Anti-Hepatocyte protein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a monoclonal antibody capable of recognizing a human Hepatocyte antigen, a preparation method of the monoclonal antibody and application of the monoclonal antibody in immunodetection. According to the technical scheme, 628-731 loci, 813-882 loci and 628-731 loci are selected to be connected in series to form the antigen peptide, codon optimization is carried out, the antigen peptide becomes a gene segment suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein contains a Hepatocyte protein segment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain capable of secreting the anti-Hepatocyte protein monoclonal antibody and the anti-Hepatocyte protein monoclonal antibody secreted by the cell strain are obtained. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing Hepatocyte protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to an anti-Hepatocyte protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Hepatocyte is the main functional cell of liver, accounting for 80% of the total number of liver cells. Hepatocyte, also known as Hep Par-1, is a specific marker of liver tissue and hepatocellular carcinoma. It can recognize the mitochondrial membrane protein of hepatocytes, and almost only exists in hepatocytes, and can recognize the liver tissue of normal adults and fetuses. Hep Par-1 reacts with a urea cycle enzyme (carbamate phosphosynthetase) located on the mitochondrial membrane of hepatocytes, and this enzyme also exists in the mitochondria of intestinal epithelial and renal tubular cells, so Hep Par-1 also has expression (usually focal) in adrenal cortical tumors and gastric and small intestinal adenocarcinoma.

[0003] In clinical pathological diagnosis, the commonly used immunohistochemical markers of hepatocellular carcinoma include hepatocyte antigen (hepatocyte paraffin, Hep Par-1), cytokeratin 18 (cytokeratin 18, CK18), glutamine synthetase (glutamine synthetase, GS) and alpha fetoprotein (alpha fetoprotein, AFP) and the like. Among them, Hep Par-1 is one of the most commonly used hepatocyte-specific proteins, and its sensitivity and specificity are both the first among various hepatocellular carcinoma-related antibodies, and the positive rate in hepatocellular carcinoma is about 80-90%. The characteristics and expression pattern of Hepatocyte are of great significance for the diagnosis, classification and prognosis evaluation of liver diseases, and can be used in combination with Arginase-1 and Glypcian-3 for differential diagnosis of primary hepatocellular carcinoma, metastatic tumor and cholangiocarcinoma. SUMMARY

[0004] The present application provides an anti-Hepatocyte protein monoclonal antibody, the heavy chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the light chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0005] Further, the monoclonal antibody is a murine monoclonal antibody, and the clone number is MX119.

[0006] The present application also provides a preparation method of an anti-Hepatocyte protein monoclonal antibody, and the antigen for immunizing mice is a recombinant protein, and the recombinant protein is expressed by E. coli.

[0007] Further, the recombinant protein comprises a Hepatocyte protein fragment and a HIS protein tag.

[0008] Further, the Hepatocyte protein fragment is the 628-731 site, the 813-882 site, and the 628-731 site in series, and is the amino acid sequence shown in SEQ ID NO. 3.

[0009] Further, the plasmid vector selected in the recombinant process is pET30a.

[0010] The inventor also provides a Hepatocyte protein immunodetection reagent, which contains the above-mentioned anti-Hepatocyte protein monoclonal antibody as an effective component.

[0011] Further, the immunodetection includes immunohistochemistry, immunoblotting, and enzyme-linked immunization.

[0012] Differing from the prior art, the beneficial technical effects of the present application are that the above-mentioned technical solution selects the Hepatocyte protein 628-731 site, the 813-882 site, and the 628-731 site in series as an antigen peptide, performs codon optimization, becomes a gene fragment suitable for expression in Escherichia coli BL21(DE3), and finally obtains a recombinant protein comprising a Hepatocyte protein fragment and a histidine protein tag. The recombinant protein is used to immunize mice, and through cell fusion, screening, and subcloning, a mouse hybridoma cell strain MX119 that secretes an anti-Hepatocyte protein monoclonal antibody is obtained, as well as the anti-Hepatocyte protein monoclonal antibody secreted by the cell strain. The antibody obtained by the present solution has high specificity and sensitivity, can specifically recognize cells expressing Hepatocyte protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 FIG. 4 is a comparison chart of immunohistochemical staining results of hepatocellular carcinoma; the left is the antibody of the present application, and the right is a commercially available antibody.

[0014] Figure 2 FIG. 5 is a comparison chart of immunohistochemical staining results of liver tissue surface epithelium; the left is the antibody of the present application, and the right is a commercially available antibody. DETAILED DESCRIPTION

[0015] In order to explain the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0016] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase "in an embodiment" in various places in the specification are not necessarily referring to the same embodiment nor are separate embodiments necessarily mutually exclusive of one another. As used in this application, the term "comprises" or "comprising" or "includes" or "including" means including but not limited to, and should not be interpreted as being restricted to names of magnitude, order or sequence. Except where otherwise expressly indicated, the use of "or" in the placing of conditions in this application shall not be interpreted as meaning that exclusive disjunction is intended; it will be interpreted to denote the existence of at least one of the conditions. Unless otherwise defined, the meaning of a technical term used herein is identical to that generally understood by a person skilled in the art to which this application belongs; the use of relevant terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the application.

[0017] Unless otherwise defined, the meaning of technical terms used herein is identical to that generally understood by a person skilled in the art to which this application belongs; the use of relevant terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the application.

[0018] In the description of the application, the phrase "and / or" is a description of the logical relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally represents that the associated objects before and after are a "or" logical relationship.

[0019] In this application, such as "first" and "second" language is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0020] In this application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent in such process, method or product.

[0021] As the same understanding as in the "Guidelines for Examination", in this application, "greater than", "less than", "exceed" and other expressions are understood as not including the number; "above", "below", "within" and other expressions are understood as including the number. In addition, the meaning of "multiple" in the description of the embodiments of the application is more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times" and the like, unless otherwise specifically limited.

[0022] Example 1 Preparation of recombinant Hepatocyte protein fragments

[0023] I. Gene optimization and synthesis

[0024] Hepatocyte protein sequence according to accession number NP_001356185.1 in NCBI database, select 628-731 sites, 813-882 sites and the tandem of 628-731 sites, and directly optimize into a gene fragment suitable for expression in E. coli BL21 (DE3). Add BamH I and Xho I enzyme sites at the 5' and 3' ends of the gene during PCR.

[0025] The PCR product was recovered after agarose gel electrophoresis, and the recovered fusion protein gene and the plasmid vector pET30a for expression were subjected to BamH I and Xho I enzyme digestion, and then recovered by electrophoresis again, and linked with T4 DNA ligase. The ligation product was transformed into competent E. coli BL21 (DE3) cells, and the colonies on the plate were inoculated and subjected to bacterial liquid PCR identification. The colonies with positive PCR results were selected for sequencing analysis, and the colonies with completely correct sequences were used.

[0026] Different antigens may be selected for immunization to prepare antibodies with different binding properties. The molecule contains multiple variants caused by variable splicing, which ultimately leads to different recognition abilities and patterns of different antibodies to cells expressing the antigen. According to the published sequence, the Hepatocyte molecule was analyzed, and based on its structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression. The sequences of 628-731 sites, 813-882 sites and 628-731 sites of Hepatocyte were selected for tandem and codon optimization, and the molecular weight was about 18 kDa. The Hepatocyte protein was obtained by sequence optimization and design of prokaryotic expression gene sequence. The recombinant immunogen is composed of the Hepatocyte protein fragment with antigenicity and the protein tag for purification of the recombinant protein, and the protein tag is HIS.

[0027] II. Protein expression and purification

[0028] The single colony culture was transferred to 100 mL of LB medium at a ratio of 1:100, kanamycin was added at a final concentration of 10 μg / mL, and the culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then 0.5 mmol / L IPTG was added, and the culture was incubated at 16°C with shaking overnight. The bacteria were collected and sonicated. The recombinant protein has a histidine tag, and nickel column was used for affinity purification of the protein. Elution was performed with 500 mmol / L imidazole, and SDS PAGE separation was performed for detection. The concentration of the recombinant Hepatocyte protein with a histidine tag was 0.5 mg / mL, which could be used for animal immunization and antibody screening and identification.

[0029] Establishment of hybridoma cell line

[0030] I. Immunization

[0031] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week old SPF female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection of 6 points per mouse with a dose of 20 μg per mouse. The mice were boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) with a dose of 20 μg per mouse. Seven days after the third boost, the serum of the mice was tested for the titer of anti-antigen polyclonal antibodies by indirect ELISA (wavelength 450 nm). The mouse with the highest titer was given a boost by tail vein injection of the antigen mixed with physiological saline at a dose of 20 μg per mouse.

[0032] II. Cell fusion

[0033] The spleen cell suspension of the immunized mice was prepared aseptically and mixed with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1, and centrifuged at 1500 rpm for 5 min. After discarding the supernatant, the centrifuge tube was placed in a 37°C water bath, and 1 mL of PEG1500 (Roche) was slowly added within 1 min, and the cells were stirred. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10 mL of serum (PAA) was added, and the cells were gently blown up, and 5 mL of 10x HAT (Sigma) mixed thymocytes was added, mixed well. Then 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed well, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in a 37°C 5% CO2 incubator.

[0034] III. Cloning and ELISA screening of positive hybridoma cells

[0035] 7 days after fusion, the size of the colony is moderate, under the dissecting microscope, suck the round, solid, large colony into the 96-well plate prepared in advance, and put it into the 37℃ 5% CO2 incubator. After 3 days, the cell amount is about 2 / 3 of the bottom area, and 100 μL of supernatant is used for ELISA screening with immunogen and synthetic polypeptide respectively. Positive clones are completely replaced with liquid, and 200 μL of complete medium containing feeder cells and 1% HT (Sigma) is added. After two days, the second ELISA screening is performed, and the positive clones are transferred into the 24-well plate prepared in advance with medium containing feeder cells and HT. After five days, 100 μL of supernatant is taken for the third ELISA screening, and the positive clones are gradually transferred into 6-well plates and cell culture bottles for expansion and cryopreservation.

[0036] Example 3 Preparation of monoclonal antibody by ascites induction method

[0037] I. Preparation of ascites

[0038] Logarithmic growth phase cells were washed with serum-free medium and suspended, counted about 5 x 10 5 , 1 mL. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites was collected 7 days later. The removed ascites was centrifuged at 4000 rpm for 10 min at 4℃. The middle ascites was carefully sucked out and collected in a centrifuge tube, which was stored at 4℃ or -20℃.

[0039] II. Purification of monoclonal antibody

[0040] The antibody was purified from ascites by HiTrap rProtein A FF (GE) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. The purified antibody was stored at -20℃.

[0041] Example 4 Identification of monoclonal antibody characteristics

[0042] I. Subclass identification

[0043] Coat goat anti-mouse IgG (Beijing Zhongshanjinqiao Biotechnology Co., Ltd.) to 0.5 μg / mL with 100 mM PBS (pH 7.4), 100 μL per well, 4°C, overnight. Pour out the liquid, wash 3 times with PBS containing 0.05% Tween (PBS-T), add 200 μL blocking solution (PBS containing 2% BSA and 3% sucrose) per well, incubate at 37°C for 1 h. Pour out the liquid, wash 3 times with PBS-T. Add 0.1 mL of hybridoma supernatant per well, incubate at 37°C for 1 h. Pour out the liquid, wash 3 times with PBS-T. Add 0.1 mL of HRP-labeled goat anti-mouse (κ, λ) antibody or 1:2000 diluted HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech Company) per well, 37°C for 1 h. Pour out the liquid, wash 3 times with PBS-T. Add 50 μL of 0.15% ABTS (Southern Biotech Company) and 0.03% H2O2 in citric acid buffer (pH 4.0) per well for color development, and measure the OD value at 405 nm within 10-20 min.

[0044] The results show that the monoclonal antibody of the application is an IgG2b type mouse monoclonal antibody.

[0045] II. Affinity constant determination

[0046] Coat the Hepatocyte recombinant protein prepared in Example 3 at a concentration of 2 μg / mL, 100 μL per well, 4°C overnight, and wash 3 times with PBS-T. Add 200 μL of blocking solution per well, 37°C for 2 h, and wash 3 times with PBS-T. Dilute the monoclonal antibody purified in Example 3 by 2-fold gradient starting from 1:200, with the last well left blank as a control, incubate at 37°C for 1 h, and wash 3 times with PBS-T. Dilute the HRP-labeled goat anti-mouse secondary antibody to 1:20,000, 100 μL per well, 37°C for 1 h, and wash 3 times with PBS-T. Add 100 μL of 0.1% TMB (Sigma Company) and 0.03% H2O2 in citric acid-phosphoric acid buffer per well for color development for 10 min, and add 50 μL of 0.5 M sulfuric acid solution to stop the reaction. Measure the absorbance value at a wavelength of 450 nm using an enzyme marker. Plot the curve of OD value versus antibody dilution factor, find the dilution factor A corresponding to half of the maximum binding OD value, and calculate the affinity constant of the antibody to be 1.92 × 10 9 .

[0047] Affinity constant

[0048] III. Specificity of monoclonal antibody reaction and application effect

[0049] To detect the recognition specificity of the monoclonal antibody of the application, the Hepatocyte recombinant protein prepared in Example 1 was used to detect the recognition specificity of the monoclonal antibody of the application by immunoblotting method, and 12% polyacrylamide gel electrophoresis was performed. The gel protein band was transferred to a PVDF membrane (Millipore Company) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in a TBS-T blocking solution containing 5% skim milk at 4°C overnight. The purified Hepatocyte protein monoclonal antibody of Example 3 (1:1000 dilution) was added and incubated at 4°C overnight. After washing the membrane with TBS-T, goat anti-mouse secondary antibody (Beijing Zhongshanjinqi Biotechnology Co., Ltd.) was added at a dilution of 1:5000, and incubated at room temperature for 1 hour. The membrane was washed again with TBST, and ECL hypersensitive developing solution (Beijing Puli Lei Gene Technology Co., Ltd.) was added. ChemiDocMP multicolor fluorescence imaging system (Bio-Rad) was used to collect the chemiluminescence image data.

[0050] Example 5 Sequence determination of the antibody

[0051] Fresh hybridoma cells were taken and the supernatant was taken for antigen binding property verification to confirm that the cell strain used for cloning can indeed secrete the required antibody. After the result was confirmed, 10 6 The above hybridoma cells. Total RNA of hybridoma cells was extracted by Trizol method, 9 µL of total RNA was taken, 2.5 µL of oligo(dT)12-18 primer (10 mM) and 5 µL of dNTPs were added, and mixed uniformly, and after 70°C incubation for 5 minutes, it was placed on ice for 5 minutes, or the denaturation operation was performed according to the reverse transcriptase used. Then 5 µL of RT buffer (5X), 2.5 µL of DTT (0.1 M) and 1 µL of reverse transcriptase were added, and 42°C reaction was performed for 1 hour. 70°C incubation for 15 minutes to terminate the reaction, the obtained cDNA was stored at -20°C. The obtained first strand cDNA was subjected to PCR amplification, 25 pmol of primer was added in a 50 µL reaction system, and the sequences of the primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the mouse monoclonal antibody primer sequence in the book “Recombinant Antibody” (Science Press, published in 2005) edited by Shen Beifen.

[0052] The rest of the dNTPs and buffers were added routinely, and finally the cDNA template 1 μL and 1 U hot start Taq DNA polymerase were added. The PCR amplification program was set as 94°C for 40 seconds, 52°C for 40 seconds, 72°C for 40 seconds, for 20 to 25 cycles, and finally 72°C for 3 minutes, and the product can be placed at 4°C for standby or directly electrophoresis. 20 μL of PCR product was taken for electrophoresis analysis, separated on a 1.5% agarose gel, the length of light chain (kappa light chain) was between 320-340 bp, and the length of heavy chain was between 340-370 bp. When there was specific product in this region, the gel was cut and recovered for cloning into T vector or expression vector for sequencing.

[0053] Example 6. Immunohistochemistry tissue chip staining and identification

[0054] I. Chip preparation process

[0055] Each sample was first stained with HE section to determine the tumor site. The full-automatic tissue chip instrument of 3DHISTECH company was used to make the tissue chip. The prepared tissue chip wax block was placed into a wax block making mold, and placed into a 68°C oven for 10 minutes, so that the wax of the tissue chip and the wax of the receptor wax block were integrated, then the mold was gently taken out of the oven, and the semi-melted paraffin was cooled at room temperature for about 30 min, then placed into a -20°C refrigerator for 6 min, and then the tissue chip wax block was taken out of the mold, sectioned or placed into a 4°C refrigerator for storage. After trimming, continuous sectioning was performed with a thickness of 3 μm, and the continuous sections were floated in 40% alcohol to naturally expand, then the separated sections were transferred to warm water at 50°C for 30 seconds, and the sections were mounted on glass slides treated with polylysine, and the prepared tissue chip was placed into a 68°C oven for 2 hours, then taken out, cooled at room temperature, and placed into a -4°C refrigerator for storage.

[0056] II. IHC staining and analysis

[0057] Routine xylene dewaxing for 3 times, each time for 6 minutes, hydration in 100%, 100%, 95%, 85% gradient ethanol for 3 minutes each time, and finally tap water washing. Antigen repair was performed, then the sections were placed into a wet box, and PBS was washed for 3 times for 3 minutes each time. 3% H2O2 was added and incubated for 10 minutes, and PBS was washed for 3 times for 3 minutes each time. The sections were spun dry, an appropriate proportion of diluted primary antibody (the first dilution was designed according to the concentration of the antibody) was added, incubated at room temperature (25°C) for 1 hour, PBS was washed for 3 times for 3 minutes each time, the secondary antibody was added and incubated at room temperature for 15-30 minutes, PBS was washed for 3 times for 3 minutes each time, the PBS was spun off, fresh DAB developing solution was used for color development for 3-10 minutes. Hematoxylin was used for re-staining for 25 seconds, and PBS was used for blue returning for 30 seconds. According to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), dehydration was performed in turn, and finally xylene was used for transparency for 3 minutes, and neutral resin was used for mounting.

[0058] The immunohistochemical staining results are divided into positive and negative. The positive expression must be at the site of cell and tissue specific antigen to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0059] 1. The sample is weakly positive, marked as "+".

[0060] 2. The sample is moderately positive, marked as "++".

[0061] 3. The sample is highly positive, marked as "+++".

[0062] 4. The sample is negative, marked as "-".

[0063] III. Data statistics

[0064] 1. Tumor tissue chip detection results:

[0065] The antibody Hepatocyte (MX119) of the application and the commercially available antibody Hepatocyte (OCH1E5) are used to synchronously detect 89 cases of hepatocellular carcinoma and compare the detection results.

[0066] The immunohistochemical results of Hepatocyte are statistically analyzed. The whole test process adopts a double-blind design, and the statistical results are as follows:

[0067]

[0068] The results show that the anti-Hepatocyte protein monoclonal antibody provided by the application has accurate staining localization, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive rate is comparable to that of the commercially available antibody, but there are 3 cases with higher positive intensity than the commercially available antibody. It shows that the antibody has higher sensitivity and effectively avoids false negative results.

[0069] Figure 1 The comparison chart of immunohistochemical staining results of one case of hepatocellular carcinoma (left: antibody of the application, right: commercially available antibody).

[0070] 2. Normal tissue chip detection results:

[0071] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0072] The present antibody (MX119) and the commercially available antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, which indicates that the specificity of the present antibody on the normal tissue is equivalent to that of the commercially available antibody. Figure 2 The following is a comparison chart of the immunohistochemical staining results of the surface epithelium of normal liver tissue (left: the present antibody, right: the commercially available antibody).

[0073] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-Hepatocyte protein monoclonal antibody, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 1; the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a murine monoclonal antibody, and the clone number is MX119.

3. A method for preparing an anti-Hepatocyte protein monoclonal antibody, characterized by, The antigen used for immunizing the mouse is a recombinant protein, which is expressed by E. coli.

4. The production method according to claim 3, characterized by, The recombinant protein comprises a Hepatocyte protein fragment and a HIS protein tag.

5. The preparation method according to claim 3, characterized in that, The Hepatocyte protein fragment is shown in SEQ ID NO. 3, which is the concatenation of the 628-731 site, the 813-882 site and the 628-731 site.

6. The preparation method according to claim 3, characterized in that, The plasmid vector used in the recombinant process is pET30a.

7. A Hepatocyte protein immunoassay reagent, characterized by, The immunoassay reagent contains the anti-Hepatocyte protein monoclonal antibody of claim 1 as an effective component.

8. The immunoassay test reagent of claim 7, wherein, The immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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