBS, pH 7.4. The positive control (the serum of the mouse with the highest serum titer after four immunizations was diluted 500 times) and PBS was used as the negative control. The OD450 of each monoclonal antibody was determined by ELISA.

[0132] Antibody titer determination criteria: Plot a curve with Log (dilution) as the horizontal axis and antibody OD450 value as the vertical axis. The curve equation is: y = min + (max - min) / (1 + 10^((LogEC50-x) × Hillslope)). Use Sigmaplot data processing software to fit the curve and obtain the median titer.

[0133] The test results are as follows:

[0134] Table 3

[0135] Antibody Median titer Commercially available antibodies 105386.8 Q38 130642.4 H15 246952.6 V07 85247.3

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

[0137] Example 4 Biochemical stability determination

[0138] The thermal stability of the antibody was detected by indirect immunofluorescence. The prepared anti-glycocholic acid antibody and the commercially available control antibody were coupled with fluorescent labels and placed in an incubator at 4°C, 25°C, 37°C, and 40°C respectively for 30 days. Then 100 μL of the supernatant was taken from each sample and placed in an ELISA instrument to detect the change in fluorescence intensity and analyze the thermal stability of the antibody. The fluorescence intensity of the freshly prepared antibody was taken as 100%.

[0139] The test results are as follows:

[0140] Table 4

[0141] Relative fluorescence intensity 4℃ 25℃ 37℃ 40℃ Commercially available antibodies 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 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] A sample of glycocholic acid of standard concentration was taken, and the H15 monoclonal antibody prepared by the present invention was used to detect the standard glycocholic acid sample. The detection results are shown in Table 5 below.

[0145] Table 5

[0146]

[0147]

[0148] Use the average value of the measured results as the ordinate to draw a graph, perform linear regression, and calculate its slope, intercept, and correlation coefficient R 2 , the results are as follows Figure 1 As shown, R 2 =0.9981, indicating that the linear correlation of the antibody H15 prepared by the present invention is extremely high.

[0149] Eight glycocholic acid analogs (cholic acid (CA), chenodeoxycholic acid, taurocholic acid, deoxycholic acid (DCA), lithocholic acid, ursodeoxycholic acid, glycodeoxycholic acid) were prepared into test samples using negative human serum to test the specificity of antibody H15. The test results showed that the cross-reaction rate between antibody H1 and glycocholic acid analogs was extremely low, which could meet the requirements of detection specificity.

[0150] 30 healthy human sera and 10 clinically diagnosed liver disease patients with abnormal glycocholic acid sera were selected, and the H15 monoclonal antibody prepared by the present invention was used to determine the content of glycocholic acid in each serum based on latex immunoturbidimetry. The results showed that the anti-glycocholic acid monoclonal antibody prepared by the present invention can clearly distinguish between normal group and case group samples, and can meet the needs of clinical detection of glycocholic acid.

[0151] Example 6 Antibody Sequencing

[0152] The hybridoma cells were preserved in lysis buffer, and the total RNA of the monoclonal antibody hybridoma cells was extracted and reverse transcribed, and then the antibody gene was cloned and sequenced. The obtained antibody sequence is 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 measured 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 FQGQIPYT 7 FR-L4 FGAGTKLELK 16

[0163] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-glycocholic acid monoclonal antibody, characterized in that: The antibody has a heavy chain variable region and a light chain variable region; Wherein, the heavy chain variable region includes the following three complementarity determining regions CDR: CDR1 shown in SEQ ID NO.: 1, CDR2 shown in SEQ ID NO.: 2, and CDR3 shown in SEQ ID NO.: 3; The light chain variable region includes the following three complementarity determining regions CDR: CDR1' shown in SEQ ID NO.:5, CDR2' shown in SEQ ID NO.: 6, and CDR3' shown in SEQ ID NO.:

7.

2. The anti-glycocholic acid monoclonal antibody according to claim 1, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:

4.

3. The anti-glycocholic acid monoclonal antibody according to claim 2, characterized in that The light chain variable region has the amino acid sequence shown in SEQ ID NO.:

8.

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

5. A carrier, characterized in that It contains the polynucleotide according to claim 2.

6. A genetically engineered host cell, characterized in that It contains the vector according to claim 3 or the polynucleotide according to claim 2 integrated into its genome.

7. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) the antibody according to claim 1; and (b) a conjugation moiety selected from the group consisting of a detectable label or an enzyme.

8. Use of the antibody according to claim 1 or the immunoconjugate according to claim 7 for preparing a reagent, a detection plate or a kit; the reagent, the detection plate or the kit is used to detect glycocholic acid in a sample.

9. A method for detecting glycocholic acid in a sample, characterized in that: The method comprises the steps of: (1) contacting a sample with the antibody according to claim 1; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of glycocholic acid in the sample.

10. A kit, characterized in that: The kit comprises the antibody according to claim 1.

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