Monoclonal antibody against human PHH3 as well as preparation method and application of monoclonal antibody

Abstract

The invention discloses an anti-human PHH3 monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of monoclonal antibodies. The anti-human PHH3 monoclonal antibody provided by the invention comprises a VHCDR1, a VHCDR2 and a VHCDR3 of which the amino acid sequences are shown as SEQ ID NO.1-3, and a VLCDR1, a VLCDR2 and a VLCDR3 of which the amino acid sequences are shown as SEQ ID NO.4-6. The anti-human PHH3 monoclonal antibody provided by the invention has the advantages that the anti-human PHH3 monoclonal antibody can be used for detecting the human PHH3; meanwhile, the invention also provides nucleotide sequences of a heavy chain variable region and a light chain variable region of the anti-human PHH3 monoclonal antibody, so that the monoclonal antibody can be prepared by utilizing a genetic engineering technology, and risk factors occurring in the production and storage process of the traditional monoclonal antibody are avoided. The monoclonal antibody prepared by the invention is stable in property, high in experimental repeatability and high in titer, and has good specificity and affinity to PHH3 protein.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody against human PHH3, a preparation method and applications thereof, and belongs to the technical field of monoclonal antibodies. Background Art

[0002] Phosphorylated histone H3 (phosph-Histone H3, PHH3) is a core histone, and it and other histones constitute the main protein components of eukaryotic cell chromatin. The expression level of PHH3 during the interphase of cell division can be negligible or non-expressed, but reaches the maximum value during the G2 and M phases of mitosis. Therefore, PHH3 can help identify cell mitosis and contribute to the identification of apoptotic bodies and nuclear fragments. In 1997, Hendzel et al. first proposed using PHH3 as a marker for nuclear division, and subsequent studies on PHH3 in different tumors have been continuously carried out. Currently, PHH3 has been regarded as a specific nuclear division marker, which can be used to determine the mitotic figures of tumor cells, especially in tissues with severely squeezed and deformed nuclei, and is very useful for distinguishing nuclear fragments and apoptotic cells in tissues. Therefore, it improves the recognition of mitotic cells and increases the specificity of nuclear division markers.

[0003] Currently, the pathological histological diagnosis and grading of various tumors are closely related to the mitotic figures of tumor tissues. Therefore, accurate mitotic counting plays a crucial role in the diagnosis and grading of tumors. Since PHH3 specifically stains cells in the G2 and M phases, it can reduce the ambiguous mitotic figures in HE staining, clearly distinguish apoptotic bodies and nuclear fragments, etc., thereby improving the accuracy of mitotic counting. In addition, due to the clear staining of mitotic figures, the hot spots of nuclear division can be quickly found, the differences between different observers can be reduced, the time for counting nuclear division can be shortened, the repeatability can be improved, and it helps to improve the accuracy of tumor diagnosis, pathological grading, and prognosis judgment.

[0004] However, at present, there are only two PHH3 antibodies available for in vitro pathological diagnosis on the domestic market: one is a rabbit anti-human polyclonal antibody reagent, and the other is a rabbit monoclonal antibody reagent (Clone: BP6092) developed by Hangzhou Bailin. The types of antibodies are few, the space for customers to choose is small, and it is easy to cause market monopoly. Summary of the Invention

[0005] The first object of the present invention is to provide a monoclonal antibody against human PHH3, and to provide a monoclonal antibody against human PHH3 with good specificity and antibody affinity.

[0006] The second object of the present invention is to provide a nucleic acid molecule, and to provide a nucleic acid molecule capable of stably expressing a monoclonal antibody against human PHH3.

[0007] The third object of the present invention is to provide an expression cassette, an expression vector, a recombinant cell or a recombinant bacterium comprising the above nucleic acid molecule, and to provide a molecular biology tool capable of stably expressing a monoclonal antibody against human PHH3.

[0008] The fourth object of the present invention is to provide a method for preparing a monoclonal antibody against human PHH3, and to provide a simple and stable method for producing a monoclonal antibody against human PHH3.

[0009] The fifth object of the present invention is to provide an application of a monoclonal antibody against human PHH3, and to provide the application of the monoclonal antibody against human PHH3 in immunohistochemistry and the preparation of immune reagents.

[0010] In order to achieve the above objects, the technical solution adopted by a monoclonal antibody against human PHH3 in the present invention is as follows:

[0011] A monoclonal antibody against human PHH3, wherein the monoclonal antibody against human PHH3 comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO.1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown in SEQ ID NO.4-6.

[0012] The beneficial effects of the above technical solution are as follows: The monoclonal antibody against human PHH3 of the present invention is a pioneering invention. Immunohistochemical studies have shown that the PHH3 antibody can specifically detect core histones when phosphorylated at serine 10 and 28. The present invention designs a polypeptide fragment, phosphorylates serine 10, conjugates HLH as an immunogen, immunizes animals to obtain hybridoma cells, and screens out a hybridoma cell line capable of secreting a specific antibody against human PHH3. The monoclonal antibody against human PHH3 secreted by it comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO.1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown in SEQ ID NO.4-6. Verified by experiments, the monoclonal antibody against human PHH3 provided by the present invention has the advantages of stable properties, good specificity and affinity, and enriches the types of domestic PHH3 antibodies.

[0013] As a further improvement, the heavy chain variable region of the monoclonal antibody against human PHH3 has an amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has an amino acid sequence shown in SEQ ID NO.8.

[0014] In order to achieve the above objects, the technical solution adopted by the nucleic acid molecule in the present invention is as follows:

[0015] A nucleic acid molecule that encodes the monoclonal antibody against human PHH3.

[0016] The beneficial effects of the above technical solution are as follows: The present invention provides a nucleic acid molecule containing the coding sequence of the monoclonal antibody against human PHH3. By using this nucleic acid molecule and through genetic engineering means, the monoclonal antibody against human PHH3 can be produced rapidly, stably, and on a large scale.

[0017] Specifically, the nucleic acid molecule can be obtained by genetic engineering recombination technology or chemical synthesis method. It is obvious to those skilled in the art that the variant sequences of the heavy chain variable region nucleotide sequence and / or the light chain variable region nucleotide sequence obtained after one or more nucleotide addition, deletion, substitution, modification and other mutations of the above nucleic acid molecule provided by the present invention, and the single-chain antibody, chimeric monoclonal antibody, modified monoclonal antibody or other forms of monoclonal antibody or antibody fragment composed of the encoded amino acid sequence still retain the ability to specifically bind to the PHH3 protein.

[0018] As a further improvement, the nucleotide sequence of the heavy chain variable region gene of the monoclonal antibody against human PHH3 is as shown in SEQ ID NO.9; the nucleotide sequence of the light chain variable region gene of the monoclonal antibody against human PHH3 is as shown in SEQ ID NO.10.

[0019] In order to achieve the above object, the technical solutions adopted for the expression cassette, expression vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule in the present invention are:

[0020] An expression cassette, expression vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule.

[0021] The beneficial effects of the above technical solution are as follows: The present invention provides a nucleic acid molecule containing the coding sequence of the monoclonal antibody against human PHH3, an expression cassette, an expression vector, a recombinant cell and a recombinant bacterium containing the above nucleic acid molecule, and preserves the monoclonal antibody against human PHH3 in the form of DNA, which is convenient for the exogenous expression and large-scale industrial production of the monoclonal antibody against human PHH3 in the later stage.

[0022] In order to achieve the above object, the technical solution adopted for the preparation method of the monoclonal antibody against human PHH3 in the present invention is:

[0023] A preparation method of a monoclonal antibody against human PHH3, which is obtained by introducing the nucleic acid molecule into a host cell, collecting the cell supernatant, and purifying and ultrafiltering.

[0024] The beneficial effects of the above technical solution are as follows: The method for preparing the anti-human PHH3 monoclonal antibody provided by the present invention has the advantages that compared with traditional monoclonal antibodies, the recombinant antibody has the advantages of known sequence, long-term preservation of the antibody gene, stable antibody properties, good experimental repeatability, etc. It is a standardized antibody production process that avoids the risk factors that occur in the production and preservation of traditional monoclonal antibodies: by using recombinant technology to prepare the anti-human PHH3 monoclonal antibody, the controllability and traceability of the antibody sequence are ensured, the batch-to-batch differences and preservation problems that may occur in traditional antibody production are solved, and a more stable and reliable antibody tool is provided, which is suitable for long-term and standardized research and applications.

[0025] As a further improvement, the host cell includes HEK293 cells and CHO cells.

[0026] In order to achieve the above object, the technical solution adopted in the application of the anti-human PHH3 monoclonal antibody in the present invention is:

[0027] An application of an anti-human PHH3 monoclonal antibody, and the application is:

[0028] (1) Prepare a PHH3 detection reagent or kit;

[0029] (2) Immunohistochemical detection, and the immunohistochemical detection is for non-diagnostic and therapeutic purposes.

[0030] The beneficial effects of the above technical solution are as follows: It has been experimentally verified in the present invention that the anti-human PHH3 monoclonal antibody has good specificity and affinity and can be used to prepare a PHH3 detection reagent or kit to detect the expression of PHH3 in tissues and cells. Specifically, the anti-human PHH3 monoclonal antibody can be used to prepare detection reagents or kits such as immunohistochemistry, Western blot, or ELISA that require the anti-human PHH3 monoclonal antibody.

[0031] Furthermore, the anti-human PHH3 monoclonal antibody developed in the present invention can achieve the same staining effect as the rabbit monoclonal antibody reagent (Clone: BP6092) in immunohistochemical detection, and has a clean background. It has higher antibody affinity and stronger antibody specificity than polyclonal antibodies, increasing the types of antibodies and providing more choices for customers. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the immunohistochemical staining effect diagram (100×) of antibody 3B10 in cervical squamous cell carcinoma tissue in Example 5 of the present invention;

[0033] Figure 2 It is the immunohistochemical staining effect diagram (100×) of antibody BP6092 in cervical squamous cell carcinoma tissue in Example 5 of the present invention;

[0034] Figure 3 This is the immunohistochemical staining effect diagram (100×) of the rabbit anti-human polyclonal antibody in cervical squamous cell carcinoma tissue in Example 5 of the present invention;

[0035] Figure 4 This is the immunohistochemical staining effect diagram (100×) of antibody 3B10 in ovarian tissue in Example 6 of the present invention;

[0036] Figure 5 This is the immunohistochemical staining effect diagram (100×) of antibody BP6092 in ovarian tissue in Example 6 of the present invention;

[0037] Figure 6 This is the immunohistochemical staining effect diagram (100×) of the rabbit anti-human polyclonal antibody in ovarian tissue in Example 6 of the present invention. Detailed implementation manners

[0038] It is reported in the literature that the phosphorylation of serine at position 10 of core histone H3 exists in most tumors. Therefore, the present invention designs a polypeptide fragment, phosphorylates serine at position 10, conjugates KLH as an immunogen to immunize animals, and obtains a monoclonal antibody against human PHH3 through screening. When this monoclonal antibody is used for immunohistochemistry, the background is clean and the specificity is good.

[0039] The following further details the present invention with specific examples. Unless otherwise specified, the equipment and reagents used in each example, experimental example and comparative example can be obtained commercially.

[0040] I. Specific example of a monoclonal antibody against human PHH3 of the present invention:

[0041] Example 1 A monoclonal antibody against human PHH3

[0042] The monoclonal antibody against human PHH3 in this example comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.7 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.8. The heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 shown in SEQ ID NOs.1-3, and the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 shown in SEQ ID NOs.4-6.

[0043] II. Specific example of a nucleic acid molecule of the present invention:

[0044] Example 2 A nucleic acid molecule

[0045] The nucleic acid molecule of this embodiment encodes the monoclonal antibody against human PHH3 in Embodiment 1. Among them, the nucleotide sequence of the heavy chain variable region gene of the monoclonal antibody against human PHH3 is as shown in SEQ ID NO.9; the nucleotide sequence of the light chain variable region gene of the monoclonal antibody against human PHH3 is as shown in SEQ ID NO.10.

[0046] III. Specific embodiment of a recombinant cell of the present invention containing the nucleic acid molecule:

[0047] Embodiment 3 A recombinant cell containing the nucleic acid molecule

[0048] The recombinant cell of this embodiment uses CHO cells as the starting cells and transfects a recombinant expression vector that externally expresses the nucleic acid molecule in Embodiment 2.

[0049] In other implementation cases, an expression vector, expression cassette or recombinant bacterium containing the expression vector in Embodiment 2 can also be prepared.

[0050] Specifically, the expression vector is selected from prokaryotic or eukaryotic expression vectors; further, the expression vector is selected from bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.

[0051] Specifically, the recombinant bacterium is selected from commonly used vector bacteria in the field of genetic engineering such as bacteria, yeasts, filamentous fungi, etc.

[0052] IV. Specific embodiment of a preparation method of a monoclonal antibody against human PHH3 of the present invention:

[0053] Embodiment 4 A preparation method of a monoclonal antibody against human PHH3

[0054] This embodiment details the acquisition of the monoclonal antibody against human PHH3 in Embodiment 1. The specific implementation operations are as follows:

[0055] 1. Acquisition of PHH3 antigen

[0056] It is reported in the literature that the phosphorylation of serine at position 10 of core histone H3 exists in most tumors. Based on means such as structural biology analysis and antigenic epitope prediction, this embodiment designs a polypeptide fragment. After phosphorylating serine at position 10 (the amino acid sequence is as shown in SEQ ID NO.11, phosphorylating the fourth amino acid), it is conjugated with KLH (Keyhole Limpet Hemocyanin) as an immunogen to immunize animals.

[0057] 2. Animal immunization

[0058] The immunogen (i.e., antigen) obtained in Step 1 is mixed with an adjuvant and then emulsified. Subsequently, New Zealand white rabbits are immunized by multi-point injection on the back, with a total of three immunizations. The second immunization is carried out 21 days after the first immunization, and then the third immunization is carried out 14 days later. On the 7th day after the third immunization, blood is collected from the marginal ear vein to evaluate the immunization effect. After achieving the desired result, the rabbits are immunized one more time. On the 6th - 10th day after the fourth immunization, 10 mL of blood is collected through the marginal ear vein.

[0059] 3. Collection of rabbit peripheral blood and isolation of PBMCs, sorting and culture of B lymphocytes

[0060] PBMCs cells (i.e., peripheral blood mononuclear cells) are isolated from the blood obtained in Step 2 (using a commercial rabbit peripheral blood lymphocyte isolation kit (Cat: P8760)). Subsequently, re-screening is carried out using antibodies against T lymphocyte surface markers CD3, CD4, and CD8 to remove T cells, monocytes, etc.

[0061] Then, activated B lymphocytes are selected using goat anti-rabbit IgG labeled with the fluorescent dye FITC.

[0062] The immunogen PHH3 is conjugated using a commercial biotin labeling kit (Cat: ARL0020S) according to the instructions, and then reacted with an avidin reagent conjugated with fluorescein (PE, APC-Cy7). Incubate at 37 °C for 1 h (shake and stir several times during this period) to obtain an antigen-biotin-avidin screening reagent. When using, add according to the cell amount (1×10 7 cells / 5 μL) to perform positive screening on the obtained B lymphocytes again to obtain antigen-specific B lymphocytes.

[0063] The sorted B lymphocytes are plated and cultured. Seed 1 - 2 cells / well into a 96-well plate pre-added with feeder cells, and at the same time add one or more cytokines such as IL-2, IL-6, IL-10, and TNF-α for co-culture.

[0064] 4. ELISA detection of B lymphocyte culture supernatant

[0065] After culturing for 7 - 12 days, take the cell supernatant for ELISA detection. Coat with the immunogen PHH3, select the serum collected before rabbit immunization as the negative control well, and define an OD450 reading value greater than 2.1 times that of the negative control well as positive, and select the screened positive wells.

[0066] 5. IHC detection of B lymphocyte culture supernatant

[0067] The supernatant of the positive wells screened by ELISA was screened by IHC again. Positive PHH3 tissue sections were selected and operated according to the immunohistochemistry experimental steps. At the same time, a commercial control antibody (Clone: BP6092) was used as a positive control to screen the clone wells with positive staining on the tissue sections.

[0068] 6. cDNA acquisition, amplification and sequencing of heavy and light chain genes of antibodies

[0069] The cells in the positive cell wells obtained by screening were lysed, total RNA was extracted and cDNA was obtained by RT-PCR. A pair of primers for the heavy chain and a pair of primers for the light chain were used to amplify the heavy chain and light chain gene bands of the cells in the cell wells by ordinary PCR. The primer sequences are shown in SEQ ID NO.12 to SEQ ID NO.15 (where SEQ ID NO.12 to SEQ ID NO.13 are heavy chain primers, and SEQ ID NO.14 to SEQ ID NO.15 are light chain primers). The obtained bands were recovered by gel extraction to obtain heavy and light chain gene products of antibodies, and a part of the obtained products was sequenced. The primer sequences used for sequencing are shown in SEQ ID NO.16 to SEQ ID NO.17.

[0070] 7. Construction of heavy chain antibody recombinant plasmid and light chain antibody recombinant plasmid

[0071] After analyzing the correctly sequenced heavy and light chain gene sequences of antibodies, the variable region sequences of the heavy and light chains were extracted. The variable region of the heavy chain and the constant region sequence of rabbit IgG, and the variable region sequence of the light chain and the constant region sequence of rabbit κ chain were inserted into the pcDNA3.4 expression vector by homologous recombination for recombination respectively.

[0072] The DH5α strain containing the pcDNA3.4 plasmid was expanded and cultured, and plasmid extraction and double digestion (EcoRI and HindIII) were carried out. Plasmid extraction was carried out according to the operation requirements of the Axygen plasmid extraction kit instructions. The double digestion reaction system was: 2 μg of pcDNA3.4 plasmid, 2 μL of EcoRI, 2 μL of HindIII, 5 μL of Buffer, 40 μL of ddH2O; the digestion condition was a water bath at 37°C for 2 h. Agarose gel electrophoresis was carried out on the digested system and the digested plasmid fragment was recovered by cutting the gel. This operation was carried out with reference to the operation requirements of the Axygen gel extraction kit instructions.

[0073] The antibody heavy and light chain variable region gene fragments were obtained by PCR. The upstream and downstream primer sequences were designed as shown in SEQ ID NO.18 to SEQ ID NO.21 (where SEQ ID NO.18 to SEQ ID NO.19 are the heavy chain sequencing primers, and SEQ ID NO.20 to SEQ ID NO.21 are the light chain sequencing primers). The PCR amplification cycle was set as follows: I: 98°C for 3 min; II: 98°C for 10 s; III: 56°C for 10 s; IV: 72°C for 10 s; II to IV: 30 cycles; V: 72°C for 5 min. A 1.5% agarose gel electrophoresis was used to obtain the target band and the target fragment was recovered by cutting the gel. This operation was also carried out with reference to the instructions of the Axygen gel recovery kit.

[0074] The target gene fragment and the double-digested plasmid fragment were subjected to homologous recombination using a seamless cloning kit. This step was carried out with reference to the operating requirements of the instructions of the Novoprotein seamless cloning kit (product number: NR005-01A).

[0075] After seamless recombination, the DH5α competent cells were transformed and screened using an ampicillin-resistant LB plate. They were cultured in an incubator at 37°C for 16 - 18 h. Five monoclonal colonies were selected for gene sequencing each. After the colonies with correct sequencing were expanded in culture, the plasmids were extracted.

[0076] 8. Plasmid Transfection

[0077] The high-concentration and high-purity antibody heavy and light chain recombinant plasmids obtained after expanded culture were respectively operated according to the operating instructions of the PEI transfection reagent and transferred into CHO-S cells. The antibody heavy chain and light chain were pre-mixed at a molar mass ratio of 1:2. The plasmid was diluted with the CHO-S cell basal medium, and at the same time, the same volume of this medium was used to dilute the PEI. The plasmid:PEI (W / W) = 1:3. It was transfected into CHO-S cells cultured to the logarithmic growth phase. 48 h after transfection, the antibody titer secreted in the supernatant was detected by the indirect ELISA method to determine the cell line with relatively high expression.

[0078] 9. Antibody Engineering Expression and Purification

[0079] The determined cell line was expanded in culture to a volume of 50 mL, the cell growth state was adjusted to the logarithmic growth phase, the plasmid was transfected, and fed the next day. The cell supernatant was harvested on the 6th day after transfection. After the supernatant was filtered through a 0.22 μm filter, it was purified by affinity chromatography through a Protein G column. The antibody obtained by binding and adsorption was eluted with a citric acid buffer solution at pH 3.0, and the eluate was collected. It was quickly neutralized to between pH 7.2 and 7.4 with a Tris-HCl solution at pH 8.0. The purified antibody was then concentrated by ultrafiltration centrifugation to make its concentration above 1 mg / mL.

[0080] IV. Specific embodiments of the application of a monoclonal antibody against human PHH3 of the present invention:

[0081] Example 5 Application of a monoclonal antibody against human PHH3 in immunohistochemical detection of cervical squamous cell carcinoma tissues

[0082] In this example, the monoclonal antibody against human PHH3 prepared in Example 4 (hereinafter referred to as antibody 3B10) was used. It was diluted into a working solution (the working concentration of the antibody was 4 μg / mL) for immunohistochemical staining of cervical squamous cell carcinoma tissues. At the same time, a comparison was made with commercially available products, rabbit anti-human polyclonal antibody and rabbit monoclonal antibody (Clone: BP6092), commonly used in the market for immunohistochemical staining. The specific implementation process is as follows:

[0083] (1) Paraffin tissue samples of cervical squamous cell carcinoma were sectioned at 3 μm per slice and baked at 65 °C for 2 h.

[0084] (2) Deparaffinization and hydration:

[0085] The paraffin sections were processed as follows: xylene for 15 min - xylene for 15 min - absolute ethanol for 5 min - absolute ethanol for 5 min - 90% ethanol for 5 min - 80% ethanol for 5 min - 70% ethanol for 5 min, and soaked in purified water for 5 min.

[0086] (3) Antigen retrieval:

[0087] The EDTA antigen retrieval solution with pH 9.0 was heated to boiling and then the tissue sections were put in, adjusted to a gentle boiling mode, timed for 20 min, naturally cooled for 5 min, then rinsed with running water to cool down to room temperature, the sections were taken out, soaked in purified water for 5 min, and rinsed and soaked in TBST for 5 min.

[0088] (4) Adding peroxidase blocker:

[0089] 100 μL per slice, incubated at room temperature for 5 min, rinsed and soaked in TBST for 5 min each time, for a total of 2 times.

[0090] (5) Incubation with primary antibody:

[0091] Add the above-mentioned antibody working solution, 100 μL per slice, incubated at 37 °C for 30 min, rinsed and soaked in TBST for 5 min each time, for a total of 2 times.

[0092] (6) Incubation with secondary antibody:

[0093] Dropwise add horseradish peroxidase-labeled goat anti-mouse / rabbit secondary antibody, 100 μL per slice, incubated at room temperature for 30 min, rinsed and soaked in TBST for 5 min each time, for a total of 2 times.

[0094] (7) Color development:

[0095] Add 100 μL of DAB chromogenic solution per slide, incubate at room temperature for 4 min, and soak in purified water twice for 5 min each time.

[0096] (8) Counterstaining:

[0097] Add 100 μL of hematoxylin staining solution per slide, incubate at room temperature for 4 min, and rinse thoroughly with purified water.

[0098] (9) Dehydration and clearing:

[0099] Dehydrate with conventional gradient ethanol and clear with xylene.

[0100] (10) Mount with neutral balsam for observation.

[0101] The staining results of the antibody 3B10 provided by the present invention, the commercial product rabbit anti-human polyclonal antibody, and the rabbit monoclonal antibody (Clone: BP6092) are shown respectively in Figure 1 、 Figure 2 and Figure 3 as shown.

[0102] It can be seen from the figure that the staining of the rabbit anti-human polyclonal antibody is weak, the positive proportion of the cell nucleus is low, and the staining effects of the antibody 3B10 and the rabbit monoclonal antibody (Clone: BP6092) are consistent.

[0103] Example 6 Application of Monoclonal Antibody Against Human PHH3 in Immunohistochemical Detection of Ovarian Tissue

[0104] In this example, the monoclonal antibody against human PHH3 prepared in Example 4 (hereinafter referred to as antibody 3B10) was used. It was diluted into a working solution (the working concentration of the antibody was 4 μg / mL) to perform immunohistochemical staining on ovarian tissue. At the same time, a comparison was made with the commonly used commercial product rabbit anti-human polyclonal antibody and the rabbit monoclonal antibody (Clone: BP6092) in immunohistochemical staining. The specific implementation process was as described in Example 5.

[0105] The staining results of the antibody 3B10 provided by the present invention, the commercial product rabbit anti-human polyclonal antibody, and the rabbit monoclonal antibody (Clone: BP6092) are shown respectively in Figure 4 、 Figure 5 and Figure 6 as shown.

[0106] It can be seen from the figure that the staining of the rabbit anti-human polyclonal antibody is weak, the positive proportion of the cell nucleus is low, and the staining effects of the antibody 3B10 and the rabbit monoclonal antibody (Clone: BP6092) are consistent.

[0107] Example 7 Consistency Evaluation of Monoclonal Antibody Against Human PHH3

[0108] In this example, immunohistochemical detection and evaluation of antibody BP6092 and antibody 3B10 were carried out in multiple tissues, and the implementation process was as described in Example 5.

[0109] Specifically, the tissues for evaluation included tonsil tissue, breast tissue, lymphoma tissue, colorectal cancer tissue, esophageal cancer tissue, gastric cancer tissue, liver tissue, pancreatic tissue, appendix, thyroid, cholecystitis, bladder cancer tissue, urothelial carcinoma tissue, placenta, sarcoma, kidney, spleen, etc. The total number of tissues for evaluation was 214, and the specific results are shown in Table 1.

[0110] Table 1 Statistical results of tissue evaluation of antibody BP6092 and antibody 3B10

[0111]

[0112] As can be seen from Table 1, in terms of the evaluation of immunohistochemical detection results, there was no difference between antibody 3B10 provided by the present invention and the control antibody BP6092.

[0113] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against human PHH3, characterized in that: The monoclonal antibody against human PHH3 comprises VH CDR1, VH CDR2 and VH CDR3 with amino acid sequences shown in SEQ ID NO.1-3, and VL CDR1, VL CDR2 and VL CDR3 with amino acid sequences shown in SEQ ID NO.4-6.

2. The monoclonal antibody against human PHH3 according to claim 1, characterized in that: The heavy chain variable region of the monoclonal antibody against human PHH3 has an amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has an amino acid sequence shown in SEQ ID NO.

8.

3. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the monoclonal antibody against human PHH3 as claimed in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein: The nucleotide sequence of the heavy chain variable region gene of the monoclonal antibody against human PHH3 is shown in SEQ ID NO.9; the nucleotide sequence of the light chain variable region gene of the monoclonal antibody against human PHH3 is shown in SEQ ID NO.

10.

5. An expression cassette, expression vector, recombinant cell or recombinant bacterium comprising the nucleic acid molecule as claimed in claim 3 or 4.

6. A method for preparing a monoclonal antibody against human PHH3 as described in claim 1 or 2, characterized in that: It is obtained by introducing the nucleic acid molecule as claimed in claim 3 or 4 into a host cell, collecting the cell supernatant, and purifying and ultrafiltering.

7. The method for preparing a monoclonal antibody against human PHH3 according to claim 6, characterized in that: The host cell includes HEK293 cells and CHO cells.

8. Use of a monoclonal antibody against human PHH3 as described in claim 1 or 2, characterized in that: The application is as follows: (1) Preparing a PHH3 detection reagent or kit; (2) Immunohistochemical detection, and the immunohistochemical detection is for non-diagnostic and therapeutic purposes.

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