Monoclonal antibodies targeting human fam227a and uses thereof

Abstract

The application relates to the field of biological medicine, and discloses a monoclonal antibody targeting human FAM227A and application thereof. Two kinds of monoclonal antibodies targeting human FAM227A are prepared for the first time, the expression level of FAM227A in tumor tissues and cells can be detected by a Western Blot method (antibody 8# is used), or the expression of FAM227A in peripheral blood serum of tumor patients can be detected by a sandwich ELISA method (two kinds of antibodies are used) to achieve the application purpose of non-invasive diagnosis. The monoclonal antibody targeting human FAM227A has high titer affinity and specificity for FAM227A.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a monoclonal antibody targeting human FAM227A and applications thereof. Background Art

[0002] Cancer stem cells are one of the main causes of malignant tumor infiltration, metastasis, and recurrence. The tumor microenvironment can promote the dedifferentiation of ordinary tumor cells and the reacquisition of stem cell properties (reprogramming), thereby becoming cancer stem cells. Elucidating the mechanisms by which the tumor microenvironment promotes tumor cell reprogramming is fundamental to the targeted elimination of cancer stem cells. However, the molecular mechanisms of tumor cell reprogramming are currently unclear. Therefore, in-depth research on the mechanisms of tumor cell stemness and the exploration of serological markers associated with tumor cell stemness are expected to serve as a means of diagnosing malignant tumors and dynamically monitoring tumor progression, and provide a theoretical basis for subsequent targeted elimination of cancer stem cells. To identify molecules that promote tumor cell reprogramming in the tumor microenvironment, the present invention team used a shRNA retroviral library to screen for a new mitochondrial molecule, FAM227A. As a member of the FAM227 family of secretory kinases, FAM227A may play a key role in the occurrence and progression of tumors.

[0003] FAM227A is a member of the FAM227 family of secretory kinases, located on human chromosome 22q13.1. It contains 570 amino acids, a molecular weight of approximately 66 kDa, and 17 exons. It has two specific peptides, located at amino acid sequences 234-242 and 428-446. FAM227A expression levels are significantly higher in the testis, epididymis, fallopian tube, and pituitary gland than in other tissues and organs, suggesting that this gene may originate early in embryonic development, have a high degree of malignant biology, and may be associated with neural differentiation. With the continuous advancement of molecular biology techniques, identifying target genes closely associated with tumor development has become a key avenue in targeted therapy research. The discovery of key targets not only aids in early diagnosis or potential risk prediction for cancer patients, but also holds promise as targets for the development of molecularly targeted drugs, providing new avenues for personalized treatment of malignant tumors. The inventors hope to identify novel target molecules involved in the development of malignant tumors through continued exploration, enabling early diagnosis and treatment interventions to improve patient outcomes. Therefore, exploring new targets for targeted early intervention in the occurrence and development of malignant tumors is an issue that needs to be addressed urgently.

[0004] Bioinformatics databases suggest that FAM227A expression varies significantly among different tumors, but there are currently no commercially available antibodies targeting human FAM227A for validation. Therefore, developing a monoclonal antibody targeting human FAM227A is a challenge that needs to be addressed. Summary of the Invention

[0005] In order to solve the above technical problems, first, the present invention provides a monoclonal antibody 8# targeting human FAM227A and a nucleotide sequence encoding a monoclonal antibody 8# targeting human FAM227A; secondly, the present invention provides a composition containing two monoclonal antibodies targeting human FAM227A; thirdly, the present invention provides the use of the above-mentioned monoclonal antibodies targeting human FAM227A and the composition thereof.

[0006] The specific technical solutions of the present invention are:

[0007] In a first aspect, the present invention provides a monoclonal antibody targeting human FAM227A, named 8#, comprising a heavy chain variable region HV and a light chain variable region LV;

[0008] The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 1: EVKLQQSGAELVRPGASVKLSCKASGYTFTNYWVNWVRQRPGQGLEWIGMIDPSDSETHYNQMFKDKAALTVDISSTTAYMQLSSLTSEDSAVYYCASYDYDSYWGQGTTLTVSS.

[0009] The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO: 2: DIQMTQTPRTLSVTIGQPASFSCTSSQSLLYSNGKTYLSWLFQRPGQSPKRLIYLVSTLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQGRHFPQTFGGGTKLEIK.

[0010] The present invention discovered for the first time that FAM227A is underexpressed in tumor tissues or cells from colorectal cancer patients compared to normal tissues, making it a key biomarker for colorectal cancer. Based on this information, the present invention developed a monoclonal antibody targeting human FAM227A, 8#, which can quantitatively detect FAM227A expression levels in tumor tissues or cells from colorectal cancer patients using Western blotting.

[0011] In a second aspect, the present invention provides a nucleotide sequence encoding the above-mentioned monoclonal antibody targeting human FAM227A, wherein:

[0012] Nucleotide sequence encoding heavy chain variable region HV such as SEQ ID NO:3 shows: GAGGTGCAGCTGCAGGAGTCAGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAACTGTCCGGCAAGGCTTCTGGCTACACCTTCACCAACTGGGTGAACTGGGTGAGACAGAGGCCTGGACAAGGCCTTGAATGGATTGGGATGATTGATCCTTCAGACAGTG AAACTCACTACAATCAAATGTTCAAGGACAAGGCCGCATTGACTGTAGACATATCCTCCACCACAGCGTACATGCAACTCAGTAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCCTCCTATGATTACGACAGCTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCC.

[0013] The nucleotide sequence encoding the light chain variable region LV is as SEQ ID NO:4 shows: GATATCCAGATGACACAGACTCCACGCACTTTGTCGGTTACCATTGGACAGCCAGCCTCTTTCTTGCACGTCAAGTCAGAGCCTCTTATATAGTAATGGCAAAACCTATTTGAGTTGGTTATTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTG GTGTCTACACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTACTGCGTGCAAGGTAGACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0014] In a third aspect, the present invention provides use of the above-mentioned monoclonal antibody targeting human FAM227A in the preparation of a product for detecting the expression level of FAM227A protein.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned monoclonal antibody targeting human FAM227A in the preparation of a colorectal cancer diagnostic product.

[0016] Furthermore, in the above application, the FAM227A is FAM227A in tumor tissue or cells.

[0017] In a fifth aspect, the present invention provides a monoclonal antibody composition targeting human FAM227A, comprising the above-mentioned monoclonal antibody targeting human FAM227A 8#; and another monoclonal antibody targeting human FAM227A 2#, wherein the amino acid sequences of the heavy chain variable region HV and the light chain variable region LV are as follows:

[0018] The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 5:

[0019] EVKLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRTKPNDHATYSAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYFCATGFFTYWGQGTLVTVSA.

[0020] The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO: 6:

[0021] QAVVTQESALTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTSLIGGTHNRTPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNQWVFGGGTKLTVL.

[0022] The nucleotide sequence encoding the heavy chain variable region HV is shown in SEQ ID NO: 7:

[0023] GAGGTCAAACTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAACCAAACCTAATGATCAT GCAACATACTCTGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGGCATTTATTTCTGTGCCACGGGCTTTTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0024] The nucleotide sequence encoding the light chain variable region LV is shown in SEQ ID NO: 8:

[0025] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCCACAA CCGAACTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCAGTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA.

[0026] The present invention discovered for the first time that FAM227A is highly expressed in the peripheral blood of many cancer patients compared to healthy controls, making it a key biomarker for these tumors. Based on this information, the present invention developed a composition containing two monoclonal antibodies targeting human FAM227A. This antibody combination can be used to quantitatively detect FAM227A expression levels in the peripheral blood of patients with these malignant tumors using an ELISA sandwich assay.

[0027] In a sixth aspect, the present invention provides use of the above-mentioned antibody composition in the preparation of a product for detecting the expression level of FAM227A protein.

[0028] In a seventh aspect, the present invention provides the use of the above-mentioned antibody composition in the preparation of a malignant tumor diagnostic product, wherein the malignant tumor is lung cancer, thyroid cancer, breast cancer, colorectal cancer, prostate cancer, liver cancer, bile duct cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, kidney cancer, gastric cancer, nasopharyngeal cancer, and bladder cancer.

[0029] Furthermore, in the above application, the FAM227A is FAM227A in peripheral blood.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention first discovered that FAM227A is lowly expressed in tumor tissues of colorectal cancer patients compared with normal tissues, and therefore can be used as an important biomarker for colorectal cancer. The present invention also first discovered that FAM227A is highly expressed in the peripheral blood of patients with various tumors compared with normal controls, and therefore can be used as an important biomarker for these tumors.

[0032] (2) Since there is currently no monoclonal antibody that can be used to detect human FAM227A, the present invention has prepared two monoclonal antibodies targeting human FAM227A for the first time. The expression level of FAM227A in tumor tissues and cells can be detected by Western Blot and other methods (using antibody 8#); or the expression of FAM227A in the peripheral blood serum of tumor patients can be detected by sandwich ELISA and other methods (using two antibodies in combination) to achieve the application purpose of non-invasive diagnosis.

[0033] (3) The monoclonal antibody targeting human FAM227A of the present invention has high titer affinity and specificity for FAM227A.

[0034] (4) The FAM227A gene can be used as a target for the specific diagnosis of malignant tumors and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the staining result of recombinantly expressed FAM227A fusion protein;

[0036] Figure 2 This is a diagram of some ELISA antibody screening processes and results;

[0037] Figure 3 To test the potency of monoclonal antibodies targeting human FAM227A;

[0038] Figure 4 Antibody pairing assay for monoclonal antibodies targeting human FAM227A;

[0039] Figure 5 To detect the specificity and sensitivity of the monoclonal antibody targeting human FAM227A;

[0040] Figure 6 To use the monoclonal antibody targeting human FAM227A in Western Blot detection of FAM227A in tumor cells and tissues;

[0041] Figure 7 The purpose is to apply monoclonal antibodies targeting human FAM227A in the serological detection of FAM227A in tumor patients. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the embodiments.

[0043] Overall embodiment

[0044] A monoclonal antibody targeting human FAM227A, designated 8#, comprising a heavy chain variable region HV and a light chain variable region LV;

[0045] The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 1: EVKLQQSGAELVRPGASVKLSCKASGYTFTNYWVNWVRQRPGQGLEWIGMIDPSDSETHYNQMFKDKAALTVDISSTTAYMQLSSLTSEDSAVYYCASYDYDSYWGQGTTLTVSS.

[0046] The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO: 2: DIQMTQTPRTLSVTIGQPASFSCTSSQSLLYSNGKTYLSWLFQRPGQSPKRLIYLVSTLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQGRHFPQTFGGGTKLEIK.

[0047] A nucleotide sequence encoding the above-mentioned human FAM227A-targeting monoclonal antibody, wherein:

[0048] Nucleotide sequence encoding heavy chain variable region HV such as SEQ ID NO:3 shows: GAGGTGCAGCTGCAGGAGTCAGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAACTGTCCGGCAAGGCTTCTGGCTACACCTTCACCAACTGGGTGAACTGGGTGAGACAGAGGCCTGGACAAGGCCTTGAATGGATTGGGATGATTGATCCTTCAGACAGTG AAACTCACTACAATCAAATGTTCAAGGACAAGGCCGCATTGACTGTAGACATATCCTCCACCACAGCGTACATGCAACTCAGTAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCCTCCTATGATTACGACAGCTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCC.

[0049] The nucleotide sequence encoding the light chain variable region LV is as SEQ ID NO:4 shows: GATATCCAGATGACACAGACTCCACGCACTTTGTCGGTTACCATTGGACAGCCAGCCTCTTTCTTGCACGTCAAGTCAGAGCCTCTTATATAGTAATGGCAAAACCTATTTGAGTTGGTTATTTCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTG GTGTCTACACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTACTGCGTGCAAGGTAGACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0050] The use of the above-mentioned human FAM227A targeting monoclonal antibody in the preparation of a product for detecting the expression level of FAM227A protein.

[0051] The use of the above-mentioned human FAM227A-targeting monoclonal antibody in the preparation of colorectal cancer diagnostic products.

[0052] Furthermore, in the above application, the FAM227A is FAM227A in tumor tissue or cells.

[0053] A human FAM227A-targeting monoclonal antibody composition comprises the aforementioned human FAM227A-targeting monoclonal antibody 8#; and another human FAM227A-targeting monoclonal antibody 2#, the amino acid sequences of the heavy chain variable region HV and the light chain variable region LV of which are as follows:

[0054] The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 5:

[0055] EVKLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRTKPNDHATYSAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYFCATGFFTYWGQGTLVTVSA.

[0056] The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO: 6:

[0057] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTSLIGGTHNRTPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNQWVFGGGTKLTVL。

[0058] The nucleotide sequence encoding the heavy chain variable region HV is shown in SEQ ID NO:7:

[0059] GAGGTCAAACTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAACCAAACCTAATGATCATGCAACATACTCTGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGGCATTTATTTCTGTGCCACGGGCTTTTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

[0060] The nucleotide sequence encoding the light chain variable region LV is shown in SEQ ID NO:8:

[0061] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCCACAACCGAACTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCAGTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA。 ​Use of the above antibody composition in preparing a product for detecting the expression level of FAM227A protein.

[0063] The antibody composition is used to prepare diagnostic products for malignant tumors, wherein the malignant tumors are lung cancer, thyroid cancer, breast cancer, colorectal cancer, prostate cancer, liver cancer, bile duct cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, kidney cancer, gastric cancer, nasopharyngeal cancer, and bladder cancer.

[0064] Furthermore, in the above application, the FAM227A is FAM227A in peripheral blood.

[0065] Example 1: Preparation of monoclonal antibodies targeting human FAM227A

[0066] (1) Expression and purification of recombinant human FAM227A

[0067] The FAM227A DNA fragment was amplified by PCR and digested with pET-28a using EcoRI and HindIII endonucleases, respectively. The mixture was then ligated using T4 ligase. The ligation product was transformed into competent Escherichia coli DH5α cells, and clones were screened. The FAM227A / pET-28a plasmid, identified by DNA sequencing, was transformed into Escherichia coli Rosetta DE3 cells, and the recombinant protein was expressed by induction with isopropyl-β-D-thiogalactose (IPTG). Inclusion bodies were collected and renatured.

[0068] Further, the cells were resuspended in phosphate buffer (pH = 8.0, 100 mM) containing 0.5% Triton X-100, lysozyme was added to a concentration of 100 μg / mL, incubated at 30°C for 15 min, ultrasonically crushed for 15 s / time for 20 times, centrifuged at 10,000 g for 10 min, and the supernatant was discarded; the suspension was resuspended in phosphate buffer (pH = 8.0, 100 mM) containing 0.5% Triton X-100, centrifuged at 10,000 g for 10 min, and the supernatant was discarded; the precipitate was weighed, the inclusion body solubilization solution was added to a concentration of 10 mg / mL, pipetted and mixed, incubated at room temperature for 15 min, centrifuged at 10,000 g for 10 min, the supernatant was collected, and dialyzed twice at 4°C for 3 h each against phosphate buffer (pH = 8.0, 50 mM) containing 0.01% dithiothreitol (DTT); the suspension was dialyzed against phosphate buffer (pH = 8.0, 50 mM) containing 1 The protein was dialyzed against phosphate buffer (pH = 8.0, 50 mM) with 0.2 mM reduced glutathione and 0.2 mM oxidized glutathione at 4°C overnight, purified by nickel column, and eluted with imidazole (pH = 8.0, 300 mM). The eluate containing the FAM227A recombinant protein was collected, dialyzed with 30% glycerol, and stored at -80°C. A portion was used for SDS-PAGE electrophoresis to determine purity. The results are shown in the table. Figure 1 , Figure 1 The figure shows the staining results of recombinantly expressed FAM227A fusion protein.

[0069] (2) Animal immunization, antibody serum acquisition and antigen stimulation

[0070] The purified FAM227A recombinant protein was mixed with Freund's complete adjuvant in a 1:1 ratio and used for the first subcutaneous multi-point immunization of Balb / c mice at a dose of 100 μg / mouse. 21 days later, the FAM227A recombinant protein was mixed with Freund's incomplete adjuvant in a 1:1 ratio and used for subsequent immunization at a dose of 50 μg / mouse for a total of 6 rounds, with an interval of 14 days between each round.

[0071] Furthermore, 50 μl of blood was collected from the submandibular vein of Balb / c mice 7 days after the fourth and sixth immunizations, and the blood was centrifuged at 10,000 g for 5 min. The supernatant was collected for subsequent titer determination.

[0072] Furthermore, mice with high serum antibody titers were intraperitoneally injected with 100 μg / mouse FAM227A recombinant protein for antigen stimulation.

[0073] (3) Cell fusion, hybridoma cell culture and limiting dilution cloning

[0074] Three days after antigen challenge, mice were sacrificed by cervical dislocation. Spleens were isolated and rinsed three times with IMEM containing 5X antibiotics. The spleens were then lavaged with 1X PBS until their color changed from dark red to off-white. The lavage fluid was collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and 5 ml of IMEM was added. The cells were resuspended and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in IMEM and counted to obtain a splenocyte suspension.

[0075] Furthermore, SP2 / 0 cells in the logarithmic growth phase were obtained, digested, and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 15 ml of prewarmed IMEM medium and centrifuged at 1500 rpm for 5 minutes. This process was repeated once. The supernatant was discarded, and the cell pellet was resuspended in IMEM medium. Cell viability and death were determined by trypan blue staining. After counting, the SP2 / 0 cells were mixed with spleen cells at a ratio of 1:10 and centrifuged at 1500 rpm for 5 minutes. The supernatant was aspirated, and 1 ml of prewarmed 50% PEG (MW 4000) was carefully added along the wall of the plate. The plate was incubated at 37°C for 1 minute. 10 ml of prewarmed IMEM medium was slowly added along the wall of the plate, and the plate was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in prewarmed DMEM medium. The hybridoma cell suspension was then plated onto a 96-well plate. Six hours later, 100 μL of 2X HAT medium was added to each well to achieve cell fusion. Ten days later, the cell culture supernatant was collected for analysis.

[0076] (4) Antibody screening and purification

[0077] 293T cells were infected with a FAM227A-overexpressing lentivirus to construct a cell line stably overexpressing FAM227A. When the cell density reached approximately 90%, the cells were harvested and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the precipitate was collected. The cell pellet was resuspended in 1 mL of 1X PBS and centrifuged again at 1000 rpm for 5 min. The supernatant was discarded and the precipitate was collected. RIPA lysis buffer containing cocktail protease inhibitors was added to the pellet and lysed on ice for 30 min. The pellet was centrifuged at 15,000 g for 15 min at 4°C, and the supernatant was collected to obtain the eukaryotic protein overexpressing FAM227A.

[0078] Dilute FAM227A recombinant protein or overexpressed FAM227A eukaryotic protein in coating buffer to a concentration of 10 μg / ml. Coat the reaction wells with 100 μl / well, seal the membrane, and incubate at 4°C overnight. The next day, tap dry the blocking buffer, add 100 μl of 5% newborn calf serum (prepared in TBST) to each well, block at 37°C for 1 hour, discard the supernatant, and tap dry. Using a multichannel pipette, pipette 30 μl of cell culture supernatant into the sample wells of the ELISA plate according to the order of clones in the 96-well plate. Fill each well with blocking buffer to 100 μl, and incubate at 37°C for 60 minutes. Wash the plate four times with 1X TBST, tap dry, and add secondary antibody for incubation at 37°C for 30 minutes. Wash the plate four times with 1X TBST, tap dry, and add TMB colorimetric solution for 10 minutes at room temperature. The colorimetric reaction is terminated by adding stop solution. Measure the absorbance at OD450 with a microplate reader and select the monoclonal wells with higher OD450 positive values ​​for subsequent eukaryotic screening. The process and results are shown in Figure 2 .

[0079] Furthermore, the monoclonal cell suspension obtained by the above screening was subjected to the in vivo induction method in mice to collect ascites, and the antibody was purified and identified by Protein A column chromatography. The purified monoclonal antibody was then concentration-measured, aliquoted, and frozen.

[0080] Finally, three monoclonal antibodies targeting human FAM227A were obtained, numbered: 2#, 5#, and 8#.

[0081] Example 2: ELISA detection of human FAM227A monoclonal antibody titer and antibody pairing

[0082] Dilute FAM227A recombinant protein to a concentration of 10 μg / ml in coating buffer, and coat the reaction wells with 100 μl / well. Seal the plate and incubate overnight at 4°C. The next day, tap dry the blocking buffer, add 100 μl of 5% newborn calf serum (prepared in TBST) to each well, and block at 37°C for 1 hour. Discard the supernatant and tap dry. Add 100 μl / well of the above-mentioned human FAM227A monoclonal antibody at various dilutions (from 1:200 to 1:25,600) and incubate at 37°C for 60 minutes. Wash the plate four times with 1X TBST and tap dry. Add the secondary antibody and incubate at 37°C for 30 minutes. Wash the plate four times with 1X TBST and tap dry. Add TMB colorimetric solution and incubate at room temperature for 10 minutes. Stop the colorimetric reaction with stop solution. The absorbance value was measured at OD450 using a microplate reader, and the titers of the three human FAM227A monoclonal antibodies were 2# (1:6400), 5# (1:800), and 8# (1:12800), respectively (the strain numbers are the strain numbers finally screened in Example 1, see Figure 2 ), see results Figure 3 .

[0083] Furthermore, two strains of human FAM227A monoclonal antibodies were diluted to 100 μl / well in coating buffer to coat the reaction microwells, and the membrane was sealed and incubated at 4°C overnight. The next day, the blocking solution was patted dry, and 100 μl of 5% newborn calf serum (prepared with TBST) was added to each well, blocked at 37°C for 1 hour, the supernatant was discarded, and the wells were patted dry. 100 μl / well of the other four human FAM227A monoclonal antibodies except the coating antibody were added, and incubated at 37°C for 60 min. After washing the plate 4 times with 1X TBST, pat dry, add the secondary antibody and incubate at 37°C for 30 min. After washing the plate 4 times with 1X TBST, pat dry, add TMB colorimetric solution, react at room temperature for 10 min, and add the stop solution to terminate the colorimetric reaction. The results showed that among the three human FAM227A monoclonal antibodies mentioned above, 2# and 8# can be preferably used for antibody pairing, see the results. Figure 4 .

[0084] Example 3: Western Blot Detection of Specificity and Sensitivity of Human FAM227A Monoclonal Antibody

[0085] FAM227A was stably transfected into 293T cells, and total protein from overexpression and control cells was extracted. The two human FAM227A monoclonal antibodies mentioned above were used as primary antibodies (1:1000 dilution) for overnight incubation. Finally, the expression of FAM227A in 293T-FAM227A cells was detected by chemiluminescence. The results showed that both human FAM227A monoclonal antibodies mentioned above could be used to detect the expression level of human FAM227A by Western Blot. Among them, 8# had the strongest affinity and the best specificity. See Results Figure 5 A.

[0086] Furthermore, the total protein of 293T cells overexpressing FAM227A was added / not added with the two human FAM227A monoclonal antibodies mentioned above for antibody blocking / not blocking, and the corresponding two human FAM227A monoclonal antibodies were used as primary antibodies (1:1000 dilution) for overnight incubation. Finally, the expression of FAM227A in 293T-FAM227A was detected by chemiluminescence. The results showed that the two human FAM227A monoclonal antibodies mentioned above had good specificity. See results Figure 5 B.

[0087] Furthermore, total protein was extracted from the stable colorectal cancer cell lines HT29-FAM227A and SW480-FAM227A that overexpress FAM227A. The cells were incubated with the two human FAM227A monoclonal antibodies at different concentrations (1:1000, 1:2000, and 1:4000) overnight. Finally, the expression of FAM227A in HT29-FAM227A and SW480-FAM227A was detected by chemiluminescence. The results showed that the optimal dilution concentration of antibody #8 was 1:1000, and the optimal dilution concentration of antibody #2 was 1:2000. See Results Figure 5 C.

[0088] Example 4: Application of Monoclonal Antibodies Targeting Human FAM227A in Western Blot

[0089] The expression level of FAM227A in various tumor cell lines was detected by Western Blot using the 8# antibody with strong specificity and high affinity. The results showed that FAM227A was highly expressed in colorectal cancer cells LOVO, RKO, and HCT116. See results Figure 6 A.

[0090] Furthermore, the expression levels of FAM227A in tumor, adjacent tissues and normal tissues of 10 pairs of colorectal cancer patients were detected by Western Blot. The results showed that the expression level of FAM227A in tumor tissues was reduced. Figure 6 B.

[0091] Example 5: Application of human FAM227A-targeted monoclonal antibody in serological detection of tumor patients

[0092] A total of 427 preoperative peripheral blood specimens were collected from patients who were first diagnosed with tumors (lung cancer, thyroid cancer, breast cancer, colorectal cancer, prostate cancer, liver cancer, bile duct cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, kidney cancer, gastric cancer, nasopharyngeal cancer, and bladder cancer) and underwent regular surgical treatment at the Second Affiliated Hospital of Zhejiang University between March 2021 and May 2021, and 200 peripheral blood specimens were collected from healthy people undergoing physical examinations. After peripheral blood specimens were collected, they were allowed to stand vertically at room temperature for 20 minutes and centrifuged at 1500 g for 10 minutes at room temperature. Serum was collected and the expression of FAM227A in serum was detected by ELISA sandwich method (8# antibody coating and 2# antibody as primary antibody co-incubation). Finally, the expression spectrum of FAM227A in the serum of patients with common tumors was drawn (see results). Figure 7 A.

[0093] Furthermore, the ROC curve method was used to determine the cut-off value of FAM227A in serum to evaluate its clinical diagnostic value (AUC = 0.71, cut-off value 0.096), see Results Figure 7 B.

[0094] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0095] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A monoclonal antibody targeting human FAM227A, characterized in that: Designated as 8#, it includes the heavy chain variable region HV and the light chain variable region LV; The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 1; The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO:

2.

2. A nucleotide sequence encoding the monoclonal antibody targeting human FAM227A according to claim 1, characterized in that: The nucleotide sequence encoding the heavy chain variable region HV is shown in SEQ ID NO: 3; The nucleotide sequence encoding the light chain variable region LV is shown in SEQ ID NO:

4.

3. Use of the monoclonal antibody targeting human FAM227A as claimed in claim 1 in the preparation of a product for detecting the expression level of FAM227A protein.

4. The use according to claim 3, characterized in that: The FAM227A is FAM227A in tumor tissues or cells.

5. The use according to claim 4, characterized in that: The tumor is a colorectal cancer tumor.

6. A monoclonal antibody composition targeting human FAM227A, characterized in that: The invention comprises the human FAM227A-targeting monoclonal antibody as claimed in claim 1, named 8#; and another human FAM227A-targeting monoclonal antibody, named 2#; the amino acid and nucleic acid sequences of the heavy chain variable region HV and the light chain variable region LV of 2# are as follows: The amino acid sequence of the heavy chain variable region HV is shown in SEQ ID NO: 5; The amino acid sequence of the light chain variable region LV is shown in SEQ ID NO: 6; The nucleotide sequence encoding the heavy chain variable region HV is shown in SEQ ID NO: 7; The nucleotide sequence encoding the light chain variable region LV is shown in SEQ ID NO:

8.

7. Use of the antibody composition according to claim 6 in the preparation of a product for detecting the expression level of FAM227A protein.

8. The use according to claim 7, characterized in that: The FAM227A is FAM227A in peripheral blood.

9. The use according to claim 8, characterized in that: The peripheral blood is the peripheral blood of a patient with a malignant tumor.

10. The use according to claim 9, characterized in that: The malignant tumor is lung cancer, thyroid cancer, breast cancer, colorectal cancer, prostate cancer, liver cancer, bile duct cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, kidney cancer, stomach cancer, nasopharyngeal cancer or bladder cancer.

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