Antibodies binding to Beclin1 and their applications

Abstract

The present invention relates to the field of antibody technology, and in particular to antibodies that bind to Beclin1 and applications thereof. The Beclin1 antibody provided by the present invention can specifically bind to Beclin1 protein, has a high affinity for Beclin1, and has a high titer. The antibody can achieve specific, sensitive and accurate detection of Beclin1, can be used for detection of Beclin1 in practice, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, in particular to antibodies binding to Beclin1 and applications thereof. Background Art

[0002] Autophagy is a form of cell self-degradation. It is a process of recycling damaged organelles or macromolecules through lysosome degradation and is regulated by autophagy-related genes (ATG). Autophagy has a dual role in regulating cell death: mild autophagy protects cells from harmful conditions to a certain extent and promotes cell survival; severe or rapid autophagy will induce programmed cell death, which is called autophagic cell death. Basal levels of autophagy are necessary to maintain cell homeostasis. At the same time, autophagy is involved in the pathological and physiological processes of anti-aging, differentiation and development, immunity, and elimination of microorganisms, tumors and other diseases.

[0003] The Beclin1 gene is the earliest discovered mammalian autophagy regulatory gene. The human Beclin1 gene encodes a protein containing 450 amino acids and a molecular weight of about 60Kd, which contains a BH3 domain (amino acids 108-127), a Bcl-2 interaction binding domain (amino acids 112-159), a nuclear export signal sequence (amino acids 180-189), a helical domain (amino acids 142-270) and an evolutionary conserved domain (amino acids 245-450). Beclin1 plays an important role in autophagy. It is the core subunit of the PI3K complex and mediates the formation of phosphatidylinositol 3-phosphate: PI3KC3-C1 is involved in the initiation of autophagosomes, and PI3KC3-C2 is involved in the maturation and endocytosis of autophagosomes. In addition, the Beclin1 gene is also considered to be a tumor suppressor gene. If the gene is missing, it may lead to malignant metastasis of cells. In mouse experiments, it was found that mice lacking beclin1 often died in the early embryonic stage, while heterozygous mice with this gene deficiency could develop and survive normally, but their probability of developing tumors was significantly increased. Beclin1 is widely expressed in tissues and cells, and is present in large quantities in mammary epithelium, glial cells, and neurons. In many malignant tumor cells, the Beclin1 gene is usually lowly expressed, but relatively highly expressed in normal tissues.

[0004] Based on the diagnostic and marker value of Beclin1 protein, Western blot (WB) and other methods based on the principle of antigen-antibody specific binding are often used in clinical practice to detect the expression level and localization information of Beclin1 in cells and tissues. Therefore, it is of great significance to develop anti-Beclin1 antibodies with good specificity and high sensitivity. However, there are few anti-human Beclin1 antibodies on the market, and the titers of existing antibodies are low and the specificity is not strong, which affects the accuracy of the test results. Summary of the invention

[0005] The present invention provides antibodies binding to Beclin1 and applications thereof.

[0006] Specifically, the present invention provides the technical solution described below.

[0007] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to Beclin1, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown as SEQ ID NO.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is LVS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.5.

[0008] The antibody or antigen-binding fragment thereof provided by the present invention can specifically bind to Beclin1, has high affinity and specificity, has high titer, and can be used to detect Beclin1 with high sensitivity and accuracy.

[0009] In the present invention, the Beclin1 is preferably human Beclin1, or rat or mouse Beclin1.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.6 or has at least 80% similarity to the sequence shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or has at least 80% similarity to the sequence shown in SEQ ID NO.7.

[0011] The above sequence similarity is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, more preferably at least 99.9%.

[0012] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0013] The above-mentioned antibody or antigen-binding fragment thereof may be a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

[0014] In some embodiments of the invention, the antibody is a monoclonal antibody.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0016] According to the amino acid sequence of the antibody or its antigen-binding fragment provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding an antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the above antibody or its antigen-binding fragment are within the scope of protection of the present invention.

[0017] In some specific embodiments of the present invention, the sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.8, and the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.9.

[0018] In a third aspect, the present invention provides a biological material, wherein the biological material contains the nucleic acid molecule described above; the biological material is an expression cassette, a vector or a host cell.

[0019] The above-mentioned expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0020] The above-mentioned vectors include but are not limited to plasmid vectors, phage vectors, virus vectors, artificial chromosome vectors and the like.

[0021] The host cells include microbial cells, insect cells or other mammalian cells. The microbial cells may be bacteria or fungi, bacteria include but are not limited to Escherichia coli, and fungi include but are not limited to yeast. The mammalian cells include but are not limited to Chinese hamster ovary cells (CHO), baby hamster embryonic kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero) and human embryonic kidney 293 cells (HEK293).

[0022] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or antigen-binding fragment thereof with a marker, and the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0023] In a fifth aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biomaterial, or the antibody conjugate:

[0024] (1) Use in the preparation of a product for detecting the presence or level of Beclin 1 in a sample;

[0025] (2) Application in the preparation of products for detecting the risk of tumor occurrence;

[0026] (3) Application in the preparation of products for detecting malignant tumors;

[0027] (4) Application in the preparation of products for detecting the risk of malignant tumor metastasis;

[0028] (5) Application in the preparation of products for prognosis assessment of malignant tumors.

[0029] In addition to the above uses, those skilled in the art can expect that the Beclin1 antibody or its antigen-binding fragment of the present invention can be used for currently known detection uses using Beclin1 as a marker, including but not limited to the construction of a liver ischemia-reperfusion injury model, the construction of a common carotid artery intima injury model, bronchial asthma, cell autophagy regulation level, etc.

[0030] In the above (1), the sample may be a body fluid sample (such as blood, serum, tissue fluid, etc.), a cell sample or a tissue sample from humans or other animals (such as rats or mice), or a cell or protein sample prepared by in vitro culture. The presence in the sample refers to whether the sample contains Beclin1, and the level refers to the content of Beclin1 in the sample.

[0031] In (2)-(5) above, Beclin1 is a known tumor suppressor gene and can be used as a biomarker for tumors, especially malignant tumors. Its expression level is significantly correlated with the occurrence, development, metastasis and prognosis of tumors. Therefore, the antibody or antigen-binding fragment thereof that binds to Beclin1 provided by the present invention can be used for diagnosis or auxiliary diagnosis, occurrence risk, metastasis risk and prognosis assessment of malignant tumors and other tumors. Among them, the malignant tumors include but are not limited to lung cancer (such as non-small cell lung cancer), cervical cancer, lymphoma (such as Hodgkin's lymphoma), gastric cancer, ovarian cancer, squamous cell carcinoma, breast cancer, melanoma, colorectal cancer, endometrial cancer, bladder cancer, etc.

[0032] In the above application, the product is a detection reagent or a detection kit.

[0033] In a sixth aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof or the antibody conjugate in detecting the presence or level of Beclin 1 in a sample for purposes other than disease diagnosis and treatment.

[0034] For detection purposes other than disease diagnosis and treatment, the sample does not include samples directly derived from living humans or animals, but may be cell or protein samples prepared by in vitro culture.

[0035] In the present invention, methods for detecting Beclin1 include, but are not limited to, immunoblotting, dot blot, ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and other immunological detection methods.

[0036] In a seventh aspect, the present invention provides a detection reagent or a kit, which comprises the above-mentioned antibody or antigen-binding fragment thereof, or comprises the antibody conjugate.

[0037] The present invention does not impose any particular limitation on the types of detection reagents and kits, including but not limited to immunoblotting detection reagents or kits, dot blot detection reagents or kits, ELISA detection reagents or kits, chemiluminescence detection reagents or kits, radioimmunoassay reagents or kits, fluorescent immunoassay reagents or kits, etc.

[0038] In some embodiments of the present invention, a immunoblotting detection kit is provided, which comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned antibody conjugate, and other reagents required for immunoblotting detection (including but not limited to blocking solution, washing solution, developing solution, etc.).

[0039] In some embodiments of the present invention, a dot blot detection kit is provided, which comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned antibody conjugate, and other reagents required for dot blot detection (including but not limited to blocking solution, washing solution, developing solution, etc.).

[0040] In an eighth aspect, the present invention provides a method for detecting Beclin1, comprising: using the above-mentioned antibody or antigen-binding fragment thereof or the antibody conjugate or the kit to detect the content of Beclin1 in a sample to be tested.

[0041] The above detection method can be selected from immunological detection methods such as immunoblotting, dot blot, ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, and immunochromatography.

[0042] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0043] The above-mentioned pharmaceutical composition is preferably used to treat autophagy-related diseases.

[0044] In a tenth aspect, the present invention also provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof.

[0045] The beneficial effects of the present invention include at least: the Beclin1 antibody provided by the present invention can specifically bind to the Beclin1 protein, has a high affinity for Beclin1, and has a high titer. The antibody can be used to achieve specific, sensitive and accurate detection of Beclin1, can be used for the detection of Beclin1 in practice, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 This is the SDS-PAGE detection result of human Beclin1 protein in Example 1 of the present invention.

[0048] Figure 2 This is the SDS-PAGE detection result of the monoclonal antibody 7A7 in Example 1 of the present invention, wherein the Marker is a protein Marker.

[0049] Figure 3This is the Western Blot detection result in Example 4 of the present invention.

[0050] Figure 4 The results are shown in Example 4 of the present invention, in which the sensitivity and detection limit of the monoclonal antibody 7A7 to human Beclin1 protein were verified by Western Blot.

[0051] Figure 5 This is a standard curve drawn using grayscale values ​​in Western Blot validation of the sensitivity and detection limit of monoclonal antibody 7A7 to human Beclin1 protein in Example 4 of the present invention.

[0052] Figure 6 This is the Dot Blot detection result in Example 5 of the present invention. DETAILED DESCRIPTION

[0053] The present invention prepares human Beclin1 antigen and immunizes mice for fusion screening to obtain a monoclonal antibody with good specificity and stability, which can specifically recognize human, rat and mouse Beclin1 (i.e. BENC1) proteins expressed in cells or tissues and has high titer and affinity; the antibody is further verified by indirect ELISA, Western blot (WB) and dot blot, and the antibody shows good specificity and sensitivity.

[0054] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1 Preparation of anti-human Beclin1 monoclonal antibody

[0056] 1. Beclin1 protein purification

[0057] The human Beclin1 gene (position 424-1350, sequence as shown in SEQ ID NO.10; amino acids corresponding to 141-450aa, sequence as shown in SEQ ID NO.11; the similarity of this segment of protein sequence to rat Beclin1 (SEQ ID NO.12) and mouse Beclin1 (SEQ ID NO.13) sequences is as high as 98.7% and 99.4%)) was synthesized into the PET-32A vector to obtain a recombinant plasmid named Beclin1-28A. The constructed recombinant plasmid was transformed into the expression strain Rosseta, and positive clones were selected for culture and sequencing. The recombinant strain was induced to express, and the uninduced strain and the empty vector strain were set as controls. The protein expression was detected by SDS-PAGE. After confirming the protein expression, the bacteria were broken by ultrasonic wave, and the supernatant and precipitate were separated. The protein was expressed in what form by SDS-PAGE. The test results showed that the Beclin1 protein was expressed in the form of insoluble inclusion bodies. The inclusion bodies were renatured and purified to obtain the target protein. The results of SDS-PAGE detection of purified protein are as follows Figure 1 shown.

[0058] 2. Animal immunization

[0059] Female BALB / c mice aged 6-8 weeks were selected and immunized with purified Beclin1 antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks. Blood was collected after three immunizations to measure the titer, and the immunization was boosted again three days before fusion.

[0060] 3. Cell fusion

[0061] The mice were killed by cervical dislocation, and the spleen was removed aseptically. The spleen was squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and the fusion promoter polyethylene glycol (PEG) was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is as follows:

[0062] (1) Myeloma (SP2 / 0) cell activation

[0063] Commercial SP2 / 0 cells were thawed and revived, then resuspended in nutrient solution (RPMI-1640 basal culture medium supplemented with 20% fetal bovine serum), and cultured in an incubator at 37°C and 5% CO2; passaged after 3-5 days.

[0064] The cells were collected and suspended in RPMI-1640 basal culture medium, and 0.5-1×10 6The cells were injected subcutaneously on the back of BALB / c mice and cultured for 9-10 days. When the tumor on the back grew to a diameter of about 0.8 cm, the mice were killed by pulling the neck, and the tumors were removed by aseptic operation after soaking in 75% alcohol for 5 minutes.

[0065] The tumor pieces were cut off and placed in a sterilized homogenizer. After adding RPMI-1640 basal culture medium and grinding thoroughly, 10 mL of RPMI-1640 basal culture medium was added and allowed to stand for 2 min. The cell suspension on the upper layer was aspirated and placed in another centrifuge tube. 10 mL of RPMI-1640 basal culture medium was added and grinding was repeated twice. The cell suspension obtained above was centrifuged at 1000 r / min for 10 min to remove the supernatant, and then resuspended in 30 mL of RPMI-1640 basal culture medium.

[0066] Add 15 mL of lymphocyte separation fluid to another centrifuge tube, and carefully place the above cell suspension on the separation fluid; then centrifuge at 1200 r / min for 15 min, aspirate the dense white cell layer at the interface with a pipette, wash the cells twice with RPMI-1640 basal culture fluid, and resuspend them in 10 mL of RPMI-1640 basal culture fluid, count and set aside.

[0067] (2) Preparation of immune spleen cells

[0068] Take a BALB / c mouse that has been boosted with immunity, bleed it from its orbits to kill it (collect serum, i.e., positive serum), and immerse it in 75% alcohol for 5-10 minutes for disinfection. Then fix it on a dissecting board for dissection, remove the spleen, cut it, and place it in a sterile homogenizer. The grinding and cell suspension preparation methods are the same as those described in SP2 / 0, and the cells are counted and set aside for later use.

[0069] (3) Preparation of feeder cells

[0070] Take an unimmunized BALB / c mouse, bleed from the eye socket, and collect the negative serum. Inject 2~3 mL of RPMI-1640 basal culture medium into the mouse's peritoneal cavity, aspirate it after blowing, and place it in another centrifuge tube for standby use. The liquid contains peritoneal macrophages. Prepare spleen cell suspension in the same way as above, and place it in the peritoneal macrophage tube. Centrifuge at 1000r / min for 10min to remove the supernatant, suspend the cells in HAT medium, and place them in a 37℃, 5% CO2 incubator for standby use.

[0071] (4) Integration

[0072] 1-2×10 7 SP2 / 0 and 10 8Mix the immune cells in a 50mL centrifuge tube, centrifuge at 1000r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 0.8 mL of 50% PEG (Sigma) preheated to 37℃, stir and let stand for 30s. After standing, add 10mL of RPMI-1640 basal culture medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, seed in a 96-well culture plate, 250μL / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HT medium and continue to culture. When the fused cell colony grows to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0073] 4. Screening of hybridoma positive clones and cell cloning

[0074] The purpose of selective culture is to screen fused hybridoma cells, using HAT selective culture medium. In HAT culture medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT culture medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells that can proliferate indefinitely.

[0075] (1) Screening of positive hybridoma cells using indirect ELISA

[0076] Coating with known antigens: dilute the purified coating antigen to 1-10 mg / mL with coating buffer; add 100 μL to each microwell, shake gently, and incubate at 4°C overnight or 37°C for 1 hour; shake off the liquid in the wells (tap dry as much as possible); wash 3 times, 2-3 minutes each time.

[0077] Block the areas in the enzyme-labeled wells that are not coated with antigens: add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each microwell, shake gently, and incubate at 37°C for 1 hour; shake off the liquid in the wells; fill each well with washing buffer, let it stand for 2 to 3 minutes, shake off the liquid in the wells, and pat dry. Use this method to wash three times with washing buffer.

[0078] Sample addition: Take 50 μL of supernatant from each well of the hybridoma to be tested and add it to the enzyme-labeled wells in order, shake gently, incubate at 37°C for 1 hour, wash, and pat dry.

[0079] Add enzyme-labeled secondary antibody: first dilute the enzyme-labeled secondary antibody to an appropriate working concentration using diluent according to the instructions, add 100 μL to each well, shake gently, and incubate at 37°C for 1 hour; then wash and pat dry.

[0080] Add color developing solution: Add 100 μL of freshly prepared color developing solution to each well, shake gently, and incubate at 37°C for 10 min.

[0081] Stop reaction: add 50 μL of stop solution to each well.

[0082] Determination result: OD of microplate reader 450 The reading was performed at 3 times that of the negative well, which was considered positive.

[0083] (2) Cloning of hybridoma cells (limiting dilution method)

[0084] Prepare a mouse feeder cell layer before cloning; gently blow the hybridoma cells to be cloned from the culture well and count the number of live cells using a hemocytometer; dilute the cells to 10 cells / mL with complete culture medium.

[0085] Add the cell suspension of the above concentration to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 1 cell. Add one drop of liquid on the 4th day of culture, and carefully observe the growth of cells in each well on the 5th-6th day and record it.

[0086] (3) Detection of specific antibodies

[0087] On the 7th to 9th day after cloning, when the cell clones cover 1 / 3-1 / 2 of the visual field, they can be detected; the cells in the positive wells can be moved to a 24-well culture plate, and when the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0088] 5. Sequence determination of the variable region of monoclonal antibody 7A7

[0089] The number of hybridoma cells collected was greater than 10 6, using a total RNA extraction kit (Solebo Technology Co., Ltd.), and according to the instructions, the total RNA of the cells was extracted respectively; the first chain of cDNA was synthesized according to the reverse transcription kit (Solebo Technology Co., Ltd.); and sent to Sangon Biotech (Shanghai) Co., Ltd. for subsequent construction and sequencing. The results of gene sequencing showed that the amino acid sequence of the heavy chain variable region of monoclonal antibody 7A7 was shown in SEQ ID NO.6, its encoding gene sequence was shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region was shown in SEQ ID NO.7, and its encoding gene sequence was shown in SEQ ID NO.9. After analysis, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 7A7 were shown in SEQ ID NO.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region was shown in SEQ ID NO.4, the amino acid sequence of CDR2 was LVS, and the amino acid sequence of CDR3 was shown in SEQ ID NO.5.

[0090] 6. Preparation of monoclonal antibody 7A7

[0091] The hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected after 7 days. The antibody was purified by Protein G affinity chromatography to obtain the monoclonal antibody 7A7. The SDS-PAGE test results are shown in Figure 2 shown.

[0092] Example 2 Indirect ELISA titer and sensitivity determination of monoclonal antibody 7A7

[0093] 1. Indirect ELISA titer determination of monoclonal antibody 7A7

[0094] Antigen coating: The antigen human Beclin1 protein was coated on the ELISA test plate with ELISA coating solution (Solarbio) at a coating concentration of 1 μg / mL, 100 μL / well, incubated at 37°C for 2 h, and washed twice with 0.01 M PBST (pH 7.4).

[0095] The coated ELISA test plate was blocked with blocking solution (0.01 M PBS solution containing 2% BSA), 200 μL / well, incubated at 37°C for 2 h, and washed three times with 0.01 M PBST (pH 7.4).

[0096] 7A7 monoclonal antibody was serially diluted with antibody diluent (0.01M PBS containing 1% BSA, pH 7.4), starting from 1:5000. The diluted primary antibody was added to the ELISA test plate with a pipette, 100 μL / well, incubated at 37°C for 1 hour, and washed 4 times with 0.01M PBST (pH 7.4).

[0097] Dilute goat anti-mouse IgG-HRP (Solarbio) 1:5000 with antibody diluent (0.01M PBS containing 1% BSA, pH 7.4), add the diluted secondary antibody to the ELISA detection plate with a pipette, 100 μL / well, incubate at 37°C for 45 minutes, and wash the plate 6 times with 0.01M PBST (pH 7.4).

[0098] Develop with TMB colorimetric kit, 100 μL / well, incubate at 25°C ± 2°C in dark for 3-5 min, add ELISA stop solution (Solarbio), 50 μL / well.

[0099] The colorimetric value was read using an ELISA reader at 450 nm. The titer of the 7A7 monoclonal antibody detected was shown in Table 1 (where K represents thousand, W represents ten thousand, and the two columns of values ​​are two parallel results). The titer was above 1:32W.

[0100] Table 1

[0101]

[0102] 2. Indirect ELISA to determine the sensitivity of monoclonal antibody 7A7

[0103] According to the above-mentioned indirect ELISA detection method, human Beclin1 protein was coated on the ELISA plate with a gradient of 0pg, 50pg, 100pg, 500pg, 1ng, 5ng, 10ng, 50ng, 100ng, and 200ng. Six replicate wells were made for each gradient, and two replicate wells for the blank control of the antibody were set. After blocking, 1mg / mL monoclonal antibody 7A7 (1:2000 diluted to prepare the primary antibody working solution) was used to allow the primary antibody to bind to the antigen. The plate was washed 4 times and sheep anti-mouse IgG-HRP (1:5000 diluted to prepare the secondary antibody working solution) was used to bind the primary antibody. After color development, the color value was read at 450nm using an ELISA reader.

[0104] The results are shown in Table 2. It can be seen that there is still a difference between the antibody detection value and the blank detection value at 50 pg. The sensitivity result can be determined as follows: 1 mg / mL monoclonal antibody 7A7 can detect 50 pg of human Beclin1 protein at a dilution ratio of 1:2000.

[0105] Table 2

[0106]

[0107] Example 3 Affinity detection of monoclonal antibody 7A7

[0108] According to the indirect ELISA method in Example 2 above, 1 μg / mL human Beclin1 was coated, the plate was washed, blocked, washed, and then the purified monoclonal antibody 7A7 (dilution ratio was 1 / 2000) was added, 100 μL / well, and incubated at 37° for 1 hour for antigen binding; after washing with PBST, different concentrations of sodium thiocyanate were used for elution, and 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mol / L sodium thiocyanate solution was added in sequence, 60 μL / well, and allowed to stand at room temperature for 15 minutes, washed with PBST, and sheep anti-mouse IgG-HRP secondary antibody (1:5000) was added for color detection. Result determination: OD after elution 450 The sodium thiocyanate concentration corresponding to the decrease to 50% of the uneluted concentration is the relative affinity constant of the antibody, expressed in mol / L. The results are shown in Table 3, and the relative affinity constant of the monoclonal antibody 7A7 is 3.25.

[0109] Table 3

[0110]

[0111] Example 4 Western Blot Application and Sensitivity Detection of Monoclonal Antibody 7A7

[0112] In this example, monoclonal antibody 7A7 was used as the primary antibody, and the Beclin1 protein in wild-type HeLa, MCF7, Jurkat and Raji cell samples and mouse brain and rat brain tissue samples was detected by western blot. The specific process is as follows.

[0113] 1. Sample preparation of cells and tissues containing Beclin1 protein

[0114] (1) Set the centrifuge parameters 30 minutes in advance: speed 1000 rpm, centrifugation time 5 minutes, temperature 4°C, cover the centrifuge lid, and the centrifuge will automatically cool down. Prepare the lysis working solution: take 10 mL of pre-cooled RIPA lysis solution and add protein inhibitor mixture and PMSF in proportion, the addition ratio is 100:1, and place it on ice for pre-cooling. Prepare scissors and disinfect them and place them on ice for pre-cooling.

[0115] (2) Wash the cell pellet collected in a 1.5 mL centrifuge tube with pre-cooled 1× PBS buffer (pH 7.2-7.4) (2×10 7cells), centrifuge at 1000 rpm and 4°C for 5 min, remove the supernatant, insert the recovered cell pellet into ice, add 1 mL of lysis working solution to the cell pellet, blow the cells evenly about 20 times with a pipette to completely disperse and suspend the cells, spread the centrifuge tube containing the cells on ice, place the ice box on a 4°C shaker, run the shaker, adjust it to slowly rock up and down, and lyse on a 4°C shaker for 20 min.

[0116] (3) Weigh 100 mg of mouse brain or rat brain tissue into a 1.5 mL centrifuge tube, add 0.5 mL of the prepared lysis working solution, and use pre-cooled scissors to cut the tissue into minced pieces on ice. Add 0.5 mL of lysis working solution again, vortex on a vortex shaker to mix thoroughly, and place on ice for later use.

[0117] (4) After 20 minutes of cell lysis, place the centrifuge tube of cells and tissues on a vortex oscillator and oscillate for 10 seconds before ultrasonic disruption. The ultrasonic conditions are: 200W power, 3 seconds of ultrasonication, 6 seconds of rest, and 1 minute of total duration each time. When changing samples, pay attention to the cleanliness of the amplitude rod. The interval between each ultrasonication is greater than 1 minute. Generally, after 3-5 ultrasonications, the liquid gradually becomes clear, and the ultrasonication is complete. To prevent protein degradation, the entire protein extraction process must be carried out on ice, and PMSF must be replenished every 20 minutes.

[0118] (5) Centrifuge the 1.5 mL centrifuge tube containing the lysed extract at 12,000 rpm and 4°C for 30 min. Prepare a new set of 1.5 mL centrifuge tubes. After the centrifugation is completed, separate the protein sample, calculate the sample volume, and use the BCA kit to detect the protein concentration. Add protein loading buffer and 1× PBS to dilute the protein to 2.5 mg / mL, and denature the protein at 100°C for 10 min. After denaturation, store the protein at -20°C.

[0119] 2. Western Blot Verification of Beclin1 Protein Expression

[0120] (1) Gel preparation, sample loading and gel running

[0121] Prepare 10% separation gel (lower layer) and 5% concentration gel (upper layer). After the gel is prepared, load it into the electrophoresis running device, add electrophoresis liquid into the inner and outer tanks, and start loading samples. The loading order is: HeLa, MCF7, Jurkat, Raji, mouse brain, rat brain. The loading amount is: 25μg. After adding electrophoresis liquid, connect to the electrophoresis instrument and run the gel at 80V for 20 minutes and 120V for 1 hour and 10 minutes.

[0122] (2) Transfer and sealing

[0123] Prepare the electrotransfer solution in advance and pre-cool it. Activate the PVDF membrane with methanol in advance, and then balance it with the electrotransfer solution for later use. Spread out the transfer clip, soak it with the electrotransfer solution, install the sponge-filter paper-gel-membrane-filter paper-sponge in sequence, fasten the clip, carefully place it in the electrotransfer device, add an ice box, fill it with electrotransfer solution, cover it according to the positive and negative poles, and transfer the membrane at a constant current of 200mA for 90 minutes.

[0124] Prepare 3% skim milk in advance for full dissolution. After transfer, wash the membrane with 1×TBST on a shaker for 5 minutes, and replace 1×TBS with 3% skim milk for blocking at room temperature for 1 hour.

[0125] (3) Primary antibody incubation, membrane washing and secondary antibody incubation

[0126] Primary antibody incubation: After blocking, dilute monoclonal antibody 7A7 with 3% skim milk at a ratio of 1:2000 and incubate at 4°C overnight.

[0127] Washing the membrane: Take it out on the next day and wash the membrane with 1×TBST for 5 min each time, wash 5 times.

[0128] Secondary antibody incubation: Use 1×TBST to dilute goat anti-rabbit IgG-HRP at a ratio of 1 / 20000 and incubate at room temperature for 1 hour. After incubation, wash the membrane with 1×TBST for 5 minutes each time, and wash 5 times.

[0129] (4) ECL luminescent liquid for development and exposure

[0130] Mix the A and B solutions of the ECL luminescent solution in a 1:1 ratio, so that the membrane is fully dipped in the luminescent solution, and then spread on the photographing area of ​​the exposure instrument, run the program for automatic exposure, and the WB result is as follows Figure 3 As shown, a single band was detected at 60 kDa in wild-type human cells HeLa, MCF7, Jurkat Raji, and mouse brain and rat brain tissue samples from rats and mice, and the protein size was consistent with the estimated 52-60 kDa, and the observed molecular weight was consistent with the literature (PMID: 35106915, PMID: 33953166, PMID: 36481655), indicating that monoclonal antibody 7A7 can specifically bind to Beclin1 protein in humans, rats, and mice.

[0131] 3. Western Blot Verification of the Sensitivity and Detection Limit of Monoclonal Antibody 7A7 for Human Beclin1 Protein

[0132] According to the above Western Blot experimental method, the immunogen human Beclin1 recombinant protein sample was used for gradient loading, blocked with 3% skim milk powder, and monoclonal antibody 7A7 (dilution ratio of 1:10000) was added as the primary antibody for antigen binding overnight. After washing the membrane, the secondary antibody goat anti-rabbit IgG-HRP (dilution ratio of 1:20000) was used to bind to the primary antibody. The results are shown in Figure 4 As shown in Table 4, grayscale values ​​were analyzed by WB test results. Since some sample wells had no significant difference with the blank wells, the wells without brightness except the blank control wells were deleted, and the grayscale values ​​were obtained as shown in Table 4. The standard curve was drawn according to the grayscale values, and a standard curve with a high R-square value was obtained, as shown in Table 4. Figure 5 As shown in the figure, by visually observing the comprehensive gray value analysis of the WB image, it can be determined that the detection sensitivity of the monoclonal purified antibody 7A7 to human Beclin1 protein is 10ng when the dilution ratio is 1:10000 in the WB application. In addition, the closer the R-square value is to 1, the better the fit between the data and the standard curve is, which can prove the accuracy and reliability of the detection sensitivity of the monoclonal antibody 7A7 to human Beclin1 protein.

[0133] Table 4 Gray value of 7A7 antibody detecting human Beclin1 protein

[0134]

[0135] Example 5 Dot Blot Application and Sensitivity Detection of Monoclonal Antibody 7A7

[0136] Monoclonal antibody 7A7 was used for Dot Blot detection of human Beclin1 protein, and its detection sensitivity was tested. The specific method and results are described as follows.

[0137] 1. Sample spotting: Prepare a nitrocellulose membrane and cut it into the required length, then draw a square grid of 1cm×1cm on the membrane. Take 1μL of human Beclin1 protein of different concentration gradients and spot it in the square grid on the membrane. The spotting gradient is: blank, 0.1ng, 0.5ng, 1ng, 5ng, 10ng, 25ng, 50ng, 100ng.

[0138] 2. Air drying: After the spotting is completed, air dry the membrane or place it in a 37°C constant temperature incubator for 30 minutes to remove excess moisture and make the spotting more uniform.

[0139] ‌3. Blocking‌: After air drying, place the membrane in blocking solution (usually 5% skim milk powder dissolved in ultrapure water) and block at room temperature for 1 hour, or overnight at 4°C to reduce nonspecific binding.

[0140] 4. Primary antibody incubation: After blocking, remove the blocking solution and add 1:2000 primary antibody working solution of monoclonal antibody 7A7, and incubate at room temperature with shaking for 1 hour.

[0141] ‌5. Washing‌: After the primary antibody incubation is completed, remove the primary antibody working solution, wash twice with TBST buffer, and then wash 3 times with TBST buffer with shaking, each time for 5 minutes, to remove unbound primary antibody.

[0142] ‌6. Secondary antibody incubation‌: After washing, incubate with 1:20,000 goat anti-mouse IgG-HRP working solution at room temperature for 1 hour with shaking to bind the primary antibody. After incubation, remove the secondary antibody working solution, wash twice with TBST buffer, and then wash 3 times with TBST buffer with shaking for 5 minutes each time to remove unbound secondary antibody.

[0143] 7. Expose with ECL luminescent liquid and observe the position and intensity of the labeled protein.

[0144] The results are as follows Figure 6 As shown, the amount of protein spotted at different gradients is positively correlated with the exposure intensity. Since the brightness of 50ng and 100ng is too high, the brightness behind cannot be seen. After blocking the 50ng and 100ng spots, it can be seen that there is a spot at 1ng. Finally, it can be determined that when the dilution ratio of monoclonal antibody 7A7 is 1:2000, 1ng / spot is detected.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to Beclin1, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NO.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is LVS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

5.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.6 or has at least 80% similarity to the sequence shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or has at least 80% similarity to the sequence shown in SEQ ID NO.

7.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.8, and the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.

9.

6. Biomaterial, characterized in that The biological material contains the nucleic acid molecule according to claim 4 or 5; The biological material is an expression cassette, a vector or a host cell.

7. An antibody conjugate, characterized in that: The antibody conjugate is obtained by conjugating the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the biomaterial according to claim 6, or the antibody conjugate according to claim 7 in the preparation of a product for detecting the presence or level of Beclin 1 in a sample.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the antibody conjugate according to claim 7 in detecting the presence or level of Beclin 1 in a sample for purposes other than disease diagnosis and treatment.

10. A detection reagent, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 7.

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