Rabbit monoclonal antibody for resisting human Acp1 protein as well as application and product of rabbit monoclonal antibody

Abstract

The invention provides an anti-human Acp1 protein rabbit monoclonal antibody as well as application and a product thereof, and relates to the technical field of biology. According to the rabbit monoclonal antibody for resisting the human Acp1 protein provided by the invention, the research of the inventor finds that the monoclonal antibody is strong in specificity and high in sensitivity, can specifically recognize the human Acp1 protein, can be used for detecting the human Acp1 protein, and is important for researching the action mechanism of the Acp1 and researching or diagnosing diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a rabbit monoclonal antibody against human Acp1 protein and an application and product thereof. Background Art

[0002] Low Molecular Weight PhosphotyrosineProtein Phosphatase (LMW-PTP), also known as Acp1 (acid phosphatase 1), is an enzyme that plays an important role in cell signaling pathways. It is involved in the dephosphorylation of tyrosine residues on proteins, a process that is essential for regulating signal transduction pathways that control a variety of processes such as cell growth, differentiation, and metabolism. The enzyme is mainly present in the cytoplasm and is sometimes associated with the cell membrane in order to interact with its substrate.

[0003] By dephosphorylating tyrosine residues on signaling proteins, Acp1 participates in regulating signaling pathways mediated by receptor tyrosine kinases (RTKs), such as the insulin receptor, platelet-derived growth factor receptor (PDGFR), and others. Acp1 is thought to play a role in immune cell signaling, particularly in T cells and macrophages, where it regulates signaling pathways that are critical for immune responses. Due to its role in cell signaling regulation, Acp1 has received much attention in cancer research. Dysregulation of phosphatases such as Acp1 may lead to aberrant signaling, thereby promoting tumorigenesis. Alterations in the expression or function of Acp1 have been associated with a variety of diseases, including cancer, diabetes, and autoimmune diseases. It is considered a potential biomarker and therapeutic target for these conditions.

[0004] In view of the diagnostic and therapeutic value of Acp1, the development of antibodies with high sensitivity and specificity against human Acp1 protein is of great significance for the diagnosis and treatment of Acp1-related diseases.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide a rabbit monoclonal antibody against human Acp1 protein to solve the problem of detecting human Acp1 protein.

[0007] A second object of the present invention is to provide a biomaterial.

[0008] The third object of the present invention is to provide a method for preparing the above-mentioned rabbit monoclonal antibody against human Acp1 protein.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned rabbit monoclonal antibody against human Acp1 protein in the preparation of a detection product for human Acp1 protein.

[0010] The fifth object of the present invention is to provide a marker for human Acp1 protein.

[0011] The sixth object of the present invention is to provide a kit for detecting human Acp1 protein.

[0012] In order to achieve the above objectives, the following technical solutions are adopted:

[0013] In the first aspect, the present invention provides a rabbit monoclonal antibody against human Acp1 protein, the variable region of the rabbit monoclonal antibody against human Acp1 protein comprising: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.5, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.6.

[0014] As a further technical solution, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0015] As a further technical solution, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0016] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac:

[0017] a. Nucleotides, comprising a nucleotide sequence encoding a rabbit monoclonal antibody against human Acp1 protein;

[0018] b. a vector carrying the nucleotides in a;

[0019] c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the rabbit monoclonal antibody against human Acp1 protein.

[0020] In a third aspect, the present invention provides a method for preparing the above-mentioned rabbit monoclonal antibody against human Acp1 protein, which is obtained by fermentation of the above-mentioned cells.

[0021] In a fourth aspect, the present invention provides use of the above-mentioned rabbit monoclonal antibody against human Acp1 protein in the preparation of a detection product for human Acp1 protein.

[0022] In a fifth aspect, the present invention provides a marker of human Acp1 protein, comprising the rabbit monoclonal antibody against human Acp1 protein and a marker;

[0023] The rabbit monoclonal antibody against human Acp1 protein is coupled to a label.

[0024] As a further technical solution, the marker includes an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

[0025] In a sixth aspect, the present invention provides a kit for detecting human Acp1 protein, the kit comprising the anti-human Acp1 protein rabbit monoclonal antibody or the human Acp1 protein marker.

[0026] As a further technical solution, the kit includes an immunochromatographic detection kit or an ELISA detection kit.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a rabbit monoclonal antibody against human Acp1 protein. The inventors have found through research that the monoclonal antibody has strong specificity and high sensitivity, can specifically identify human Acp1 protein, can be used for the detection of human Acp1 protein, and is important for studying the mechanism of action of Acp1 and in disease research or diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The results of Western blot detection of rabbit monoclonal antibody 1G9 against human Acp1 protein in different cell samples;

[0031] Figure 2 The results of immunohistochemistry detection of rabbit monoclonal antibody 1G9 against human Acp1 protein in human kidney tissue;

[0032] Figure 3 The results of immunohistochemistry detection of rabbit monoclonal antibody 1G9 against human Acp1 protein in human gastric tissue;

[0033] Figure 4The results of immunohistochemistry detection of rabbit monoclonal antibody 1G9 against human Acp1 protein in human testicular tissue;

[0034] Figure 5 This is a diagram of sorting positive cell populations;

[0035] Figure 6 These are the results of the binding experiment between the rabbit monoclonal antibody 1G9 against human Acp1 protein and Acp1 protein. DETAILED DESCRIPTION

[0036] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0038] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain antigen binding sites. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also called hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held together closely by FRs to form the variable region. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0039] The term "vector" refers to a nucleic acid delivery vehicle into which a nucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0040] The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0041] In the first aspect, the present invention provides a rabbit monoclonal antibody against human Acp1 protein, the variable region of the rabbit monoclonal antibody against human Acp1 protein comprising: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.5, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.6.

[0042] The amino acid sequences of SEQ ID NO.1 to SEQ ID NO.6 are shown in Table 1.

[0043] Table 1

[0044] Highly variable region sequence serial number CDR1-VH SYYMN SEQ ID NO.1 CDR2-VH IMFPNGKIYYATWAKG SEQ ID NO.2 CDR3-VH DDSGDVNI SEQ ID NO.3 CDR1-VL QSSQSVYTNNRLA SEQ ID NO.4 CDR2-VL GASTLPS SEQ ID NO.5 CDR3-VL LGTYDCLSADCLA SEQ ID NO.6

[0045] In some optional embodiments, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0046] QSVKESGGRLVTPGTPLTLTCKVSGFSLSSYYMNWVRQAPGKGLE WIGIMFPNGKIYYATWAKGRFTISKTSTTVDLKIISPTTEDTATYFCTGDDS GDVNIWGPGTLVTVSS (SEQ ID NO. 7).

[0047] In some optional embodiments, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0048] DIVLTQTASPVSAAVGGTVTINCQSSQSVYTNNRLAWYQQKPGQPA KEMIYGASTLPSGVSSRFKGSGSGTQFALTISDVQADDAATYYCLGTYDC LSADCLAFGGGTKLEIK (SEQ ID NO. 8).

[0049] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac:

[0050] a. Nucleotides, comprising a nucleotide sequence encoding a rabbit monoclonal antibody against human Acp1 protein;

[0051] b. a vector carrying the nucleotides in a;

[0052] c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the rabbit monoclonal antibody against human Acp1 protein.

[0053] In some alternative embodiments, the cell comprises a hybridoma cell.

[0054] In a third aspect, the present invention provides a method for preparing the above-mentioned rabbit monoclonal antibody against human Acp1 protein, which is obtained by fermentation of the above-mentioned cells.

[0055] The preparation method is simple and efficient, and a large amount of rabbit monoclonal antibodies against human Acp1 protein can be obtained through fermentation.

[0056] In a fourth aspect, the present invention provides use of the above-mentioned rabbit monoclonal antibody against human Acp1 protein in the preparation of a detection product for human Acp1 protein.

[0057] The rabbit monoclonal antibody against human Acp1 protein provided by the present invention can specifically recognize human Acp1 protein, and thus can be used for the detection of human Acp1 protein.

[0058] In a fifth aspect, the present invention provides a marker of human Acp1 protein, comprising the rabbit monoclonal antibody against human Acp1 protein and a marker;

[0059] The rabbit monoclonal antibody against human Acp1 protein is coupled to a label.

[0060] The marker can be used for specific labeling of human Acp1 protein.

[0061] In some optional embodiments, the marker includes but is not limited to enzymes, fluorescent molecule labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

[0062] In a sixth aspect, the present invention provides a kit for detecting human Acp1 protein, the kit comprising the anti-human Acp1 protein rabbit monoclonal antibody or the human Acp1 protein marker.

[0063] In some optional embodiments, the kit includes but is not limited to an immunochromatographic detection kit or an ELISA detection kit.

[0064] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0065] Example 1 Preparation of rabbit monoclonal antibodies against human Acp1 protein

[0066] 1. Preparation of immunogen

[0067] In this example, human Acp1 protein (Uniprot protein number: P24666) 1-158aa was selected as the immunogen after prokaryotic expression in E. coli. The amino acid sequence of human Acp1 protein 1-158aa is: MAEQATKSVLFVCLGNICRSPIAEAVFRKLVTDQNISENWRVDSAATSGYEIGNPPDYRGQSCMKRHGIPMSHVARQITKEDFATFDYILCMDESNLRDLNRKSNQVKTCKAKIELLGSYDPQKQLIIEDPYYGNDSDFETVYQQCVRCCRAFLEKAH (SEQ ID NO.9).

[0068] 2. Rabbit Immunization

[0069] Reagents: Adjuvant, 75% alcohol.

[0070] Consumables: Syringe.

[0071] Sample: Human Acp1 protein immunogen.

[0072] The steps for rabbit immunization are as follows:

[0073] Step a): Animal selection: New Zealand white rabbits are used, weighing about 2.5 kg, young and strong. The animals are selected to be healthy animals with shiny fur and free movement. The selected animals are raised for about 2 weeks to adapt to the environment.

[0074] Step b): Prepare the rabbits before the experiment and mark them.

[0075] Step c): Take the antigen out of the -20℃ freezer and dissolve it at room temperature to avoid repeated freezing and thawing. Mark the syringe with the project number and animal number.

[0076] Step d): extract antigen (mix antigen completely), the antigen concentration for the first immunization is 1 mg / ml, 0.5 ml / rabbit, and the antigen amount is halved for the second to sixth immunizations.

[0077] Step e): extract the adjuvant, with the adjuvant and antigen being extracted in a volume ratio of 1:1. Complete adjuvant is used for the first immunization, and incomplete adjuvant is used for the second to sixth immunizations. When extracting, the adjuvant should be fully mixed before being drawn into the syringe.

[0078] Step f): two syringes are connected by a syringe connecting tube and then completely emulsified. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37° C. water.

[0079] Step g): The rabbits were injected subcutaneously at multiple points, with 0.2 ml at each point. The second immunization was performed on the 14th day after the first immunization, and the interval between the second and third immunizations was 7 days. After the fourth immunization, a booster immunization was performed according to the titer of the animal serum, generally once a month. After the rabbits were immunized six times, blood was collected for titer testing.

[0080] Step h): Steps for collecting small serum samples: Fix the experimental rabbit in a fixed frame, tap the ear with your hand to dilate the central artery of the ear. Disinfect with 75% alcohol, fix the rabbit's ear with your left hand, hold the syringe with your right hand, insert the needle into the central artery of the ear 1 / 3 of the distance from the end, parallel to the artery, and pierce the artery in the centripetal direction. Take 8 ml of blood at a time, and use cotton balls to press and stop bleeding after blood collection.

[0081] 3. Rabbit serum titer test

[0082] The rabbit serum was tested for the immunogen potency by indirect ELISA method. Rabbits with OD450nm values ​​exceeding 0.6 under the serum dilution condition of 1:64000 were considered to have qualified potency and could proceed to the next step.

[0083] Reagents: goat anti-rabbit-HRP (Hua'an Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Bioengineering: A501492), glycine (Shanghai Bioengineering: GB0235), BSA (Shanghai Bioengineering: A500023-0100), Tween-20 (Shanghai Bioengineering: A600560), NaHCO3 (Shanghai Bioengineering: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Bioengineering and were domestically produced analytical grade.

[0084] Consumables: ELISA plate (Hangzhou Shengyou).

[0085] Equipment: Electric constant temperature incubator (Shanghai Senxin: DRP-9162), ELISA instrument (MD: Cmax plus).

[0086] The indirect ELISA procedure is as follows:

[0087] Step a): The antigen was diluted to 2 μg / mL with coating buffer, and 50 μL / well was added to the ELISA plate. The plate was covered and coated at 4°C overnight.

[0088] Step b): The liquid in the wells was shaken dry, 1% BSA / TBS was added to the ELISA plate at a volume of 150 μL / well, and the plate was placed in a 37° C. electric thermostatic incubator for 1 hour.

[0089] Step c): Shake off the liquid in the wells, add 50 μL / well of serum with different dilution ratios to the ELISA plate, cover the plate, and incubate in a 37°C constant temperature incubator for 30 min.

[0090] Step d): Dry the primary antibody mixture, add washing solution (1×TBST) to the ELISA plate at a volume of 180 μL / well, and wash the ELISA plate twice. Dilute the goat anti-rabbit-HRP to a working concentration (1:30000) with 1% BSA, add 50 μL / well to the ELISA plate, cover the lid, and incubate in a 37°C constant temperature incubator for 45 minutes.

[0091] Step e): color development, termination and reading: discard the liquid in the wells, add washing solution to the ELISA plate at an amount of 180 μL / well, and wash the ELISA plate 3 times; add 100 μL of freshly prepared TMB color development substrate to each reaction well, incubate at 37°C for 10 min; then add 90 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the ELISA reader. The results are shown in Table 2.

[0092] Table 2 Animal titer test data

[0093]

[0094]

[0095] The results showed that the serum titer of A0071 rabbits was higher, so the following experiments were all conducted with A0071 rabbits.

[0096] 4. Isolation of rabbit peripheral blood lymphocytes (PBMC)

[0097] Reagents: rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis solution (Solarbio: R1010), PBS (BasalMedia: B310KJ-500ml).

[0098] Consumables: 50 mL centrifuge tube (BD: 352070), 10 mL pipette (Hangzhou Lanjieke: 352070).

[0099] Equipment: biological safety cabinet (Sujing: BSC-1300IIB2), centrifuge (Thermo: THM#75004530).

[0100] The isolation steps of rabbit peripheral blood lymphocytes (PBMC) are as follows:

[0101] Step a): Take 20 ml of anticoagulated blood from the rabbit with the best titer after the sixth immunization, place it on ice, and take an equal volume of PBS to dilute the whole blood.

[0102] Step b): Add an equal volume of rabbit peripheral lymphocyte separation fluid into a centrifuge tube, and spread the diluted blood evenly above the surface of the separation fluid.

[0103] Step c): Centrifuge at room temperature for 30 min in a horizontal rotor at 1800 rpm.

[0104] Step d): After centrifugation, there will be obvious stratification: the top layer is the diluted plasma layer, the middle layer is the transparent separation liquid layer, the white film layer between the plasma and the separation liquid is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes. Carefully pipette the white film layer cells into a 50mL clean centrifuge tube, wash the white film layer cells with 10mL PBS, and then centrifuge at 1500rpm for 5min.

[0105] Step e): discard the supernatant, resuspend the cells in 20 mL of PBS, centrifuge at 1500 rpm for 5 min. Repeat step e) and discard the supernatant for later use.

[0106] Step f): Add 5 ml of red blood cell lysis solution, vortex or invert gently to mix. Place at 37°C for 5 minutes. After the red blood cells are lysed, the solution is clear and transparent.

[0107] Step g): Centrifuge at 1500 rpm for 5 minutes to precipitate leukocytes, and carefully discard the supernatant.

[0108] Step h): Resuspend the cells in 20 mL of PBS and centrifuge at 1500 rpm for 5 min.

[0109] Step i): after repeating step h), discard the supernatant and separate the peripheral blood lymphocytes for later use.

[0110] 5. Antigen-specific B cell sorting

[0111] Antigen-positive B cells are negatively selected by T cell surface markers, positively selected by B cell surface immunoglobulin, and then specific B cells are sorted from peripheral blood mononuclear cells using fluorescent dye-labeled sorting antigens.

[0112] Reagents: PBS (Basal Media: B310KJ-500ml), fetal bovine serum (Sijiqing: 11011-8611), mouse anti-rabbit CD4 antibody-FITC, mouse anti-rabbit IgG-PE, mouse anti-rabbit IgM-Biotin, streptavidin-PE / Cy7, BrilliantViolet 421 TM -Streptavidin, Alexa 647-streptavidin, biotin-labeled m6A-BSA.

[0113] Consumables: 15mL centrifuge tube (BD: 352097), EP tube (AXYGEN: MCT-150-C).

[0114] Equipment: biological safety cabinet (Sujing: BSC-1300IIB2), flow sorter (Sony: MA900), centrifuge (Thermo: THM#75004530).

[0115] The steps for the specific binding of human Acp1 protein immunogen to B cell sorting are as follows:

[0116] Step a): Set the flow cytometry indexes (IgG, IgM, m6A-BSA) and experimental groups (blank cell control group, compensation single positive tube control group, minus one fluorescence control group, experimental group) for this experiment.

[0117] Step b): IgM and antigen (m6A-BSA) positive indicators need to firstly react IgM-Biotin and antigen (m6A-BSA)-Biotin with streptavidin-PE / Cy7 and Brilliant Violet421 respectively. TM / Alexa After pre-incubation with 647-streptavidin for 20 min, the cells were blocked with excess BSA-Biotin for 20 min before use for sorting and detection.

[0118] Step c): peripheral blood lymphocytes were taken, resuspended with 10 mL PBS, and after cell counting, the cells were divided into 1.5 mL EP tubes and centrifuged at 1500 rpm for 5 min.

[0119] Step d): Carefully discard the supernatant and resuspend with 100uL PBS.

[0120] Step e): Add corresponding antibodies according to the experimental groups set in step a, and vortex gently to mix.

[0121] Step f): After incubation at room temperature for 20 min, add 800 uL PBS for re-suspending and centrifuge at 1500 rpm for 5 min.

[0122] Step g): Carefully discard the supernatant, resuspend the cells in 1 mL of PBS, and centrifuge at 1500 rpm for 5 min.

[0123] Step h): After repeating step g, the supernatant was discarded carefully, and 1 mL of PBS containing 2% FBS was added to the experimental group for re-suspending, and 200 uL of PBS containing 2% FBS was added to the other groups for re-suspending.

[0124] Step i): Place on ice and load onto the machine.

[0125] Step j): Adjust the fluorescence compensation on the flow cytometer through the blank cell control group and the compensation single positive tube control group, and gate through the minus one fluorescence control group. Finally, add the experimental group samples and sort out the antigen double positive cell population, such as Figure 5 As shown. (The distribution diagram of PBMC in SSC-A and FSC-Az, the lymphocyte population is selected according to experience as shown in the figure. Among them, A: the selected rabbit PBMC lymphocyte population is removed by FSC-W and FSC-A to remove the adherent cells; B: FITC and PE-Cy7 double negative cells are obtained; C: PE positive cells are selected; D: antigen positive cells are selected for single cell separation and collection).

[0126] 6. Cloning and production of rabbit monoclonal antibodies

[0127] In order to obtain rabbit monoclonal antibodies that recognize Acp1 protein, the positive B cells obtained by sorting were reverse transcribed to obtain cDNA and then sequenced, and the selected rabbit monoclonal antibody heavy chain variable region (VH) and light chain variable region (VL) were constructed into expression vectors (wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8). Finally, it was expressed in mammalian cells Expi293F for production.

[0128] Reagent: Expi293 TM Expression system kit (Thermo, A14635).

[0129] Consumables: 96-well deep-well plates, pipette tips.

[0130] Equipment: Constant temperature shaker, electronic pipette.

[0131] Antibody plasmid cell transfection:

[0132] Step a): Place the Expi293F culture medium in a 37°C water bath in advance. Observe the cell status and cell growth under a microscope. Determine the cell expansion experiment subculture ratio on the day based on the cell growth on the day and the needs of the subsequent transfection experiment. Generally, the subculture density is controlled at 2x10 6 Take out the cell solution in the CO2 cell shaker, count it, and calculate the amount of cells required for transfer.

[0133] Select the most suitable cell shake flask according to the amount of cells transferred. Generally, the culture volume should not exceed 1 / 3 of the shake flask volume and should not be less than 1 / 5 of the shake flask volume. Add preheated Expi293 culture medium according to the cell density to make the final cell density at 2x10 6 About 100 living cells / mL, used for transfection operation on the next day.

[0134] Step b): On the day of transfection, preheat the Expi293 medium at room temperature and away from light. Determine the number of cells to be transfected and prepare the corresponding number of 96-well deep-well plates. Determine the cell density based on the cell counting operation, add the preheated medium to dilute the cells to a final cell density of 3x10 6 Live cells / mL, use a pipette to dispense into a deep-well plate at 1ml / well, and carefully place it in a cell shaker: 37℃, 8% CO2, 900rpm, 80% humidity for culture. Take the corresponding number of 96-well cell culture plates, and dispense into the culture plates according to the ratio of 1ug DNA + 60ul Opti-MEM (cold reagent) in 1ml cells, and mix them by blowing with a pipette (recommended antibody ratio HC:LC=1:2). Take another centrifuge tube with the most suitable volume, dilute the transfection reagent in the ratio of 3.2ul Expifectamine293 + 60ul Opti-MEM (cold reagent) in 1ml cells, gently invert and mix, and let it stand at room temperature for 5min. After 5min, move the diluted transfection reagent to a disposable reservoir, use a pipette to move it to the corresponding position of the 96-well cell culture plate, and let it stand for 10-20mins. Use a pipette to add the resting ExpiFectamine 293 / DNA complex to the cell suspension and carefully place it in a shaker: 37°C, 8% CO2, 900rpm, 80% humidity. 18-22 hours after transfection, add 6ul Enhancer 1 and 60ul Enhancer 2 to 1ml. After 4-7 days, harvest the supernatant for purification. The antibody in the supernatant is named rabbit monoclonal antibody 1G9.

[0135] Example 2 Application and Identification of Rabbit Monoclonal Antibody 1G9

[0136] 1. Western Blot (WB) analysis

[0137] In order to detect the recognition specificity of the rabbit monoclonal antibody of the present invention to the Acp1 protein, the human liver cancer cell line HepG2, the human cervical cancer cell line HeLa, the human acute T lymphocytic leukemia cell line Jurkat and the African green monkey kidney cell line COS-1 expressing the Acp1 protein were selected as samples for detection. The experimental steps are as follows: the above cells are lysed respectively, the protein is extracted, and 10% polyacrylamide gel electrophoresis is performed. Subsequently, the protein in the gel is transferred to a PVDF membrane according to conventional methods. The PVDF membrane is placed in a TBST blocking solution containing 5% skimmed milk powder and incubated at room temperature for 1 hour. Thereafter, an anti-Acp1 protein rabbit monoclonal antibody with a dilution ratio of 1:60000 is added and incubated at 4°C overnight. Subsequently, the membrane is washed with TBST, and then a goat anti-rabbit secondary antibody is added and incubated at room temperature for 1 hour. The membrane is washed with TBST again, and then an ECL ultrasensitive color developing solution is added for development. The results are as follows. Figure 1shown.

[0138] 2. Immunohistochemistry (IHC)

[0139] Before staining paraffin sections (human kidney tissue samples, human stomach tissue samples, human testicular tissue samples), dewaxing treatment is required to ensure smooth staining of the sections. The process is: xylene I 10 minutes; xylene II 10 minutes; xylene III 10 minutes; anhydrous ethanol I 3 minutes; anhydrous ethanol II 3 minutes; 95% alcohol 3 minutes; running water rinse 3 minutes.

[0140] Antigen repair: Pour about 1L 1×Tris-EDTA (pH 9.0) into a high-temperature container and heat it with an induction cooker to boil for 3-5 minutes to ensure that there are no bubbles at low heat; put the tissue slices into the container, adjust the induction cooker to low power to maintain the temperature at 90-95℃ and cook for 20 minutes, cover the pot. After cooling at room temperature for 10 minutes, rinse with running water and cool to room temperature.

[0141] Immunohistochemical staining:

[0142] 1. Wash the sections three times with pure water, 5 minutes each time.

[0143] 2. Blocking: Place the slices in 3% hydrogen peroxide solution and incubate for 10 minutes.

[0144] 3. Wash the sections twice with pure water, 5 minutes each time.

[0145] 4. After the slices are slightly dried, use filter paper to wipe off the water around the tissue slices, use a histochemical pen to draw a circle around the tissue, add 10% negative goat serum prepared with 1% BSA into the circle, cover the tissue, and block at room temperature for 1 hour.

[0146] 5. Remove the blocking solution, then add the primary antibody diluted with the recommended antibody diluent to each section (usually 100 μl / tissue) and incubate in a humidified chamber at room temperature for 1 hour.

[0147] 6. Remove the antibody solution and wash with 1×TBST three times, 5 min each time.

[0148] 7. After patting the slices dry, add goat anti-rabbit or mouse secondary antibody (Hua'an Biotechnology, Cat. No. HA1119) in the circle to cover the tissue and incubate in a wet box at room temperature for 20 minutes.

[0149] 8. Wash three times with 1×TBST, 5 min each time.

[0150] 9. DAB color development: After patting the slices dry, add freshly prepared DAB color development solution in the circle. Control the color development time under a microscope. The positive color is brown-yellow. Observe until a positive signal appears or the color development takes 5 minutes. Rinse the slides with a washing bottle filled with tap water or a special DAB beaker to stop the color development. Rinse with running tap water for 5-10 minutes.

[0151] Re-staining and dehydration: The process is hematoxylin for 10 minutes; running water washing for 3 minutes; blueing for 1 minute; running water washing for 3 minutes; 95% alcohol I for 1 minute; 95% alcohol II for 1 minute; 95% alcohol III for 1 minute; anhydrous ethanol I for 1 minute; anhydrous ethanol II for 1 minute; xylene I for 1 minute; xylene II for 1 minute; oven at 65℃ for 2 minutes. Then seal the slides with neutral gum. Finally, scan the slides and determine the results.

[0152] The results of immunohistochemical staining showed that the anti-human Acp1 rabbit monoclonal antibody 1G9 was accurately localized in the cytoplasm with a clean background, such as Figure 2-Figure 4 shown.

[0153] 1G9 antibody specificity test:

[0154] The indirect ELISA experiment of antigen-antibody binding confirmed that the binding of 1G9 antibody to Acp1 protein was specific.

[0155] Reagents: goat anti-rabbit-HRP (Hua'an Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Bioengineering: A501492), glycine (Shanghai Bioengineering: GB0235), BSA (Shanghai Bioengineering: A500023-0100), Tween-20 (Shanghai Bioengineering: A600560), NaHCO3 (Shanghai Bioengineering: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Bioengineering and were domestically produced analytical grade.

[0156] Consumables: ELISA plate (Hangzhou Shengyou).

[0157] Equipment: Electric constant temperature incubator (Shanghai Senxin: DRP-9162), ELISA instrument (MD: Cmax plus).

[0158] Indirect ELISA procedure:

[0159] Step a): Coating: Acp1 protein was diluted to 1 μg / mL with coating buffer, and 50 μL / well was added to the ELISA plate, the lid was closed, and the plate was coated at 4°C overnight.

[0160] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the ELISA plate at a volume of 150 μL / well, and place in a 37°C electric thermostatic incubator for constant temperature blocking for 1 hour.

[0161] Step c): Sample addition: Shake off the liquid in the wells, dilute the anti-Acp1 monoclonal antibody 1G9 in a gradient manner with PBS (starting from 8ug / ml and then diluted twice), then add it to the ELISA plate and react at 37°C for 45 minutes.

[0162] Step d): Add secondary antibody: Drain the primary antibody mixture, add washing solution (1×TBST) to the ELISA plate at a volume of 180 μL / well, and wash the ELISA plate twice. Dilute goat anti-rabbit-HRP to a working concentration (1:30000) with 1% BSA, add 50 μL / well to the ELISA plate, cover the lid, and incubate in a 37°C constant temperature incubator for 45 minutes.

[0163] Step e): Color development, termination and reading: discard the liquid in the wells, add washing solution to the ELISA plate at a volume of 180 μL / well, and wash the ELISA plate 3 times; add 100 μL of freshly prepared TMB color development substrate to each reaction well, incubate at 37°C for 10 minutes; then add 90 μL / well of stop solution to terminate the reaction, and measure the OD value at 450nm on the ELISA reader.

[0164] The results show that Figure 6 As shown, the 1G9 antibody presents an S-shaped curve, indicating that it has specific recognition for the Acp1 protein.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rabbit monoclonal antibody against human Acp1 protein, characterized in that: The variable region of the rabbit monoclonal antibody against human Acp1 protein includes: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.5, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.

6.

2. The rabbit monoclonal antibody against human Acp1 protein according to claim 1, characterized in that: The variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.

7.

3. The rabbit monoclonal antibody against human Acp1 protein according to claim 1, characterized in that: The variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.

8.

4. Biomaterial, characterized in that The biological material is selected from any one of ac: a. Nucleotides, comprising a nucleotide sequence encoding a rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3; b. a vector carrying the nucleotides in a; c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3.

5. The method for preparing the rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3, characterized in that: Obtained by fermentation of the cell described in claim 4.

6. Use of the rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3 in the preparation of a detection product for human Acp1 protein.

7. A marker of human Acp1 protein, characterized in that: A method comprising a rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3 and a marker; The rabbit monoclonal antibody against human Acp1 protein is coupled to a label.

8. The marker of human Acp1 protein according to claim 7, characterized in that The markers include enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

9. A kit for detecting human Acp1 protein, characterized in that: The kit comprises the rabbit monoclonal antibody against human Acp1 protein according to any one of claims 1 to 3 or the marker of human Acp1 protein according to claim 7 or 8.

10. The kit according to claim 9, characterized in that The kit includes an immunochromatographic detection kit or an ELISA detection kit.

Images