Antibody 3E11, antibody composition and application of human Golgi membrane protein GP73

Abstract

The present invention relates to the technical field of immunological detection methods, and in particular to an antibody, antibody composition and application of human Golgi membrane protein GP73. The antibody provided by the present invention has a high relative affinity constant with human Golgi membrane protein GP73, can bind to the target protein with high sensitivity and high specificity, and has high stability; based on the antibody, the present invention provides a method for detecting human Golgi membrane protein GP73 using a double antibody sandwich ELISA method, which can accurately, quickly and in large quantities detect the content of human Golgi membrane protein GP73 in serum or related products, and has good application prospects in the content detection of human Golgi membrane protein GP73.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection methods, and in particular to an antibody, an antibody composition and an application of human Golgi membrane protein GP73. Background Art

[0002] GP73 is a type II Golgi membrane protein with a molecular weight of 73 kDa. The GP73 gene has 3042 base pairs, contains 9 introns and 10 exons, and is located on human chromosome 9q21.33. GP73 contains 401 amino acids, which mainly include an N-terminal domain, a transmembrane domain, and a coiled-coil domain. It is only expressed in bile duct epithelial cells, and has low or no detectable expression in hepatocytes of healthy liver. GP73 is highly expressed in the colon, stomach, prostate, and trachea, but is rarely or not expressed in muscle, lymphoid tissue, and leukocytes. At the cellular level, GP73 is mainly present in epithelial cells. GP73 is a marker for hepatocellular carcinoma, and is also associated with the occurrence of other types of cancer, including melanoma, glioblastoma, prostate cancer, lung cancer, breast cancer, and colorectal cancer.

[0003] The currently commonly used detection method for GP73 is the ELISA method. The ELISA method has the characteristics of simple operation, no special equipment requirements, low cost, and little subjective influence. It can also automatically analyze the results, so it is particularly important for a wide range of practical applications in clinical practice.

[0004] In addition to the ELISA method, the Real-time Quantitative PCR (qRT-PCR) method can also be used to detect GP73. The qRT-PCR method refers to adding fluorescent markers to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally using the amplification curve to quantitatively analyze the unknown template. Its disadvantage is that the required test specimens—PBMCs—are difficult to obtain, and the entire operation process requires high technical conditions and is expensive.

[0005] Other detection methods include Western blot, which has high sensitivity and specificity for detecting the expression of GP73 in serum. However, due to its complex operation and high requirements for laboratory conditions and testing personnel, it has limited its large-scale promotion in clinical diagnostic applications.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an antibody to human Golgi membrane protein 1 (GP73), an antibody composition and applications thereof.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against human Golgi membrane protein GP73, 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 against human Golgi membrane protein GP73 are GFTFSDYG, IYNSGGGP, and TRDIGNFYVPFDY, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are QDIRNY, YTS, and QQGNTLPPT, respectively.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody against human Golgi membrane protein GP73 is as shown in SEQ ID NO: 8, or has at least 80% sequence similarity to SEQ ID NO: 8; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7, or has at least 80% sequence similarity to SEQ ID NO: 7.

[0011] Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof against the human Golgi membrane protein GP73.

[0013] Preferably, the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 6, or has at least 80% sequence similarity to SEQ ID NO: 6; the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO: 5, or has at least 80% sequence similarity to SEQ ID NO: 5.

[0014] In a third aspect, the present invention provides a biological material, which contains the nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.

[0015] In a fourth aspect, the present invention provides an antibody conjugate of human Golgi membrane protein GP73, which is obtained by conjugating the antibody or antigen-binding fragment of the human Golgi membrane protein GP73 with a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0016] In a fifth aspect, the present invention provides a composition of antibodies to human Golgi membrane protein GP73, the composition comprising the antibody to human Golgi membrane protein GP73 or its antigen-binding fragment; further comprising the following antibody to human Golgi membrane protein GP73 or its antigen-binding fragment: the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFTFSNYV, ISRGGTYI, and TREGIFFTAVGTGEDYSAMDY, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are KSLLHSNGITS, QMS, and AQNLELYT, respectively.

[0017] In a sixth aspect, the present invention provides any one of the following (1) to (5) uses of the antibody or antigen-binding fragment thereof against human Golgi membrane protein GP73, the nucleic acid molecule, the biomaterial, the antibody conjugate against human Golgi membrane protein GP73, or the composition:

[0018] (1) Use in the preparation of a product for detecting the presence or level of human Golgi membrane protein GP73 in a sample;

[0019] (2) Use in detecting the presence or level of human Golgi membrane protein GP73 in a sample for non-diagnostic and therapeutic purposes;

[0020] (3) Use in the preparation of a product for neutralizing the activity of human Golgi membrane protein GP73 in a sample;

[0021] (4) Use in the preparation of drugs for neutralizing the activity of human Golgi membrane protein GP73 in vivo;

[0022] (5) Application in quality control or production of products containing human Golgi membrane protein GP73.

[0023] In a seventh aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof against human Golgi membrane protein GP73, or the antibody conjugate, or the composition.

[0024] Preferably, the kit is an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0025] Beneficial effects:

[0026] The antibody provided by the present invention has a high relative affinity constant with human Golgi membrane protein GP73, can bind to the target protein with high sensitivity and high specificity, and has high stability; based on the antibody, the present invention provides a method for detecting human Golgi membrane protein GP73 using a double antibody sandwich ELISA method, which can accurately, quickly and in large quantities detect the content of human Golgi membrane protein GP73 in serum or related products, and has good application prospects in the detection of the content of human Golgi membrane protein GP73. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0028] Figure 1 This is a diagram of the Protein G affinity chromatography purification of antibodies 6G12 and 3E11 in Example 1 of the present invention.

[0029] Figure 2 This is the standard curve drawn in the double antibody sandwich ELISA method in Example 3 of the present invention, the horizontal axis is the concentration of the GP73 standard, and the vertical axis is the absorbance OD value. DETAILED DESCRIPTION

[0030] The present invention aims to develop a kit that can accurately, quickly and in large quantities detect the content of human GP73 in samples. The present invention first screened and obtained two antibodies 6G12 and 3E11 with excellent affinity for human Golgi membrane protein GP73. The present invention further used these two antibodies to establish an enzyme-linked immunosorbent assay based on the double antibody sandwich method, and provided a corresponding ELISA kit. The ELISA kit provided by the present invention can accurately and high-throughput detect the content of human serum or other samples containing human Golgi membrane protein GP73.

[0031] The present invention adopts the enzyme-linked immunosorbent assay technology based on the double antibody sandwich method to detect the content of human GP73. The principle is as follows: anti-human GP73 monoclonal antibody is coated on an ELISA plate; gradient dilution standard and pre-diluted sample are added respectively, and human GP73 in the standard and sample will fully bind to the coated antibody on the ELISA plate; after washing the plate, biotin-labeled anti-human GP73 antibody is added, and the antibody will specifically bind to human GP73 in the standard and sample captured by the coated antibody on the plate; after washing the plate, horseradish peroxidase (HRP)-labeled streptavidin is added, and biotin and streptavidin will have high-intensity non-covalent binding; after washing the plate, the color developer substrate TMB is added, and if different concentrations of human GP73 exist in the sample in the reaction well, HRP will turn the colorless TMB into different shades (positively correlated) of blue substances, and the reaction well will turn yellow after adding the stop solution; finally, at λmax=450 nm (OD=450 The absorbance (OD) of the reaction well sample is measured at 100 nm. The concentration of human GP73 in the sample is proportional to the OD. The concentration of human GP73 in the sample can be calculated through the standard curve. This method uses an enzyme colorimetric amplification system, which has a high detection sensitivity and can detect samples with relatively low content.

[0032] Compared with other methods such as qRT-PCR (μg level), the kit of the present invention has high detection sensitivity (pg level), ranging from 0.625-40ng / mL, with a sensitivity of 13pg / mL and R 2 =0.9999. In the 37°C accelerated stability test, the kit of the present invention had no significant changes within 13 days, and the stability performance was good.

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. 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.

[0034] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0036] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0037] In the following embodiments, the sequence information involved is as follows.

[0038] The coding gene sequence of the light chain variable region of monoclonal antibody 6G12 (SEQ ID NO.1):

[0039] GATATCGTGATGACGCAGGCTGCATTGTCCAATCCAGTCACTCTTGGAACATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTCTTTGTTTTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGAT GTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTACTGTGCTCAAAATCTAGAACTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0040] The coding gene sequence of the heavy chain variable region of monoclonal antibody 6G12 (SEQ ID NO.2):

[0041] GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGTCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGGGGTGGTACTTACATCTATTATCCAGACAGTGTG AAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTACAAGAGAGGGGATTTTCTTTACTGCGGTAGGAACCGGGGAGGATTACTCTGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0042] The amino acid sequence of the light chain variable region of monoclonal antibody 6G12 (SEQ ID NO.3):

[0043] DIVMTQAALSNPVTLGTSVSISCRSSKSLLHSNGITSLFWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELYTFGGGTKLEIK.

[0044] The amino acid sequence of the heavy chain variable region of monoclonal antibody 6G12 (SEQ ID NO.4):

[0045] EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYVMSWVRQTPEKRLEWVATISRGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCTREGIFFTAVGTGEDYSAMDYWGQGTSVTVSS.

[0046] The coding gene sequence of the light chain variable region of monoclonal antibody 3E11 (SEQ ID NO.5):

[0047] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGAAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGATCAGATTATTCTCTCACCATTAGCAACCTGGACCAAGCAGATATTGCCACTTACTTTTGTCAACAGGGTAATACGCTTCCTCCGACGTTCGGTGGAGGCACCAGGGTGGAAATCAAA。

[0048] Coding gene sequence of the heavy chain variable region of monoclonal antibody 3E11 (SEQ ID NO.6):

[0049] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTTGGTCGCAACCATTTATAATAGTGGTGGTGGCCCCTCTTATTCAAACAGTGTGAAGGGCCGATTTACCATCTCCAGAGACAATGCCAAGAACACCCTGTTCCTGCAAATGAACAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGTACAAGGGATATTGGTAACTTTTACGTTCCTTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA。

[0050] Amino acid sequence of the light chain variable region of monoclonal antibody 3E11 (SEQ ID NO.7):

[0051] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGSDYSLTISNLDQADIATYFCQQGNTLPPTFGGGTRVEIK。

[0052] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3E11 (SEQ ID NO.8):

[0053] EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYGMSWVRQTPDKRLELVATIYNSGGGPSYSNSVKGRFTISRDNAKNTLFLQMNSLRSEDTAMYYCTRDIGNFYVPFDYWGQGTTLTVSS.

[0054] Complementarity determining region CDR1 of the light chain variable region of monoclonal antibody 6G12 (SEQ ID NO.9):

[0055] KSLLHSNGITS.

[0056] Complementarity determining region CDR2 of the light chain variable region of monoclonal antibody 6G12 (SEQ ID NO.10):

[0057] QMS.

[0058] Complementarity determining region CDR3 of the light chain variable region of monoclonal antibody 6G12 (SEQ ID NO.11):

[0059] AQNLELYT.

[0060] Complementarity determining region CDR1 of the heavy chain variable region of monoclonal antibody 6G12 (SEQ ID NO.12):

[0061] GFTFSNYV.

[0062] Complementarity determining region CDR2 of the heavy chain variable region of monoclonal antibody 6G12 (SEQ ID NO.13):

[0063] ISRGGTYI.

[0064] Complementarity determining region CDR3 of the heavy chain variable region of monoclonal antibody 6G12 (SEQ ID NO.14):

[0065] TREGIFFTAVGTGEDYSAMDY.

[0066] The complementary determining region CDR1 of the light chain variable region of monoclonal antibody 3E11 (SEQ ID NO.15):

[0067] QDIRNY.

[0068] Complementarity determining region CDR2 of the light chain variable region of monoclonal antibody 3E11 (SEQ ID NO.16):

[0069] YTS.

[0070] Complementarity determining region CDR3 of the light chain variable region of monoclonal antibody 3E11 (SEQ ID NO.17):

[0071] QQGNTLPPT.

[0072] The complementarity determining region CDR1 of the heavy chain variable region of the monoclonal antibody 3E11 (SEQ ID NO. 18): GFTFSDYG.

[0073] The complementarity determining region CDR2 of the heavy chain variable region of the monoclonal antibody 3E11 (SEQ ID NO. 19): IYNSGGGP.

[0074] Complementarity determining region CDR3 of the heavy chain variable region of monoclonal antibody 3E11 (SEQ ID NO.20):

[0075] TRDIGNFYVPFDY.

[0076] Example 1

[0077] Preparation of anti-human GP73 monoclonal antibody.

[0078] 1. Animal immunization.

[0079] Female BALB / c mice aged 6-8 weeks were selected and immunized with recombinant human GP73 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.

[0080] 2. Cell fusion.

[0081] 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:

[0082] Myeloma (SP2 / 0) cell activation:

[0083] Commercial SP2 / 0 cells were thawed and revived, then resuspended in nutrient solution (RPMI 1640 medium supplemented with calf serum) and placed in a 37°C, 5% CO 2 Culture in an incubator under normal conditions; subculture after 3-5 days.

[0084] The cells were collected and suspended in RPMI 1640 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 their necks, and the tumors were removed by aseptic operation after soaking in 75% alcohol for 5 minutes.

[0085] The tumor pieces were cut off and placed in a sterile homogenizer. RPMI 1640 medium was added and fully ground. 10 mL of RPMI 1640 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 medium was added and the 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 basic RPMI 1640 medium.

[0086] Add 15 mL of lymphocyte separation medium to another centrifuge tube, and carefully place the above cell suspension on the separation medium; then centrifuge at 1200 r / min for 15 min, pipette out the dense white cell layer at the interface, wash the cells twice with RPMI 1640 medium, resuspend them in 10 mL of RPMI 1640 medium, count and set aside.

[0087] Preparation of immune splenocytes:

[0088] 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.

[0089] Preparation of feeder cells:

[0090] Take an unimmunized BALB / c mouse, bleed from its orbit, and collect the negative serum. Inject 2-3mL of RPMI 1640 culture medium into the mouse's peritoneal cavity, then aspirate and place in another centrifuge tube for later use. The liquid contains peritoneal macrophages. Prepare spleen cell suspension in the same way as above and place in the peritoneal macrophage tube. Centrifuge at 1000r / min for 10min to remove the supernatant. After the cells are suspended in HAT culture medium, place at 37°C and 5% CO 2 Place in incubator until use.

[0091] Fusion:

[0092] 1-2×10 7 SP2 / 0 and 1×10 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 0.8mL of 50% PEG (sigma) preheated to 37℃, stir and let stand for 30 s. After standing, add 10mL of RPMI 1640 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 and seed in a 96-well culture plate, 250μL / well, at 37℃, 5%CO 2 Culture in an incubator. On the 4th day after fusion, change to HT medium and continue culturing. When the fused cell colonies grow to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0093] 3. Screening of hybridoma positive clones and cell cloning.

[0094] 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. The specific operation is as follows:

[0095] The steps for screening positive hybridoma cells using indirect ELISA are as follows:

[0096] Coating with known antigens: dilute the purified coating antigen to 1-10 μg / mL with coating buffer; add 100 μL to each microwell, shake gently, and store in a 4°C refrigerator overnight or at 37°C for 1 hour; shake off the liquid in the wells (try to pat dry the liquid in the wells); wash 3 times, 2-3 minutes each time.

[0097] Block the uncoated areas in the ELISA wells: add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each well, shake gently, and incubate at 37°C for 1 hour; discard the liquid in the wells; fill each well with washing buffer (containing 1× PBST, Na 2 HPO 4 8MM, NaCl 0.136M, KH 2 PO 42MM, KCL 2.6MM, Tween-20 0.05% (V / V)), let stand for 2-3min, shake off the liquid in the well, pat dry, and use this method to wash 3 times with washing buffer. Sample addition: Take 50μL of supernatant from each well of the hybridoma to be tested and add it to the enzyme-labeled wells in sequence, and shake gently. 37℃ 1h, wash, and pat dry.

[0098] Add enzyme-labeled anti-antibody: First dilute the enzyme-labeled secondary antibody to an appropriate working concentration with diluent according to the instructions, add 100μL to each well, shake gently, and place at 37℃ for 1h; then wash and pat dry. Add color development solution: Add 100μL of freshly prepared color development solution to each well, shake gently, and place at 37℃ for 10min. Stop reaction: Add 50μL of stop solution to each well.

[0099] Determination result: OD of microplate reader 450nm The reading was performed at 3 times that of the negative well, which was considered positive.

[0100] Cloning of hybridoma cells (limiting dilution method):

[0101] 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 with complete culture medium to 5, 10, or 30 cells / ml.

[0102] Add the three concentrations of cell suspension to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 0.5, 1 and 3 cells, respectively. 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.

[0103] Detection of specific antibodies: On the 7th to 9th day after cloning, when the cell clones cover 1 / 3 to 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.

[0104] 4. Determination of variable region sequences of monoclonal antibodies 6G12 and 3E11.

[0105] The number of hybridoma cells collected was greater than 10 6, and sent to Shenggong Bioengineering for subsequent construction and sequencing. The gene sequencing results were obtained: the light chain variable region sequence of the 6G12 cell line was 333bp long, encoding 111 amino acids, the DNA sequence was shown in SEQ ID NO 1, and the protein sequence was shown in SEQ ID NO 3; the heavy chain variable region sequence was 384bp long, encoding 128 amino acids, the DNA sequence was shown in SEQ ID NO 2, and the protein sequence was shown in SEQ ID NO 4. The light chain variable region sequence of the 3E11 cell line was 321bp long, encoding 107 amino acids, the DNA sequence was shown in SEQ ID NO 5, and the protein sequence was shown in SEQ ID NO 7; the heavy chain variable region sequence was 360bp long, encoding 120 amino acids, the DNA sequence was shown in SEQ ID NO 6, and the protein sequence was shown in SEQ ID NO 8.

[0106] 5. Large-scale preparation of monoclonal antibodies 6G12 and 3E11.

[0107] After establishment, the hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected after about 7 days. The antibodies were purified by Protein G affinity chromatography ( Figure 1 ).

[0108] Example 2

[0109] Determination of relative affinity constants between monoclonal antibodies 6G12 and 3E11.

[0110] GP73 antigen was coated on the ELISA plate and blocked. After washing the plate with PBST, monoclonal antibodies 6G12 and 3E11 were diluted to saturation concentration and added to the ELISA plate, 100 μL / well, and incubated at room temperature for 2 hours. After washing the plate with PBST, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mol / L NaSCN (sodium sulfhydrate) solution was added in sequence at 60 μL / well, and incubated at room temperature for 15 minutes. After washing the plate with PBST, HRP-labeled goat anti-mouse IgG was added, and the plate was incubated at room temperature for 45 minutes for color development detection. The sodium thiocyanate concentration corresponding to the OD value at 450 nm after elution dropped to 50% of that without elution is the relative affinity constant of the antibody, expressed in mol / L.

[0111] Table 1

[0112]

[0113] The results showed that the relative affinity constants of monoclonal antibodies 6G12 and 3E11 were both greater than 2.5 mol / L (Table 1), indicating good affinity.

[0114] Example 3

[0115] Establishment of a double antibody sandwich ELISA kit for human GP73 content.

[0116] 1. Biotin labeling of monoclonal antibody 3E11.

[0117] Add antibody 3E11 to the dialysis bag and dialyze in 0.1M CB buffer at 4°C overnight. Take 2.2mg NHS-D-Biotin and dissolve it in 1mL DMSO. Take 100μL of NHS-D-Biotin and dissolve it in 1mL of antibody 3E11, stir and mix at room temperature for 4h. Take it out and put it in the dialysis bag, dialyze it in 0.01M PBS, change the solution after 2h, and dialyze it overnight at 4°C. Take out the labeling solution that has been dialyzed overnight, add an equal volume of glycerol, and add 1% BSA and 0.03%-0.05% proclin 300 and store it at -20°C.

[0118] 2. Preparation of monoclonal antibody 6G12 ELISA plate.

[0119] Dilute monoclonal antibody 6G12 to 2μg / mL with 0.05M pH9.6 carbonate coating buffer. Add 0.1mL / well to the reaction wells of the 96-well polystyrene reagent plate and store at 4℃ overnight. The next day, discard the solution in the wells and wash 3 times with washing buffer, 3 minutes each time. After the above steps, block each well of the reaction plate with 2% BSA solution, 0.3mL per well, for 2h. Discard the solution in the wells, dry in a drying room, evacuate, and store at 4℃.

[0120] 3. Establishment of double antibody sandwich ELISA method.

[0121] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6G12, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human GP73 standard with different dilution concentrations. The dilution concentrations of human GP73 are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL and set up a blank control. After sealing the plate, shake and incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of biotin-labeled antibody 3E11 working solution to the reaction well, shake and incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction well, shake and incubate at room temperature for 30 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color develop at room temperature in the dark for 15 minutes, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value (within 5 minutes) at a wavelength of 450 nm using an ELISA reader. Draw a standard curve with different concentrations of human GP73 standards as the horizontal axis and the corresponding OD value as the vertical axis, and establish a regression equation. The results show that its detection range is 0.625-40 ng / mL, R 2 is 0.9999 ( Figure 2 ).

[0122] 4. Double antibody sandwich ELISA stability test.

[0123] The ELISA plate coated with monoclonal antibody 6G12, biotin-labeled monoclonal antibody 3E11, and standard human GP73 were placed at 37°C for accelerated stability test and placed for 13 days (about 19.5 months at 4°C). Then take out the test, the test method is to take out the ELISA plate, wash the plate 3 times and spin dry. Add 100μL of human GP73 standard with different dilution concentrations, and the dilution concentrations of human GP73 are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL and set up a blank control. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and spin dry. Add 100μL of biotin-labeled antibody 3E11 working solution to the reaction well, seal the plate, incubate at room temperature for 60min, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at room temperature for 30 minutes, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, color develop at room temperature in the dark for 15 minutes, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength (within 5 minutes) with an ELISA reader. Draw a standard curve with different concentrations of human GP73 standards as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation.

[0124] Table 2

[0125]

[0126] The results showed that the OD value was greater than 1.5 on day 13, and the gradient was good, indicating that the kit had good stability (Table 2).

[0127] Example 4

[0128] Determination of spike recovery results.

[0129] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6G12, return to room temperature, wash the plate 3 times and spin dry. Select 5 serum / plasma samples (numbered 1, 2, 3, 4, and 5) from voluntary blood donors in the laboratory, add 100 μL of human serum samples / standards with different dilution multiples, dilute the samples 80 times, add 15ng / mL, 5ng / mL, and 0.5ng / mL standards on this basis, and the dilution concentrations of human GP73 standards are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, and 0.625ng / mL, and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of biotin-labeled antibody 3E11 working solution to the reaction well, seal the plate, incubate at room temperature for 60 minutes, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at room temperature with shaking for 30 min, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, color develop at room temperature in the dark for 15 min, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength with a microplate reader (within 5 min).

[0130] Table 3

[0131]

[0132] The spike recovery was calculated, and the test results showed that this method could determine that human GP73 had a good recovery within a certain range (a range of 70%-130% indicated a good result) (Table 3).

[0133] Example 5

[0134] Linear recovery results were determined.

[0135] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6G12, return to room temperature, wash the plate 3 times and spin dry. Select 5 serum / plasma samples (numbered 1, 2, 3, 4, and 5) from voluntary blood donors in the laboratory, add 100 μL of human serum samples / standards with different dilution multiples, and the sample dilution multiple is 80 times. On this basis, 10 times, 20 times, 40 times and 80 times dilutions are performed. The dilution concentrations of human GP73 standard are 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and a blank control is set. After sealing the plate, shake and incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of biotin-labeled antibody 3E11 working solution to the reaction well, shake and incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at room temperature with shaking for 30 min, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, color develop at room temperature in the dark for 15 min, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength with a microplate reader (within 5 min).

[0136] Table 4

[0137]

[0138] The linear recovery rate was calculated, and the test results showed that this method could determine that human serum GP73 had a good linear relationship within a certain range (a range of 70%-130% indicated a good result) (Table 4).

[0139] Example 6

[0140] Sample result determination.

[0141] Comparison of sample measurement results: 8 human serum / plasma samples were randomly selected for simultaneous measurement (unit: ng / mL). According to literature reports (Wenli S, Li W, Wenjie Z, et al. Abnormal Expression of GolgiProtein 73 in Clinical Values ​​and Their Role inhBV-RelatedhepatocellularCarcinoma Diagnosis and Prognosis. [J]. hepatitisMonthly, 2015, 15 (12): e32918.; Hongshan W, Boan L, Renwen Z, et al. Serum GP73, aMarker forevaluating progression in patients with chronichBV infections. [J]. PloS one, 2013, 8 (2): e53862.), the GP73 content in normal human serum is less than 60 ng / mL (the content varies according to age and individual).

[0142] Table 5 Test sample results

[0143]

[0144] The test results showed that the content determined by this kit was less than 60ng / mL (Table 5).

[0145] Example 7

[0146] The results of quantitative comparison of freeze-dried standard samples were determined.

[0147] The freeze-dried and 4°C human GP73 standards were quantitatively compared. The detection method was to take out the ELISA plate, wash the plate 3 times and spin dry. Add 100 μL of human GP73 standards of different dilution concentrations, and the dilution concentrations of human GP73 were 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of biotin-labeled antibody 3E11 working solution to the reaction well, seal the plate, incubate at room temperature for 60 minutes, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction well, seal the plate, incubate at room temperature for 30 minutes, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 min at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm with an ELISA reader (within 5 min). Draw a standard curve with different concentrations of human GP73 standards as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation.

[0148] Table 6 Quantitative results of standard products

[0149]

[0150] The results showed that the standard curve after lyophilization was well parallel to the standard curve at 4°C, with a good gradient and good stability of the kit (Table 6).

[0151] Example 8

[0152] Sensitivity results determination

[0153] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6G12, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human GP73 standard with different dilution concentrations. The dilution concentrations of human GP73 are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL and set 20 blank controls. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of biotin-labeled antibody 3E11 working solution to the reaction well, incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of enzyme conjugate working solution to the reaction well, incubate at room temperature for 30 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength (within 5 minutes) with an ELISA reader. Draw a standard curve with different concentrations of human GP73 standards as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation. Determine the average value of 20 blank ODs plus two standard deviations, and substitute them into the standard curve to calculate the corresponding detectable concentration.

[0154] Table 7 Sensitivity test results

[0155]

[0156] The sensitivity was calculated and the test results showed that the sensitivity of this kit was 13 pg / mL (Table 7).

[0157] 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 against human Golgi membrane protein GP73, 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 antigen-binding fragment thereof of the human Golgi membrane protein GP73 are GFTFSDYG, IYNSGGGP and TRDIGNFYVPFDY, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are QDIRNY, YTS and QQGNTLPPT, respectively.

2. The antibody or antigen-binding fragment thereof against human Golgi membrane protein GP73 according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody against human Golgi membrane protein GP73 is shown in SEQ ID NO:8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

3. The antibody or antigen-binding fragment thereof against human Golgi membrane protein GP73 according to claim 1 or 2, characterized in that: The antigen binding fragment is Fab, Fab', F(ab')2, Fv or single-chain antibody.

4. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof against the human Golgi membrane protein GP73 according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:6; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

5.

6. Biomaterial, characterized in that It 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 of human Golgi membrane protein GP73, characterized in that: The antibody or antigen-binding fragment thereof of the human Golgi membrane protein GP73 according to any one of claims 1 to 3 is coupled with a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling and radioactive labeling.

8. A composition of antibodies against human Golgi membrane protein GP73, characterized in that: The composition comprises an antibody or an antigen-binding fragment thereof against the human Golgi membrane protein GP73 according to any one of claims 1 to 3; and further comprises an antibody or an antigen-binding fragment thereof against the human Golgi membrane protein GP73 as follows: the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFTFSNYV, ISRGGTYI, and TREGIFFTAVGTGEDYSAMDY, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are KSLLHSNGITS, QMS, and AQNLELYT, respectively.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4 or 5, the biomaterial of claim 6, the antibody conjugate of human Golgi membrane protein GP73 of claim 7, or the composition of claim 8 in any one of the following (1) to (3): (1) Use in the preparation of a product for detecting the presence or level of human Golgi membrane protein GP73 in a sample; (2) Use in detecting the presence or level of human Golgi membrane protein GP73 in a sample for non-diagnostic and therapeutic purposes; (3) Application in quality control or production of products containing human Golgi membrane protein GP73.

10. A kit, characterized in that It comprises the antibody or antigen-binding fragment thereof against human Golgi membrane protein GP73 as described in any one of claims 1 to 3, or the antibody conjugate as described in claim 7, or the composition as described in claim 8; the kit is an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

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