CN113087794B

CN113087794B - Monoclonal antibody for resisting HNF1 beta protein, cell strain, preparation method and application thereof

Info

Publication number: CN113087794B
Application number: CN202110518492.3A
Authority: CN
Inventors: 杨清海; 陈惠玲; 赵普; 林洋; 李萍
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-04-12
Anticipated expiration: 2041-05-12
Also published as: CN113087794A

Abstract

The invention relates to a monoclonal antibody capable of identifying a human HNF1 beta antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. According to the technical scheme, 23 rd to 132 th amino acids at the C-terminal of the HNF1 beta protein are selected as antigen peptides, codon optimization is carried out, the antigen peptides become gene fragments suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein comprises an HNF1 beta protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 11A1 capable of efficiently secreting the anti-HNF 1 beta protein monoclonal antibody and the anti-HNF 1 beta protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing HNF1 beta protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

Monoclonal antibody for resisting HNF1 beta protein, cell strain, preparation method and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-HNF 1 beta protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

HNF1 β is a transcription factor involved in the development of multiple organs, particularly liver, kidney, pancreas and muller tube embryos, and is also a regulator of glucose homeostasis. Tsuchyia is equal to the difference gene expression of clear cell carcinoma and non-clear cell carcinoma of ovary researched by using oligonucleotide array technology in 2003, and the result shows that the expression of HNF1 beta in clear cell carcinoma is obviously up-regulated, and the HNF1 beta is proved to belong to a very specific marker of clear cell carcinoma of ovary by respectively applying RT-PCR, immunoblotting and immunohistochemical methods. Kato et al analyzed the expression of HNF1 β in 30 ovarian clear cell tumors (clear cell carcinoma 26, borderline tumor 3, and benign 1) and showed that HNF1 β was expressed in all cases and HNF1 β was also expressed in the endometrium epithelium of 9 of 12 cases with endometriotic epithelium, but HNF1 β was rarely expressed in other types of ovarian epithelial tumors (endometrioid, serous, mucinous, and Brenner), which further confirmed that HNF1 β was a good marker for ovarian clear cell tumors, including benign, borderline, and malignant clear cell tumors. Since clear cell alteration can be seen in other types of ovarian cancer, Kao et al applied immunohistochemistry to analyze 178 ovarian cancers with clear cell alteration (80 clear cell cancers, 60 high-grade serous carcinomas, 25 endometrioid adenocarcinomas and 13 mixed endometrioid + clear cell carcinomas) and showed that most clear cell carcinomas diffusely strongly positively expressed HNF1 β, that HNF1 β positive intensity was not correlated with tumor histological configuration, that high-grade serous and endometrioid adenocarcinomas normally did not or weakly expressed HNF1 β, that 13 mixed carcinomas strongly expressed HNF1 β only in clear cell carcinomas, that 9 of endometrioid carcinomas did not or weakly expressed HNF1 β, and that 4 of carcinomas moderately expressed HNF1 β, which again confirmed the specificity of HNF1 β in the diagnosis of clear cell ovarian carcinomas. Higashiguchi et al also found that HNF1 beta specifically expressed in clear cell carcinoma could be used as differential diagnosis marker of clear cell carcinoma and mesothelial cell in cytological specimens.

Disclosure of Invention

The inventor provides an anti-HNF 1 beta monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

Further, the monoclonal antibody specifically recognizes HNF1 β protein.

Further, the monoclonal antibody is mouse IgG₁Subtype monoclonal antibodies.

Further, the hybridoma cell line is produced by a hybridoma cell line with the preservation number of CGMCC NO 20765.

The inventor also provides a preparation method of the anti-HNF 1 beta protein monoclonal antibody, the antigen used for immunizing mice is recombinant protein, the recombinant protein is expressed by escherichia coli recombination, and comprises an HNF1 beta protein fragment and a histidine protein tag. The HNF1 beta protein fragment is the amino acid fragment from the 23 rd position to the 132 th position of HNF1 beta protein, and the amino acid sequence of the HNF1 beta protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting HNF1 beta-resistant protein molecules, wherein the cell strain is a mouse hybridoma cell strain 11A1 which is preserved in China general microbiological culture Collection center (CGMCC NO 20765) in 9 and 17 months of 2020, and the accession number is CGMCC NO 20765, and the cell strain is the institute of microbiology of China academy of sciences No. 3, North Jing China, Naja province, North Cheng West Lu No.1 institute of south China, North Jing City.

The inventor also provides the application of the monoclonal antibody in the HNF1 beta protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor finally provides an HNF1 beta protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID 4; the amino acid sequence of the variable region of the monoclonal antibody light chain is the amino acid sequence shown in SEQ ID 5.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, 23 rd to 132 th amino acids at the C-terminal of the HNF1 beta protein are selected as antigen peptides, codon optimization is carried out, the antigen peptides become gene fragments suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein comprises an HNF1 beta protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 11A1 capable of efficiently secreting the anti-HNF 1 beta protein monoclonal antibody and the anti-HNF 1 beta protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing HNF1 beta protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of purified pectin from the recombinant HNF 1. beta. protein fused with a histidine tag of example 1, wherein M represents Marker; 1 is a whole fungus sample after ultrasonic treatment; 2, a supernatant sample after ultrasonic treatment; 3, precipitating a sample after ultrasonic treatment; 4 is an uninduced control sample.

FIG. 2 is a graph comparing immunohistochemical staining results for renal clear cell carcinoma; the left is HNF1 beta secreted by 11A1, and the right is commercial HNF1 beta.

FIG. 3 is a graph comparing the results of immunohistochemical staining of the appendix; the left is HNF1 beta secreted by 11A1, and the right is commercial HNF1 beta.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1 preparation of recombinant HNF1 beta protein fragment

First, gene optimization and synthesis

Selecting a protein fragment at 23-132 th position of HNF1 beta according to a protein sequence with the accession number of P35680 in a Uniprot database, wherein the protein fragment is an amino acid sequence shown as SEQ ID NO. 1; directly optimizing into a gene fragment suitable for being expressed in Escherichia coli BL 21. EcoRI and XhoI cleavage sites were added to the 5 'and 3' ends of the gene during PCR, respectively.

And separating and recovering the PCR product through agarose gel electrophoresis, carrying out EcoR I and XhoI enzyme digestion on the recovered fusion protein gene and a plasmid vector Pet30a for expression respectively, carrying out electrophoresis recovery again, and connecting with T4 DNA ligase. And transforming the connecting product into escherichia coli competent cells BL21, selecting clones on a plate for inoculation, and performing PCR identification on bacterial liquid. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. HNF1 beta molecules were analyzed according to published sequences, based on structure on cell membrane, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure, selecting regions suitable for soluble expression and good immunogenicity for recombinant expression, and selecting amino acid residues 23-132 of HNF1 beta for codon optimization, with a molecular weight of about 19 kDa. The HNF1 beta protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of an HNF1 beta protein fragment with antigenicity and a protein tag for purifying recombinant protein, wherein the protein tag is HIS.

II, protein expression and purification

The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, shaking culture is carried out at 37 ℃ until OD600 is 0.6-0.8, 1mmol/L IPTG is added, shaking culture is carried out at 16 ℃ for overnight, and the strain is collected and then is crushed by ultrasound. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 500mmol/L imidazole and SDS PAGE separation was carried out.

FIG. 1 is a diagram of purified pectin of recombinant HNF1 beta protein. The protein concentration is 0.5mg/mL, and the protein can be used for requirements of animal immunization and antibody screening and identification.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptides of example 1 were emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) were immunized and injected ventrally at 6 o' clock per mouse at a dose of 20. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, the round, solid and large clone mass is absorbed under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and 5 percent CO at 37 ℃ is put in₂Culturing in an incubator. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening is carried out, and the positive clone is transferred intoA24-well plate of culture medium (containing feeder cells and HT) was prepared beforehand. After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is IgG₁Murine monoclonal antibodiesAnd (3) a body.

Second, determination of affinity constant

The HNF1 beta recombinant protein prepared in example 1 was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 3.84 × 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The HNF1 β recombinant protein prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 11A1 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequences of antibodies

Collecting and culturing fresh hybridomaThe cells were collected, the supernatant was subjected to antigen binding property verification, and it was confirmed that the cell line used for cloning did secrete the desired antibody, and after confirmation, the cell line was collected by centrifugation 10⁶The hybridoma cells described above. Total RNA from hybridoma cells was extracted by Trizol method, and 9. mu.L of total RNA was added with 2.5. mu.L of oligo (dT) 12-18 primer (10mM) and 5. mu.L of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of LRT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added, and the reaction was carried out at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA thus obtained was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized based on the sequence of the murine monoclonal antibody primer as described in "recombinant antibody" by Hippo Seiyaku (science publishers, 2005 publications).

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ L of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, the length of light chain (. kappa.light chain) was between 320-340bp and the length of heavy chain was between 340-370bp, and the region-specific product was recovered by gel cutting and cloned into T-vector or expression vector for sequencing.

Example 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody HNF1 beta (11A1) and the commercial antibody HNF1 beta (rabbit polyclonal antibody) are synchronously detected on different human tumor tissue chips (including 18 cases of renal clear cell carcinoma and 27 cases of ovarian clear cell carcinoma) and the detection results are compared.

Immunohistochemical results for HNF1 β were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-HNF 1 beta protein secreted by the 11A1 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In immunohistochemical detection, the positive rate is equivalent to that of a commercially available antibody, and the positive intensity of most samples is higher than that of the commercially available antibody, so that the sensitivity is higher, and false negative results are effectively avoided.

FIG. 2 is a graph comparing the immunohistochemical staining results of renal clear cell carcinoma (left is HNF 1. beta. secreted by 11A1 cell line, right is commercially available HNF 1. beta.).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (11A1) and the commercial antibody (TA347) were detected simultaneously on the normal tissue chip, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

FIG. 3 is a comparison of the results of immunohistochemical staining of fallopian tubes (left is HNF 1. beta. secreted by 11A1 cell line, right is commercially available HNF 1. beta.).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims

1. An anti-HNF 1 beta monoclonal antibody, characterized in that, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

2. The monoclonal antibody of claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

3. The monoclonal antibody of claim 1, which specifically recognizes HNF1 β protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG₁Subtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20765.

6. A hybridoma cell strain capable of secreting anti-HNF 1 beta protein molecules is a mouse hybridoma cell strain 11A1, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20765.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of an HNF1 β protein immunoassay reagent.

8. The use according to claim 7, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

9. An HNF1 beta protein immunohistochemical detection reagent, characterized in that, the immunohistochemical detection reagent contains the anti-HNF 1 beta protein monoclonal antibody of claim 1 as an effective component.