HCBP6 monoclonal antibody and its application

Abstract

This invention relates to the field of biomedical technology, specifically to HCBP6 monoclonal antibodies and their applications. This invention utilizes bioinformatics databases and online tools to analyze the structure of the HCBP6 protein, screening for highly immunogenic HCBP6 antigen peptides. Antibody 1 and Antibody 2 are obtained through immunization. The combination of Antibody 1 and Antibody 2 is used for the detection of HCBP6, exhibiting high sensitivity and good detection results. It has significant development and application value and long-term implications for the auxiliary diagnosis of fatty liver.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to HCBP6 monoclonal antibody and its applications. Background Technology

[0002] Hepatitis C virus (HCV), a major pathogen of transfusion-transmitted non-A, non-B hepatitis (NANB) H, is a single-stranded positive-sense RNA virus belonging to the Flaviviridae genus. Therefore, the pathogenesis of HCV infection differs significantly from that of hepatitis B virus (HBV), a DNA virus. The interaction between HCV and hepatocytes is likely a crucial part of the HCV infection pathogenesis. The HCV core protein influences the gene expression profile of infected hepatocytes. Simultaneously, the HCV core protein binds to itself to form homodimer structures and can also bind to other types of proteins in hepatocytes to form heterodimers or multimers, severely interfering with signal transduction in hepatocytes. Besides causing acute and chronic viral hepatitis, liver fibrosis, and hepatocellular carcinoma (HcC), HCV infection can also lead to hepatic steatosis, B-cell lymphoma, and cryoglobulinemia.

[0003] Fatty liver disease is named by Rokitansky based on the excessive lipid deposition in the liver parenchymal cells of obese patients. It is classified into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) based on the presence or absence of a history of long-term heavy alcohol consumption. NAFLD refers to a clinicopathological syndrome characterized by excessive fat deposition in hepatocytes, without a history of heavy alcohol consumption (equivalent to ethanol intake of <30g / day for men and <20g / day for women) or other clearly defined liver-damaging factors (hepatitis viruses, autoimmune diseases, drugs, etc.).

[0004] In recent years, due to changes in people's dietary structure and lifestyle, the global incidence of NAFLD has continued to rise. However, the pathogenesis of NAFLD remains unclear, and there is no ideal treatment. Hepatitis C virus core-binding protein 6 (HCBP6) is one of the proteins that can bind to the core protein of hepatitis C virus, screened using yeast two-hybrid technology. HCBP6 plays an important role in inhibiting the occurrence of fatty liver disease and glucose and lipid metabolism.

[0005] Analyzing the differences in serum HCBP6 expression between patients with fatty liver disease and healthy controls lays the foundation for research on HCBP6 as a therapeutic target for NAFLD. There is a severe shortage of effective raw materials for detecting HCBP6 levels both domestically and internationally, making the development of a raw material for detecting hepatitis C virus core protein-binding protein 6 (HCBP6) an urgent priority. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an HCBP6 monoclonal antibody and its application.

[0007] This invention provides an HCBP6 monoclonal antibody, comprising antibody 1 and / or antibody 2.

[0008] The heavy chain variable region of antibody 1 has at least one of the amino acid sequences shown in SEQ ID NO:1, 2 or 3;

[0009] The light chain variable region of antibody 1 has at least one of the amino acid sequences shown in SEQ ID NO:4, 5 or 6;

[0010] The heavy chain variable region of antibody 2 has at least one of the amino acid sequences shown in SEQ ID NO:9, 10 or 11;

[0011] The light chain variable region of antibody 2 has at least one of the amino acid sequences shown in SEQ ID NO:12, 13 or 14.

[0012] Furthermore, the HCBP6 monoclonal antibody described in this invention,

[0013] The heavy chain variable region of antibody 1 has an amino acid sequence as shown in SEQ ID NO:7;

[0014] The light chain variable region of antibody 1 has an amino acid sequence as shown in SEQ ID NO:8;

[0015] The heavy chain variable region of antibody 2 has an amino acid sequence as shown in SEQ ID NO:15;

[0016] The light chain variable region of antibody 2 has an amino acid sequence as shown in SEQ ID NO:16.

[0017] Furthermore, the HCBP6 monoclonal antibody described in this invention,

[0018] The heavy chain constant region of antibody 1 is mouse IgG1 subtype; the light chain constant region is mouse κ1 type.

[0019] The heavy chain constant region of antibody 2 is mouse IgG1 subtype; the light chain constant region is mouse κ1 type.

[0020] The antibodies described in this invention were obtained by screening 38 antibodies from immunized mice after antigen screening. These 38 antibodies were individually coated onto an NC membrane or labeled with colloidal gold and then combined and arranged in 1444 possible combinations. Colloidal gold test strips prepared from these 1444 combinations were used for the detection of quality control samples and clinical samples. Twenty combinations showed good detection results, with antibody number 38 labeled and antibody number 9 coated showing the best performance.

[0021] This invention provides an antigenic polypeptide for immunizing to obtain the HCBP6 monoclonal antibody described herein, said antigenic polypeptide having at least one of the amino acid sequences shown in I) to III) below:

[0022] I) An amino acid sequence as shown in SEQ ID NO:18; or

[0023] II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0024] III) An amino acid sequence having at least 90% sequence identity with the amino acid sequence described in (I) or (II).

[0025] The HCBP6 antigen peptide described in this invention was obtained by analyzing protein results using bioinformatics databases and online tools. Experimental results show that, compared with other sequences, the HCBP6 antigen peptide with the amino acid sequence shown in SEQ ID NO:18 has the best immunogenicity, higher serum titer after immunization, and is more conducive to antibody production.

[0026] The present invention provides a recombinant antigen comprising the antigenic polypeptide and carrier protein described herein.

[0027] Furthermore, the carrier protein includes at least one of hemocyanin, bovine serum albumin, and / or chicken ovalbumin.

[0028] In a specific embodiment of the present invention, the carrier protein is hemocyanin, which serves as an antigen carrier protein to enhance the immunogenicity of the HCBP6 antigenic polypeptide.

[0029] This invention provides biological materials comprising at least one of the following: i) to iv)

[0030] i) The HCBP6 monoclonal antibody, antigen polypeptide, or recombinant antigen nucleic acid described in this invention;

[0031] ii) A recombinant vector containing a vector backbone and nucleic acids as shown in i);

[0032] iii) Transfect or transform host cells with the recombinant vector shown in ii);

[0033] iv) Culture products obtained by culturing host cells as shown in iii).

[0034] In this invention, the nucleic acid can be DNA, RNA, cDNA, or PNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0035] In this invention, the recombinant vector, the source of the vector skeleton includes plant vectors, animal vectors, bacterial vectors, fungal vectors, bacteriophage vectors, or viral vectors, etc., and this invention does not limit the source.

[0036] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operablely linked coding gene in a specific host organism.

[0037] This invention provides a host cell that is transfected or transformed with the recombinant vector as described in this invention.

[0038] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0039] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit the sources.

[0040] This invention provides a method for preparing the HCBP6 monoclonal antibody, the antigenic peptide, or the recombinant antigen, which obtains the HCBP6 monoclonal antibody, antigenic peptide, or recombinant antigen by culturing host cells in biological materials as described in this invention.

[0041] The present invention provides labeled antibodies, which include the HCBP6 monoclonal antibody and labeling described herein.

[0042] Furthermore, the markers include chemical markers and / or biological markers; the biological markers include at least one of biotin, avidin, or enzymes; the chemical markers include isotopes and / or chemical drugs.

[0043] The present invention provides a conjugate comprising the HCBP6 monoclonal antibody and conjugation medium described herein.

[0044] Furthermore, the coupling medium includes nanoparticles, including gold nanoparticles, but this invention does not limit the scope of the invention.

[0045] This invention provides the use of at least one of the following (A) to (F) in the preparation of products for HCBP6 detection or fatty liver diagnosis:

[0046] A) The HCBP6 monoclonal antibody described in this invention;

[0047] B) The antigenic polypeptide described in this invention;

[0048] C) The recombinant antigen described in this invention;

[0049] D) The biomaterials described in this invention;

[0050] E) The labeled antibody described in this invention;

[0051] F) The coupling compound described in this invention.

[0052] This invention provides a product for HCBP6 detection or fatty liver diagnosis, the raw materials of which include at least one of the following: a) to d)

[0053] a) The HCBP6 monoclonal antibody described in this invention;

[0054] b) The biomaterials described in this invention;

[0055] c) The labeled antibody described in this invention;

[0056] d) The coupling compound described in this invention.

[0057] The product described in this invention includes a test kit and / or a test strip;

[0058] The kit may also include solvents or solutions for auxiliary detection, positive controls, negative controls, etc. In practical applications, the kit described in this invention can be an enzyme-linked immunosorbent assay (ELISA) kit;

[0059] The test strip also includes a carrier matrix, such as an NC membrane, a gold label pad, an absorbent pad, and a sample pad. Specifically, the test strip of the present invention can be a colloidal gold test strip.

[0060] This invention provides a method for HCBP6 detection or fatty liver diagnosis, which involves testing a sample using the product described in this invention.

[0061] Furthermore, the sample includes serum and / or plasma.

[0062] This invention uses bioinformatics databases and online tools to analyze the structure of HCBP6 protein, screens out HCBP6 antigen peptides with high immunogenicity, and obtains antibody 1 and antibody 2 through immunization. The combination of antibody 1 and antibody 2 is used for the detection of HCBP6, which has high detection sensitivity and good detection effect, and has high development and application value. It has long-term significance in the field of auxiliary diagnosis of fatty liver. Attached Figure Description

[0063] Figure 1 Protein structure analysis;

[0064] Figure 2 HPLC detection of synthesized antigen peptides;

[0065] Figure 3 MS detection of synthesized antigen peptides;

[0066] Figure 4 Tail blood titer detection;

[0067] Figure 5 The colloidal gold was detected. Detailed Implementation

[0068] This invention provides an HCBP6 monoclonal antibody and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0069] The amino acid sequence of CDR1 in the heavy chain of antibody 1 (38# antibody) is: NYGMN (SEQ ID NO:1);

[0070] The amino acid sequence of CDR2 in the heavy chain of antibody 1 (38# antibody) is: WINTYTGEPTYADDFKG (SEQ ID NO:2);

[0071] The amino acid sequence of CDR3 in the heavy chain of antibody 1 (38# antibody) is: LDY (SEQ ID NO:3);

[0072] The amino acid sequence of CDR1 in the light chain of antibody 1 (38# antibody) is: SASSVSYMH (SEQ ID NO:4);

[0073] The amino acid sequence of CDR2 in the light chain of antibody 1 (38# antibody) is: DTSKLAY (SEQ ID NO:5);

[0074] The amino acid sequence of CDR3 in the light chain of antibody 1 (38# antibody) is: QQWGSNPWT (SEQ ID NO:6);

[0075] The amino acid sequence of the heavy chain variable region of antibody 1 (antibody 38#) is: MDWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTN YGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAY LQINNLKNEDMATYFCVRLDYWGQGTTLTVSS (SEQ ID NO:7);

[0076] The amino acid sequence of the light chain variable region of antibody 1 (antibody 38#) is: MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSASPGEKVTMTCSASSSVSY MHWYQQKSGTSPKRWIYDTSKLAYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWGSNPWTFGGGTKLEIK (SEQ ID NO:8);

[0077] The amino acid sequence of CDR1 in the heavy chain of antibody 2 (9# antibody) is: SYGMS (SEQ ID NO: 9);

[0078] The amino acid sequence of CDR2 in the heavy chain of antibody 2 (9# antibody) is: SISSGGSYTYYPDSVKG (SEQ ID NO:10);

[0079] The amino acid sequence of CDR3 in the heavy chain of antibody 2 (9# antibody) is: HYDYDY (SEQ ID NO:11);

[0080] The amino acid sequence of CDR1 in the light chain of antibody 2 (9# antibody) is: KASQDINTYLS (SEQ ID NO:12);

[0081] The amino acid sequence of CDR2 in the light chain of antibody 2 (antibody 9#) is: RANRLVD (SEQ ID NO:13);

[0082] The amino acid sequence of CDR3 in the light chain of antibody 2 (9# antibody) is: LQYDEFPWT (SEQ ID NO:14);

[0083] The amino acid sequence of the heavy chain variable region of antibody 2 (9# antibody) is: MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSSYG MSWVRQTPDKRLEWVASISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQM SSLKSEDTAMYYCASHYDYDYWGQGTSVTVSS (SEQ ID NO:15);

[0084] The amino acid sequence of the light chain variable region of antibody 2 (9# antibody) is: MRTPAQFLGILLLWFPGIRCDINMTQSPSSMYASLGERLTITCKASQDINTYL SWFQQKPGKSPKSLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPWTFGGGTKLEIK (SEQ ID NO:16);

[0085] The amino acid sequence of HCBP6 is: METSAPRAGSQVVATTARHSAAYRADPLRVSSRDKLTEMAASSQGNFEGNFESLDLAEFAKKQPWWRKLFGQESGPSAEKYSVATQLFIG GVTGWCTGFIFQKVGKLAATAVGGGFFLLQLANHTGYIKVDWQRVEKDMKKAKEQLKIRKSNQIPTEVRSKAEEVVSFVKKNVLVTGGFFGGFLLGMAS(SEQ ID NO:17);

[0086] The amino acid sequence of polypeptide 2 is: YRADPLRVSSRDKLTEMAASSQGNFEGNFESLDLAEFAKKQPWWRKLFGQESGPSAEKYS (SEQ ID NO:18);

[0087] The amino acid sequence of polypeptide 3 is: CNHTGYIKVDWQRVEKDMKKAKEQLKIRKSNQIPT (SEQ ID NO:19);

[0088] The nucleotide sequence of the heavy chain variable region of Antibody 1 (38# antibody) is: atggattggctgtggaacttgctattcctgatggcagctgcccaaagtgcccaagcacagatccagttggtgcagtctggacctgagttgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgctgatgacttcaagggacggtttgccttctctctggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacatggctacatatttctgtgtaagactggactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO:20);

[0089] The nucleotide sequence of the light chain variable region of Antibody 1 (38# antibody) is: atggattttcaagtgcagattttcagcttcctgctaatcagtgcctcagtcataatatccagaggacaaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacttccaaactggcttatggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggggtagtaatccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO:21);

[0090] The nucleotide sequence of the heavy chain variable region of antibody 2 (antibody 9#) is: atgaacttcgggctcagcttgattttccttgccctcattttaaaaggtgtccagtgtgaggtgcagctggtggagtctggggggag acttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctatggcatgtcttgggttcgccagactccagacaagaggctg gagtgggtcgcaagcattagtagtggtggtagttacacctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtattt gcaaatgagcagtctgaagtctgaggacacagccatgtattactgtgcaagccactatgattacgactactggggtcaaggaacctcagtcaccgtctcctca(SEQ IDNO:22);

[0091] The nucleotide sequence of the light chain variable region of antibody 2 (antibody 9#) is: atgaggacccctgctcagtttcttggcatcttgttgctctggtttccaggtatccgatgtgacatcaacatgacccagt ctccatcttccatgtatgcatctctaggagagagactcactatcacttgcaaggcgagtcaggacattaatacctatttaagctggttccagcagaaaccag ggaaatctcctaagtccctgatctatcgtgcaaacagattggtagatggggtcccatcaaggttcagtggcagtggatctgggcaagattattctctcacca tcagcagcctggaatatgaagatatgggaatttattattgtctacagtatgatgagtttccgtggacgttcggtggaggcaccaagctggaaatcaaa(SEQ ID NO:23).

[0092] The reagents and consumables used in this invention are all commercially available products that can be purchased on the market.

[0093] The present invention will be further illustrated below with reference to the embodiments:

[0094] Example 1: Antigenic polypeptide of hepatitis C virus core protein binding protein 6 (HCBP6) and its application

[0095] I. Antigenic polypeptide of hepatitis C virus core protein binding protein 6 (HCBP6)

[0096] The amino acid sequence of hepatitis C virus core protein binding protein 6 (HCBP6) is shown in SEQ ID NO:1. The protein structure was analyzed using bioinformatics databases and online tools to predict relevant parameters such as hydrophobicity, surface accessibility, and antigenicity. Figure 1 Following the principles of peptide design and based on the requirements of immunogens, peptides 2 and 3, with amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3, were selected.

[0097] The polypeptide 2 (SEQ ID NO:2) antigen polypeptide was obtained by a commercial company through chemical synthesis and analyzed by HPLC. Figure 2 ) and MS ( Figure 3 The purity and molecular weight of the synthesized antigen peptides were found to fully meet the design requirements. The antigen peptide 3 (SEQ ID NO:3) was obtained by a commercial company through chemical synthesis. The molecular weight of SEQ ID NO:3 was 3.5KD, and it was coupled with KLH protein (Abcam ab285712).

[0098] II. Immunogens of Hepatitis C Virus Core Protein Binding Protein 6 (HCBP6)

[0099] Appropriate amounts of HCBP6-synthesized immunogens SEQ ID NO:2, SEQ ID NO:3 protein, and SEQ ID NO:3-conjugated KLH protein were emulsified with an equal volume of Freund's complete adjuvant (Sigma catalog number: F5881-10X10ml). These were then subcutaneously injected into the axillary and abdominal sites of Balb / C mice (Hunan Slack Jingda Experimental Animal Co., Ltd., 6 mice aged 6-8 weeks, 0.5 mL / mouse). Six mice were immunized with each immunogen. A second intraperitoneal booster immunization was administered 21 days later, followed by a third intraperitoneal booster immunization 14 days after the second immunization (for the second and third immunizations, the immunogen dosage was halved and emulsified with an equal volume of Freund's incomplete adjuvant, 0.5 mL / mouse / administration). Tail blood was collected after the three booster immunizations for immunotiter testing. Mice whose titers met the fusion requirements were given a final intraperitoneal immunization 3 days before fusion, using the same antigen dose as the initial immunization, 0.5 mL / mouse.

[0100] III. Tail Blood Serum Titer Detection

[0101] Prepare CB buffer (1.69 g sodium carbonate, 2.86 g sodium bicarbonate) and bring the volume to 1 L, setting the pH to 9.51. Dilute the immunogen with the above CB buffer to a final concentration of 2 μg / mL, and add 0.1 mL / well to a 96-well ELISA plate. Incubate overnight at 4°C. The next day, wash the ELISA plate twice with 1×PBST, blot dry, add 0.2 mL / well of 1×PBS containing 1% BSA, block at 37°C for 1 hour, blot dry, and use for detection.

[0102] The dilution factors of mouse tail blood serum were as follows: row A - 10-fold, row BC - 100-fold, row DE - 1000-fold, row F - 10,000-fold, row G - 100,000-fold, and row H - blank. 1#-6# represent the numbers of the 6 mice immunized with each immunogen. 0.1 mL / well of serum was incubated in the reaction plate at 37°C for 1 hour. The plate was then washed 6 times with 1×PBST, patted dry, and 0.1 mL / well of 1.5K diluted horseradish peroxide-labeled goat anti-mouse (SIGMA) was added. After incubation at 37°C for 1 hour, the plate was washed 6 times with 1×PBST. 0.1 mL / well of ELISA development solution (containing 0.015% (M / V) 3,3',5,5'-tetramethylbenzidine, 0.03% (V / V) hydrogen peroxide, and 0.16% (M / V) citrate buffer) was added. After development at 37°C for 10 minutes, 1M sulfuric acid solution was added to stop the reaction. The absorbance at 450 nm was measured. The results are shown in Tables 1 and 2, with the dilution factor on the x-axis and the average absorbance on the y-axis, after subtracting the blank. Figure 4 .

[0103] Table 1. Immunological effects of peptide 2 and peptide 3

[0104]

[0105]

[0106] Table 2. Immunological effects of peptide 3-KLH

[0107]

[0108] Figure 4 This is a serum titer curve obtained using ELISA. The curve visually reflects the overall serum titer situation, as shown in Tables 1 and 2. Figure 4 It can be determined that, among the synthesized peptides, only peptide 2 (SEQ ID NO:2) showed good immunogenicity, meeting the immunogenic requirements for its monoclonal antibody development. Peptide 3 (SEQ ID NO:3), whether on its own or conjugated with KLH protein, did not meet the experimental requirements for immunogenicity; therefore, peptide 2 was primarily used in subsequent experiments.

[0109] IV. Preparation of Monoclonal Antibodies

[0110] 4.1 Preparation of feeder cells

[0111] One to two days before fusion, macrophages were aseptically extracted from the peritoneal cavity of Kunming rats, placed in HAT medium, counted, and the cell concentration was adjusted to 102. 4 ~10 5 Add 0.1 mL of the sample to a 96-well cell culture plate and incubate at 37°C in a 5% CO2 incubator for later use.

[0112] 4.2 Preparation of SP2 / 0 cells

[0113] SP2 / 0 cells cultured to 70%–90% were prepared into a suspension and counted.

[0114] 4.3 Preparation of spleen cells

[0115] Three to five days after the final immunization, spleen cells were removed under sterile conditions, prepared into a suspension, and counted.

[0116] 4.4 Cell Fusion and Culture

[0117] Mix the above-mentioned spleen cells with SP2 / 0 cells at a ratio of (2-5) / 1, centrifuge at 1500 rpm for 5 min, discard the supernatant, thoroughly disperse the cells, slowly add 1 mL of fusion agent (SIGMA), let stand for 1 min, then slowly add 30-50 mL of basal culture medium incubated at 37℃, centrifuge at 1200 rpm for 8 min, discard the supernatant. Add an appropriate amount of HAT medium and mix carefully. Add the fused cell suspension to a 96-well cell culture plate containing feeder cells, 0.1 mL / well, and incubate at 37℃ in a 5% CO2 incubator. Observe the cells for 4-7 days, then replace with HT medium.

[0118] 4.5 Cell supernatant detection and limiting dilution

[0119] Cell supernatant was analyzed using the tail blood titer assay. When fused cells were cultured for 8–14 days, 0.1 mL of cell supernatant was added to each well for enzyme immunoassay. OD was selected. 450nm Positive cell wells with an OD value greater than 2.0 were subjected to limiting dilution. Single-clone wells with an OD value greater than 2.0 in the cell supernatant were selected for expansion culture, cell cryopreservation, and ascites fluid preparation.

[0120] 4.6 Ascites preparation

[0121] Male Bacb / C mice aged 6–8 weeks were intraperitoneally injected with 0.5 mL of Freund's complete adjuvant (Sigma product code: F5506-10X10ml) for 7 days, followed by cellular immunization with 500,000–1,000,000 cells per mouse. The growth of ascites fluid was observed and collected after 9–15 days.

[0122] 4.7 Antibody Preparation

[0123] After ascites treatment, antibodies were recovered using a protein A affinity chromatography column. The recovered antibodies were dialyzed and stored in 1×PBS, and antibody concentration was detected using a Thermo Scientific NAN0DR0P ONE.

[0124] 4.8 Reproducibility Experiment of Synthetic Peptides as Immunogens

[0125] Following the method described in this invention, the synthesized polypeptide 2 of this invention was used as an immunogen to immunize 60 mice, resulting in the preparation of 38 monoclonal strains. The concentrations are shown in Table 3 below.

[0126] Table 3. Preparation of monoclonal antibodies

[0127] serial number Cloned ID Antibody concentration (mg / ml) serial number Cloned ID Antibody concentration (mg / ml) serial number Cloned ID Antibody concentration (mg / ml) 1 7E3E9 2.91 14 5C2D8 6.49 27 5C2E3 5.09 2 3F8G8 5.05 15 3F8F6 1.87 28 7B5D3 1.34 3 3F8G3 4.47 16 5C2G7 3.05 29 7D9C9 3.2 4 16H4H9 3.85 17 16D10C3 5.66 30 10G5D9 7.41 5 16F3H7 7.45 18 43G6G8 7.05 31 3F11H9 4.41 6 16A2F3 8.09 19 16H4F1 3.14 32 9F9D6 1.36 7 5G4B6 6.51 20 9F9H8 3.83 33 37C9E4 4.22 8 16F3B7 12 21 3F11C4 2.94 34 10G4E6 2.24 9 35G8A6 3.43 22 14F4A5 8.65 35 7F3C5 4.58 10 7E3D4 1.45 23 10G4G6 3.35 36 37C9H7 11.57 11 40G4F7 2.56 24 16A2B9 6.28 37 3F11H9 7.33 12 10G5E6 5.96 25 16F3D5 8.42 38 14F4G6 10.88 13 5C2C6 12.6 26 3F11H4 11.47

[0128] Table 3 shows that the prepared antibody concentration was good, and the enzyme immunoassay titers of the 38 antibodies developed in this invention were all good. The 38 antibodies were individually coated onto an NC membrane or labeled with colloidal gold and then combined, resulting in 1444 possible arrangements. Colloidal gold test strips prepared from these 1444 arrangements were used for the detection of quality control samples and clinical samples. Among these, 20 combinations showed good detection results, with antibody number 38 labeled and antibody number 9 coated showing the best effect. Figure 5 (Only the best pair is listed here). This demonstrates that the HCBP6 immunogen has good reproducibility in the preparation of hybridoma cells and monoclonal antibodies, and can be applied industrially.

[0129] V. Application of Monoclonal Antibodies

[0130] 5.1 Preparation of colloidal gold test strips

[0131] Coating: Dilute the antibody (antibody number 9) to 0.5-2 mg / ml with 1×PBS containing 2% sucrose, streak it onto an NC membrane, and dry it overnight at 37°C (antibody performance testing, no control line).

[0132] Labeling: Take an appropriate amount of 0.02% colloidal gold, add 10 μL / mL of 0.1M potassium carbonate, mix well, and react at room temperature for 10 min. Add 10 μg / mL of antibody (antibody number 38), mix well, and react at room temperature for 10 min. Add 50 μL / mL of 10% BSA (w / v), react at room temperature for 10 min, centrifuge (12000 rpm, 10 min), and discard the supernatant. Resuspend in 0.01M PB (pH=7.4) containing 3%–5% (w / v) sucrose, 0.5%–3% (w / v) inositol, 1%–2% (w / v) BSA, and 0.1%–0.5% (w / v) Tween-20, to 1 / 3 of the original volume.

[0133] Preparation of gold pads: For each 0.6cm×30cm gold pad, 750μL of colloidal gold suspension was evenly coated and dried overnight at 37℃.

[0134] Sample pad preparation: Soak a 2.8cm×30cm sample pad in 1×PBS containing 0.5% BSA and 0.2% Tween-20, and dry overnight at 37°C.

[0135] Test strip assembly: Assemble the NC membrane, gold label pad, absorbent pad, and sample pad in sequence, cut into 3.5mm strips, and attach the base plate and cover plate. The samples tested by the test strips were: pure water, 1×PBS (zero value), reference standard 100ng / mL, 1μg / mL, 10μg / mL, serum from 3 clinically diagnosed cases of fatty liver (40#, 42#, 38#), and serum from clinically non-fatty liver cases (48#, 31#). The interpretation results are shown in Table 4, and the corresponding color development of the test strips is as follows. Figure 5 .

[0136] According to Table 4 and Figure 5 (Antibody No. 38 is labeled, antibody No. 9 is coated) The colloidal gold detection results show that the color development of the antibody detection quality control product of this invention becomes more and more obvious from low to high values, and the detection of clinical fatty liver indicators is also very clear.

[0137] Table 4. Test Results

[0138]

[0139] Currently, there are no reagent kits developed in my country specifically for HCBP6 protein. This invention provides significant value for the development of HCBP6 detection kits and offers excellent assistance in the diagnosis of fatty liver.

[0140] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An HCBP6 monoclonal antibody, characterized in that, Including antibody 1 and / or antibody 2, The amino acid sequence of CDR1 of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of CDR1 of the light chain variable region of antibody 1 is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

6. The amino acid sequence of CDR1 of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO:10, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

11. The amino acid sequence of CDR1 of the light chain variable region of antibody 2 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

14.

2. The HCBP6 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:7; The amino acid sequence of the light chain variable region of antibody 1 is shown in SEQ ID NO:8; The amino acid sequence of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:15; The amino acid sequence of the light chain variable region of antibody 2 is shown in SEQ ID NO:

16.

3. The HCBP6 monoclonal antibody according to claim 1 or 2, characterized in that, The heavy chain constant region of antibody 1 is mouse IgG1 subtype; the light chain constant region is mouse κ1 type. The heavy chain constant region of antibody 2 is mouse IgG1 subtype; the light chain constant region is mouse κ1 type.

4. A biomaterial, characterized in that, Includes at least one of the following: i) to iii) i) The nucleic acid encoding the HCBP6 monoclonal antibody according to any one of claims 1 to 3; ii) A recombinant vector containing a vector backbone and nucleic acids as shown in i); iii) Transfecting or transforming host cells with the recombinant vector shown in ii); iv) Culture products obtained by culturing host cells as shown in iii).

5. The method for preparing the HCBP6 monoclonal antibody according to any one of claims 1 to 3, characterized in that, The HCBP6 monoclonal antibody is obtained by culturing host cells in the biomaterial as described in claim 4.

6. A labeled antibody, characterized in that, Includes the HCBP6 monoclonal antibody and marker as described in any one of claims 1 to 3.

7. The labeled antibody according to claim 6, characterized in that, The marker is a chemical marker and / or a biological marker; the biological marker is at least one of biotin, avidin, or an enzyme. The chemical markers are isotopes and / or chemical drugs.

8. A coupling, characterized in that, Includes the HCBP6 monoclonal antibody and conjugation medium as described in any one of claims 1 to 3; The coupling medium is colloidal gold.

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