Monoclonal antibody against CK18, detection reagent based on monoclonal antibody, and preparation method and application of monoclonal antibody

Abstract

The invention relates to an anti-CK18 monoclonal antibody or an antigen binding fragment thereof, a detection reagent based on the anti-CK18 monoclonal antibody, and a preparation method and application of the anti-CK18 monoclonal antibody or the antigen binding fragment. According to the present invention, the anti-CK18 monoclonal antibody or the antigen binding fragment thereof can specifically bind with the CK18 protein or the fragment thereof with the high affinity, such that the low detection lower limit and the high detection sensitivity can be provided when the anti-CK18 monoclonal antibody or the antigen binding fragment thereof is used for the immunological detection of CK18; therefore, the monoclonal antibody or the antigen binding fragment of the monoclonal antibody has extremely high potential to be developed into an immunodetection antibody or a kit of the CK18 protein, or a diagnostic antibody or a diagnostic kit of diseases (such as NASH) related to abnormality of the CK18 protein (such as abnormal expression level), and has relatively great clinical application prospect and value.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies and immunological detection, and in particular to an anti-cytokeratin 18 (CK18) monoclonal antibody or an antigen-binding fragment thereof, a detection reagent based thereon, and a preparation method and application thereof. Background Art

[0002] Cytokeratin (CK) is a class of intermediate filament proteins, which is an important component of the cytoskeleton and plays a vital role in maintaining cell morphology, mechanical stability of cells, and cell-to-cell interactions. In particular, cytokeratin 18 (CK18), as a representative member of this family, is mainly expressed in epithelial cells and acts as the main skeleton protein in hepatocytes, protecting hepatocytes from mechanical and non-mechanical stress damage, thereby maintaining the stability of cell shape.

[0003] In the field of disease diagnosis, the application value of CK18 is particularly significant. In particular, in the diagnosis of liver diseases, CK18 has become a key biomarker. In the process of transformation from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), the expression level of CK18 in hepatocytes is significantly increased, and its cleavage fragments are also released into the blood. There are two main forms of CK18 in serum: total CK18 protein (CK18-M65) and cysteine-cleaved CK18 fragments (CK18-M30). By monitoring the concentration of CK18 in serum and its fragment forms, early screening of liver disease, disease progression monitoring, and prognosis assessment can be effectively performed. In the course of liver diseases such as alcoholic liver disease, drug-induced liver injury, and various types of viral hepatitis, the expression of CK18 also changes significantly, and the degree of change is closely related to the scope and severity of liver cell damage, thus providing an important reference for clinicians to accurately judge the type and stage of liver disease.

[0004] However, although CK18 has shown great potential in the field of disease diagnosis, we still face many challenges in the preparation of CK18 recombinant antibodies, such as the optimization and selection of antigen epitopes, the construction of efficient expression systems, and the purification and identification of antibodies. Therefore, it is urgent to develop CK18 antibodies with high sensitivity and good specificity to promote the further development and application of CK18 in disease diagnosis and related medical research fields. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an anti-CK18 monoclonal antibody or its antigen-binding fragment which can specifically bind to the CK18 protein with high affinity, its related detection products, and its preparation method and application.

[0006] The inventors of the present invention obtained the monoclonal antibody against CK18 protein of the present invention after a large number of experimental screenings, which can specifically recognize and bind to CK18 protein with high affinity. Therefore, the monoclonal antibody of the present invention is particularly suitable for immunological detection of CK18 protein and diagnosis and / or prognosis evaluation of diseases related to abnormal CK18 protein levels.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides an anti-CK18 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises:

[0009] (1) the three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:7, and the three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:8; or,

[0010] (2) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:17, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:18.

[0011] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0012] (1) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; wherein the amino acid sequence of SEQ ID NO:5 is SAS; or,

[0013] (2) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively; wherein the amino acid sequence of SEQ ID NO:15 is WAS.

[0014] Further preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0015] (1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8; or

[0016] (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:17 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:17; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:18 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:18.

[0017] More preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0018] (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or

[0019] (2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 18.

[0020] In addition, the monoclonal antibody or its antigen-binding fragment further comprises a constant region; preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody; further preferably, the constant region is a constant region of an IgG1, IgG2a, IgG2b or IgG3 type.

[0021] In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof further has a leader sequence (eg, a signal peptide sequence) at the N-terminus of the heavy chain variable region and / or the light chain variable region.

[0022] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fv, scFv antibody, humanized antibody, chimeric antibody, bispecific or multispecific antibody.

[0023] The monoclonal antibody or antigen-binding fragment thereof of the present invention can bind to CK18 protein with high affinity and specificity, and thus can be used for immunological detection of CK18 protein or its fragment (i.e., a fragment covering the antigen-binding epitope of the antibody) and diagnosis and / or prognosis assessment of diseases associated with abnormal CK18 protein (e.g., abnormal expression level).

[0024] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0025] In a possible embodiment, the polynucleotide is a set of polynucleotides.

[0026] In some preferred embodiments, the polynucleotide set comprises:

[0027] (I) a first polynucleotide encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 9 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 10 or its corresponding mRNA molecule; or

[0028] (II) a first polynucleotide encoding a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 19 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 18, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 20 or its corresponding mRNA molecule.

[0029] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above and at least one expression control element operably linked to the polynucleotide.

[0030] In a fourth aspect, the present invention provides a vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0031] The vector of the present invention may be a cloning vector or an expression vector; for example, it may be a plasmid, a cosmid, a phage, etc.

[0032] In some preferred embodiments, the vector is an expression vector, preferably a eukaryotic expression vector.

[0033] In a fifth aspect, the present invention provides a host cell transformed with the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the vector as described in the fourth aspect;

[0034] The host cells include, but are not limited to, prokaryotic cells and eukaryotic cells; the prokaryotic cells are, for example, Escherichia coli cells, and the eukaryotic cells are, for example, yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0035] The host cell may also be a cell line, such as a 293T cell line.

[0036] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0037] In a sixth aspect, the present invention provides a detection reagent for detecting CK18 protein, which comprises the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.

[0038] In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in certain preferred embodiments, the kit further comprises a second antibody, which specifically recognizes the monoclonal antibody or antigen-binding fragment thereof of the present invention or an anti-idiotypic antibody; preferably, the second antibody further comprises a detectable label; such detectable labels are well known to those skilled in the art, and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

[0039] In a preferred embodiment, the detection reagent is a double antibody sandwich immunodetection reagent, which comprises two monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of those defined in items (1) and (2) of the first aspect above.

[0040] In the seventh aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect and / or the detection reagent as described in the sixth aspect in the preparation of a product for detecting the presence or level of CK18 protein or its fragment in a sample and / or for the diagnosis and / or prognosis assessment of diseases associated with abnormal CK18 protein levels.

[0041] In a possible embodiment, the sample is a biological sample from a subject.

[0042] In a feasible embodiment, the disease associated with abnormal CK18 protein level is a liver disease, preferably non-alcoholic fatty liver disease, alcoholic liver disease, drug-induced liver injury, and liver cancer.

[0043] In an eighth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0044] In a ninth aspect, the present invention provides a method for detecting the presence or level of CK18 protein in a sample, the method comprising using the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect and / or the detection reagent as described in the sixth aspect.

[0045] In some preferred embodiments of this method, the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable label.

[0046] In some other preferred embodiments of the method, the method further comprises: using a secondary antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0047] The method can be used for diagnostic purposes (eg, when the sample is a sample from a patient), or for non-diagnostic purposes (eg, when the sample is a cell sample rather than a sample from a patient).

[0048] Therefore, in some specific embodiments, the present invention provides a method for diagnosing and / or evaluating the prognosis of a disease associated with abnormal CK18 protein levels in a subject, comprising: using the monoclonal antibody or antigen-binding fragment thereof of the present invention as described in the first aspect above to detect the presence or absence of CK18 protein in a sample from the subject. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in other preferred embodiments, the method further comprises using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present invention. In a feasible embodiment, the subject is a mammal, such as a human.

[0049] The general method of using monoclonal antibodies or their antigen binding fragments to detect the presence of target antigens in samples or their levels is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method and similar detection methods.

[0050] Beneficial Effects

[0051] The inventors have confirmed through experiments that the monoclonal antibody of the present invention can specifically bind to the CK18 protein or its fragment with high affinity, and therefore, when used for the immunological detection of CK18, it can provide a lower detection limit and a higher detection sensitivity; in view of this, the monoclonal antibody or its antigen-binding fragment of the present invention has a very high potential to be developed as an immunological detection antibody or kit for CK18 protein, or a diagnostic antibody or diagnostic kit for diseases (such as NASH) associated with abnormal CK18 protein (for example, abnormal expression level), and has great clinical application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0053] Figure 1 This is the plasmid map for expressing the recombinant CK18 antigen protein constructed in Example 1.

[0054] Figure 2 This is the gel electrophoresis diagram of the recombinant CK18 antigen protein prepared in Example 1.

[0055] Figure 3 The figure is a binding curve diagram of the 10 screened monoclonal antibodies and the recombinant CK18 antigen protein detected by ELISA, as detected in Example 4.

[0056] Figure 4 The figures are the test results of 35 patients with non-alcoholic steatohepatitis (NASH) and 35 healthy subjects using the chemiluminescent detection reagent based on the two monoclonal antibodies of the present invention; wherein, Figure A is the standard curve, and Figure B is the test results. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Unless explicitly stated otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] The term "and / or" should be understood to mean that a single option can be selected, or a combination of two or more options.

[0060] The expression "percent (%) sequence identity" or simply "identity" with respect to an amino acid sequence means the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence when the amino acid sequences are aligned (and gaps are introduced, if necessary) to obtain the maximum percentage sequence identity (and any conservative substitutions are not considered part of the sequence identity). Sequence alignments can be performed using various methods in the art to determine the percentage (%) sequence identity.

[0061] The term "antigen binding fragment" refers to an antigen binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen binding region or variable region of the parent antibody, such as one or more CDRs. The antigen binding fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, etc.

[0062] The "Fab" fragment consists of a light chain and the CH1 and variable regions of a heavy chain.

[0063] A "Fab'" fragment contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0064] The "F(ab')2" fragment contains two light chains and portions of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0065] The "Fv" region contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0066] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0067] "Bispecific antibodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0068] "Affinity" or "binding affinity" refers to the binding force between an antibody and its antigen. The affinity of an antibody for its antigen is usually represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0069] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.

[0070] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0071] Example 1: Preparation of CK18 monoclonal antibody

[0072] Step 1: Immunogen preparation

[0073] First, the human CK18 gene fragment was amplified using the HEK293 cell cDNA library as a template; the amplified product was subjected to nucleic acid electrophoresis and gel recovery steps to obtain the target gene nucleic acid fragment, the recovered target gene nucleic acid fragment was mixed with the pET32a vector after NcoI / XhoI double restriction digestion, and the target gene nucleic acid fragment and the vector were recombined using the Gibison Assembly method; the recombined reaction product was transformed into TOP10 competent cells; the positive plaques with good growth status were picked, the plasmids were extracted and the sequences were verified by Sanger sequencing, and the CK18ED-pET32a plasmid with correct sequence (plasmid map as shown in Figure 1) was transformed into BL21 (DE3) expression strain; 2 plaques with good growth status were picked, inoculated into 20 ml ampicillin-resistant LB medium, and cultured overnight in a 100 ml Erlenmeyer flask at 37°C 220 rpm, the bacterial solution cultured in the previous step was transferred to 1000 ml ampicillin-resistant LB medium, and cultured in a 5000 ml Erlenmeyer flask at 37°C 220 rpm until OD600 = 0.6, then the shaking temperature was adjusted to 20°C, and 0.5 mM IPTG was added for induction for 16 h; after the expression was completed, the bacteria were collected and resuspended in 50 mM Tris-HCl 500 mM NaCl lysis buffer, and the bacteria were broken by ultrasonication on ice at 60% power for 1 hour, and then centrifuged at 12000 rpm for 30 min, the precipitate was removed, and the supernatant was retained. The supernatant of the bacterial lysate was loaded onto a Ni-NTA metal chelate chromatography column with a total filler volume of 5 ml. The affinity chromatography column was equilibrated and washed using the above-mentioned lysis buffer. The above-mentioned lysis buffer was added with 400 mM imidazole to prepare an elution buffer to elute the target protein (i.e., the recombinant CK18 antigen protein, whose amino acid sequence is shown in SEQ ID NO: 21). The collected target protein eluate was dialyzed into a storage buffer and identified by SDS-PAGE.

[0074] The SDS-PAGE results of the recombinant CK18 antigen protein are as follows Figure 2 As shown; Figure 2 The results showed that the obtained electrophoresis band was consistent with the molecular weight of CK18 protein, which suggested that the above expression and purification steps indeed obtained CK18 protein, which can be used as an immunogen.

[0075] Step 2: Immunize mice with antigen

[0076] The recombinant CK18 antigen protein produced and purified as described above was used to immunize female BALB / C mice aged 6-8 weeks; specifically, immunization was performed by subcutaneous multiple-point injection; for the first immunization, the immunogen was mixed with an equal volume of Freund's complete adjuvant to prepare an emulsion, and for the second and third immunizations, the immunogen was mixed with an equal volume of Freund's incomplete adjuvant to prepare an emulsion; three weeks after the first immunization, the second immunization was performed, and the third immunization was performed two weeks after the second immunization; one week after the last immunization, the mouse orbital blood was collected for antibody titer detection, and the two mice with the highest titer were selected according to the titer test results for shock immunization before cell fusion.

[0077] Step 3: Cell fusion

[0078] Preheat the prepared PEG solution in a 37°C cell culture incubator in advance; collect SP2 / 0 cells in a 50ml centrifuge tube before fusion; take the spleen of the immunized mouse under sterile conditions, grind and filter the spleen of the mouse to make a single cell suspension; mix myeloma cells SP2 / 0 and immune mouse spleen cells in a ratio of 1:5, add preheated PEG and stir gently, incubate at 37°C for 5 minutes for cell fusion, add the fused cells to a 96-well plate with a feeder cell layer, 100ul / well. Place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0079] Step 4: Hybridoma cell cloning and screening

[0080] The fused cells were cultured for one week before testing; specifically, positive clones reactive with the CK18 recombinant antigen were selected, and then the cells were subcloned using the limiting dilution method, and finally the selected positive monoclones were expanded and cultured in a plate.

[0081] Example 2: Obtaining monoclonal antibody variable region gene sequences and expressing antibodies

[0082] For the 5 positive monoclonal clones screened in Example 1, the total RNA of hybridoma cells was extracted using the Trizol method, and reverse transcribed to synthesize cDNA. Mouse-specific light and heavy chain amplification primers were used to amplify the gene sequences of the light chain variable region and heavy chain variable region of the antibody using cDNA as a template.

[0083] The amplified light and heavy chain variable region gene fragments are connected to the framework plasmid with the desired constant region, and the resulting recombinant plasmid is transfected into mammalian cells to express the antibody. In this way, the present embodiment expresses the above 5 positive monoclonal complete antibodies with IgG1 constant region, which are numbered 1#, 2#, 3#, 4#, and 5# respectively.

[0084] Example 3: Immune library construction and antibody screening

[0085] In this embodiment, a phage antibody library screening method is used to screen positive monoclonal antibodies, and the specific steps are as follows:

[0086] (1) According to the method described in Example 2, RNA was extracted from the spleen cells of the mice immunized in Example 1 and the light and heavy chain variable region gene sequences were amplified;

[0087] (2) Partial linker fragments were introduced into the 3' end of the light chain variable region and the 5' end of the heavy chain variable region obtained by the above amplification, and the light chain variable region fragment, the heavy chain variable region fragment, and the phagemid vector were homologously recombined to construct the scFv recombinant plasmid by homologous recombination. The constructed plasmid was electrotransferred into the competent medium and the library capacity was calculated. The helper phage was added and the phage immune library was prepared by PEG8000 precipitation. One day before screening, the CK18 recombinant antigen was coated in an immune tube, and the phage immune library was added to bind at 37°C. The unbound phages were washed away with PBS and PBST, and then the bound phages were eluted with a low pH Gly-HCl buffer. The above steps were repeated for enrichment 3 times, and the selected monoclonal clones were identified by ELISA. A total of 5 positive monoclonal clones were obtained. The positive clones were sequenced to obtain the gene sequences of the light and heavy chain variable regions of the positive antibodies. Subsequently, according to the method described in Example 2, the five positive monoclonal complete antibodies (with a constant region of IgG1) obtained from the above screening were expressed in mammalian cells and numbered 6#, 7#, 8#, 9#, and 10#, respectively.

[0088] Example 4: Antibody affinity testing

[0089] Through the above examples, a total of 10 monoclonal antibodies against CK18 protein were screened and obtained; the affinity of these 10 monoclonal antibodies to CK18 antigen was tested by ELISA, and the specific steps are as follows:

[0090] (1) Antigen coating: dilute the CK18 recombinant antigen protein (prepared in Example 1) to an appropriate concentration using PBS, add 100 μL / well to the ELISA plate, place the ELISA plate in a 4° C. refrigerator, and incubate for 12-14 h.

[0091] (2) Blocking: Add 3% skimmed milk powder to the ELISA plate for blocking, 200 ul / well, and incubate at 37°C for 2 h.

[0092] (3) Adding primary antibody: dilute the antibody to be tested in a gradient manner with PBS, add the diluted antibody to the ELISA plate at 100 μL / well, incubate at 37°C for 45 min, wash the plate three times and pat dry.

[0093] (4) Adding secondary antibody: Add diluted enzyme-labeled secondary antibody to the ELISA plate at 100 μL / well, incubate at 37°C for 45 min, wash the plate three times and pat dry.

[0094] (5) Color development and reading: Add color developing solution to the ELISA plate, incubate at room temperature for 10 min, and then terminate the reaction. Use an ELISA reader to read the value.

[0095] (6) Data analysis.

[0096] The binding curves of the above 10 monoclonal antibodies and CK18 recombinant antigen protein are shown in Figure 3 shown. Figure 3 The results show that the average EC50 of the 10 monoclonal antibodies binding to the CK18 antigen is 48 ng / ml, indicating that the 10 monoclonal antibodies all have good antigen binding activity; in particular, the EC50 values ​​of 2# and 4# are the smallest, indicating that they have the highest affinity with the antigen. These two monoclonal antibodies are the monoclonal antibodies for which protection is sought in the present invention.

[0097] Sequence analysis and identification of these two monoclonal antibodies showed that the heavy chain variable region and light chain variable region gene sequences were as follows, where the underlined regions are the CDR regions:

[0098] >2#-Ab-VH (i.e., SEQ ID NO: 7)

[0099] QVQLKESGPGLVAPSQSLSITCTVS GFSLTSYG VHWVRQPPGKGLEWLGV IWAG GST NYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYC ARDWDWYFYV WGQ GTSVTVSS; wherein the underlined parts are SEQ ID NOs: 1, 2, 3 respectively;

[0100] >2#-Ab-VL (i.e., SEQ ID NO: 8)

[0101] DIVMTQSHKFMSTSIGDRVSITCKAS QDV VAWSQQKPGQSPKLLIY SAS YRY TGVPDRFTGSGSGTTDFTFTISSVQAEDLAVYYC QQQHYSTPWT FGGGTKLEIK; wherein the underlined parts are SEQ ID NO: 4, 5, 6 respectively;

[0102] >4#-Ab-VH (i.e., SEQ ID NO: 17)

[0103] QVQLKESGPGLVAPSQSLSITCTVS GFSLTGYG VNWVRQPPGKGLEWLGM IWGD GST DYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYC ARNDRGDPY WGQGTS VTVSS; wherein the underlined parts are SEQ ID NOs: 11, 12, 13 respectively;

[0104] >4#-Ab-VL (i.e., SEQ ID NO: 18)

[0105] NIMMTQSPSSLAVSAGEKVTMSCKSS QSVLYNSNQKNY LAWYQQKPGQSPKLLI Y WAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC HQYLSSWT FGGGTKLEIK; wherein the underlined parts are SEQ ID NOs: 14, 15, and 16 respectively.

[0106] Example 5: Application of chemiluminescent detection reagent based on CK18 antibody

[0107] In this example, antibody #2 was selected as the capture antibody and coupled to magnetic beads, and antibody #4 was selected as the labeling antibody and coupled to alkaline phosphatase. Serum samples from 35 patients diagnosed with non-alcoholic steatohepatitis (NASH) and 35 healthy subjects were tested using a semi-automatic or fully automatic chemiluminescence immunoassay.

[0108] First, the CK18 recombinant antigen prepared in Example 1 was diluted in a gradient manner to obtain a series of CK18 standard concentrations; the concentrations of these CK18 standards were detected using the chemiluminescent reagent, and a standard curve was established based on the detected concentration and the theoretical concentration. The standard curve is as follows: Figure 4 As shown in Figure A, the standard curve R 2 =0.9983, indicating that the detected concentration of the CK18 standard is highly consistent with the theoretical concentration, proving that the chemiluminescent detection reagent based on the monoclonal antibody of the present invention has high detection accuracy.

[0109] Then, the CK18 content was tested in serum samples collected from 35 patients diagnosed with non-alcoholic steatohepatitis (NASH) and 35 healthy subjects. During the test, the serum sample loading volume was 50-200μl, the incubation time was 10-60 minutes, and the number of washes was 3-5 times.

[0110] The test results of 35 patients with nonalcoholic steatohepatitis (NASH) and 35 healthy subjects were as follows Figure 4 As shown in Figure B in the figure, the results show that there is a significant difference in the serum CK18 content level between the NASH patient group and the healthy subject group, indicating that there is a significant correlation between the serum CK18 content and NASH; this is consistent with the recognized fact in the art that serum CK18 can be used as a diagnostic marker for NASH.

[0111] The above results fully demonstrate that the chemiluminescent detection reagent based on the CK18 monoclonal antibody of the present invention has good detection performance and can be used for the diagnosis of NASH.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-CK18 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) the three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:7, and the three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:8; or, (2) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:17, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:

18.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; wherein the amino acid sequence of SEQ ID NO:5 is SAS; or, (2) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively; wherein the amino acid sequence of SEQ ID NO:15 is WAS.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8; or (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:17 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:17; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:18 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

18.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or (2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

18.

5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The monoclonal antibody or antigen-binding fragment thereof further comprises a constant region; Preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody; More preferably, the constant region is of IgG1, IgG2a, IgG2b or IgG3 type.

6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv antibody, humanized antibody, chimeric antibody, bispecific or multispecific antibody.

7. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The polynucleotide according to claim 7, wherein The polynucleotide is a polynucleotide group, and the polynucleotide group includes: (I) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:9 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:10 or its corresponding mRNA molecule; or (II) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 19 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 20 or its corresponding mRNA molecule.

9. A nucleic acid construct comprising the polynucleotide according to any one of claims 7 to 8 and at least one expression control element operably linked to the polynucleotide.

10. A vector comprising the polynucleotide according to any one of claims 7 to 8, or the nucleic acid construct according to claim 9.

11. A host cell transformed with the polynucleotide according to any one of claims 7 to 8, the nucleic acid construct according to claim 9 or the vector according to claim 10.

12. An immunoassay reagent for detecting CK18 protein, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

13. The immunoassay reagent according to claim 12, characterized in that: It is a double antibody sandwich immunoassay reagent, and comprises two monoclonal antibodies or antigen-binding fragments thereof selected from the group consisting of those defined in items (1) and (2) of any one of claims 1 to 6.

14. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to any one of claims 7 to 8, the nucleic acid construct according to claim 9, the vector according to claim 10, the host cell according to claim 11 and / or the immunodetection reagent according to claim 12 or 13 in the preparation of a product for detecting the presence or level of CK18 protein or its fragment in a sample and / or for the diagnosis and / or prognosis assessment of a disease associated with abnormal CK18 protein levels.

15. The use according to claim 14, characterized in that The sample is a biological sample of a subject; And / or, the disease associated with abnormal CK18 protein level is a liver disease, preferably non-alcoholic fatty liver disease, alcoholic liver disease, drug-induced liver injury, or liver cancer.

16. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the method comprising: The host cell of claim 11 is allowed to express the monoclonal antibody or the antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or the antigen-binding fragment thereof, and the expressed monoclonal antibody or the antigen-binding fragment thereof is recovered from the culture of the host cell.

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