HCBP6 monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an HCBP6 monoclonal antibody and application thereof. Bioinformatics database information and an online tool are adopted to analyze an HCBP6 protein structure, the HCBP6 antigen polypeptide with high immunogenicity is obtained through screening, the antibody 1 and the antibody 2 are obtained through immunization, the combination of the antibody 1 and the antibody 2 is used for HCBP6 detection, the detection sensitivity is high, the detection effect is good, the development and application value is high, and the application prospect is wide. The method has long-term significance in the field of auxiliary diagnosis of fatty liver.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to HCBP6 monoclonal antibodies and applications thereof. Background Art

[0002] Hepatitis C virus (HCV), the main pathogen of transfusion-transmitted non-A, non-B viral hepatitis (NANB)H, is a single-stranded positive-strand RNA virus belonging to the genus Flavivirus. Therefore, the pathogenesis of HCV infection is very different from that of DNA viruses such as hepatitis B virus (HBV). The interaction between HCV and hepatocytes may be an important part of the pathogenesis of HCV infection. HCV core protein affects the gene expression profile in infected hepatocytes. At the same time, HCV core protein combines with itself to form a homodimer structure, and can also combine with other types of proteins in hepatocytes to form heterodimers or multimers, which seriously interfere with signal transduction in hepatocytes. In addition to causing acute and chronic viral hepatitis, liver fibrosis, and hepatocellular carcinoma (Hc)C, HCV infection also includes liver steatosis, B cell lymphoma, cryoglobulinemia, etc.

[0003] Fatty liver disease was named by Rokitansky based on the large amount of lipid deposits in the hepatocytes of the liver tissue of obese patients. It is divided into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) according to whether the patient has a history of long-term heavy drinking. NAFLD refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes in patients without a history of heavy drinking (equivalent ethanol volume <30g / d for men and <20g / d for women) and other clear liver damage factors (hepatitis virus, autoimmunity, drugs, etc.).

[0004] In recent years, due to changes in people's diet and lifestyle, the global incidence of NAFLD has continued to rise, but the pathogenesis of NAFLD has not yet been clarified, and there is no ideal treatment plan. Hepatitis C virus core-binding protein 6 (HCBP6) is one of the proteins that can bind to the hepatitis C virus core protein screened by yeast two-hybrid technology. HCBP6 plays an important role in inhibiting the occurrence of fatty liver disease or glucose and lipid metabolism.

[0005] By analyzing the difference in serum HCBP6 expression between patients with fatty liver disease and healthy controls, the foundation for the study of HCBP6 as a therapeutic target for NAFLD was laid. There is a severe shortage of effective raw materials for detecting HCBP6 levels at home and abroad, and it is urgent to develop a raw material for detecting hepatitis C virus core protein binding protein 6 (HCBP6). Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide HCBP6 monoclonal antibodies and applications thereof.

[0007] The present invention provides a HCBP6 monoclonal antibody, which comprises antibody 1 and / or antibody 2.

[0008] The heavy chain variable region of the 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 the 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 of the present invention,

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

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

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

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

[0017] Furthermore, the HCBP6 monoclonal antibody of the present invention,

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

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

[0020] The antibodies described in the present invention are obtained by screening from 38 antibodies obtained by immunizing mice after antigen screening; the 38 antibodies are individually coated on NC membranes or labeled with colloidal gold and then arranged in combination, with a total of 1444 combinations. The colloidal gold test strips prepared from the 1444 combinations are used for the detection of quality control products and clinical samples, among which 20 combinations have better detection results, among which the effect of labeling with antibody number 38 and coating with antibody number 9 is the best.

[0021] The present invention provides an antigenic polypeptide for obtaining the HCBP6 monoclonal antibody of the present invention by immunization, wherein the antigenic polypeptide has at least one of the amino acid sequences shown in I) to III) below:

[0022] 1), the amino acid sequence shown in SEQ ID NO: 18; or

[0023] II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having 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 polypeptide of the present invention is obtained by screening after analyzing protein results using bioinformatics database information and online tools. The test results show that compared with other sequences, the HCBP6 antigen polypeptide with an amino acid sequence as shown in SEQ ID NO: 18 has the best immunogenicity, a higher serum titer after immunization, and is more conducive to the production of antibodies.

[0026] The present invention provides a recombinant antigen, which comprises the antigen polypeptide of the present invention and a carrier protein.

[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 is used as an antigen carrier protein to improve the immunogenicity of the HCBP6 antigen polypeptide.

[0029] The present invention provides a biomaterial comprising at least one of the following i) to iv):

[0030] i) the HCBP6 monoclonal antibody of the present invention, the antigen polypeptide of the present invention, or the nucleic acid of the recombinant antigen of the present invention;

[0031] ii), a recombinant vector comprising a vector backbone and the nucleic acid as shown in i);

[0032] iii), transfecting or transforming a host cell with the recombinant vector as shown in ii);

[0033] iv) culturing the host cell as shown in iii) to obtain a culture product.

[0034] In the present 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. 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). Nucleic acid can be linear or circular in topology. Nucleic acid can be a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0035] In the present invention, the sources of the recombinant vector and the vector skeleton include plant vectors, animal vectors, bacterial vectors, fungal vectors, phage vectors, or viral vectors, etc., and the present invention does not limit this.

[0036] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences or elements that are essential for the expression of an operably linked coding gene in a specific host organism.

[0037] The present invention provides a host cell which is transfected or transformed with the recombinant vector of the present invention.

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

[0039] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, but the present invention is not limited thereto.

[0040] The present invention provides a method for preparing the HCBP6 monoclonal antibody or the antigenic polypeptide or the recombinant antigen, which comprises culturing host cells in the biomaterial as described in the present invention to obtain the HCBP6 monoclonal antibody or the antigenic polypeptide or the recombinant antigen.

[0041] The present invention provides a labeled antibody, which comprises the HCBP6 monoclonal antibody of the present invention and a label.

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

[0043] The present invention provides a conjugate, which comprises the HCBP6 monoclonal antibody of the present invention and a conjugation medium.

[0044] Furthermore, the coupling medium includes nanoparticles, and the nanoparticles include gold nanoparticles, which is not limited in the present invention.

[0045] The present invention provides the use of at least one of the following A) to F) in the preparation of a product for HCBP6 detection or fatty liver diagnosis:

[0046] A), the HCBP6 monoclonal antibody of the present invention;

[0047] B), the antigenic polypeptide of the present invention;

[0048] C), the recombinant antigen of the present invention;

[0049] D), the biomaterial of the present invention;

[0050] E), the labeled antibody of the present invention;

[0051] F), the conjugate of the present invention.

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

[0053] a), the HCBP6 monoclonal antibody of the present invention;

[0054] b) the biomaterial of the present invention;

[0055] c) the labeled antibody of the present invention;

[0056] d) The conjugate of the present invention.

[0057] The product of the present invention comprises a detection kit and / or a test strip;

[0058] The kit may also include a solvent or solution for auxiliary detection, a positive control substance, a negative control substance, etc. In practical applications, the kit of the present invention may be an enzyme-linked immunosorbent assay kit;

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

[0060] The present invention provides a method for detecting HCBP6 or diagnosing fatty liver, which comprises using the product of the present invention to detect a sample.

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

[0062] The present invention uses bioinformatics database data and online tools to analyze the HCBP6 protein structure, screens and obtains HCBP6 antigen polypeptides with high immunogenicity, obtains antibodies 1 and 2 through immunization, and the combination of the antibodies 1 and 2 is used for the detection of HCBP6, has high detection sensitivity, good detection effect, high development and application value, and has long-term significance in the field of auxiliary diagnosis of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Protein structure analysis;

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

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

[0066] Figure 4 Show tail blood titer test;

[0067] Figure 5 Colloidal gold detection. DETAILED DESCRIPTION

[0068] The present invention provides HCBP6 monoclonal antibodies and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. 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 the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

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

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

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

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

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

[0074] The amino acid sequence of CDR3 of the light chain of antibody 1 (antibody #38) 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 of the heavy chain of antibody 2 (antibody #9) is: SYGMS (SEQ ID NO: 9);

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

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

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

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

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

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

[0084] The amino acid sequence of the light chain variable region of antibody 2 (antibody #9) 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 the present invention are all common commercial products and can be purchased in the market.

[0093] The present invention will be further described below in conjunction with embodiments:

[0094] Antigen Polypeptide of Hepatitis C Virus Core Protein Binding Protein 6 (HCBP6) and Its Application

[0095] I. Antigen 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 as shown in SEQ ID NO: 1. Using bioinformatics database materials and online tools to analyze the protein structure, relevant parameters such as the hydrophobicity, surface accessibility, antigenicity of the protein are predicted as Figure 1 . Following the principles of polypeptide design and the requirements of the immunogen, polypeptides 2 and 3 with the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 were selected.

[0097] The antigen polypeptide of polypeptide 2 (SEQ ID NO: 2) was obtained by chemical synthesis from a commercial company and was detected by HPLC( Figure 2 ) and MS( Figure 3 ). The purity and molecular weight of the synthesized antigen polypeptide and other properties fully met the design requirements; the antigen polypeptide of polypeptide 3 (SEQ ID NO: 3) was obtained by chemical synthesis from a commercial company. The molecular weight of SEQ ID NO: 3 was 3.5 KD, and it was conjugated to KLH protein (Abcam ab285712).

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

[0099] An appropriate amount of the synthesized immunogen of HCBP6, SEQ ID NO: 2, SEQ ID NO: 3 protein, and SEQ ID NO: 3 conjugated to KLH protein were emulsified with an equal amount of Freund's complete adjuvant (sigma catalog number: F5881 - 10X10ml) respectively, and were subcutaneously injected into the axilla and abdominal sites of Balb / C mice (Hunan Slack Jingda Experimental Animal Co., Ltd., 6 - 8 week-old female, 6 mice) at a dose of 0.5 mL / mouse. Each immunogen was used to immunize 6 mice. After 21 days, a second intraperitoneal booster immunization was carried out. 14 days after the second immunization, a third intraperitoneal booster immunization was carried out (at the second and third immunizations, the dosage of the immunogen was halved and emulsified with an equal volume of Freund's incomplete adjuvant, 0.5 mL / mouse / time). After three booster immunizations, tail blood was collected for detection of the immune titer. For mice whose titers reached the fusion requirements, 3 days before fusion, a final intraperitoneal immunization was carried out with the antigen dosage of the first immunization, 0.5 mL / mouse.

[0100] III. Detection of Tail Blood Serum Titer

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

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

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

[0104]

[0105]

[0106] Table 2. Peptide 3-KLH immune effect

[0107]

[0108] Figure 4 It is a curve chart of serum titer detected by ELISA, which directly reflects the overall situation of serum titer. Figure 4 It can be judged that among the synthesized peptides, only peptide 2 (SEQ ID NO: 2) has a good immune effect as an immunogen, which meets the requirements of the immunogen required for the development of its monoclonal antibody. The immune effect of peptide 3 (SEQ ID NO: 3) does not meet the experimental requirements whether it is alone or coupled to KLH protein, and peptide 2 is mainly used in subsequent experiments.

[0109] 4. Preparation of Monoclonal Antibodies

[0110] 4.1 Preparation of feeder cells

[0111] 1 to 2 days before fusion, aseptically remove macrophages from the peritoneal cavity of Kunming mice and place them in HAT medium, count them, and adjust the cell concentration to 10 4 ~10 5 Add 0.1 mL / well to a 96-well cell culture plate at 37°C and 5% CO 2 Keep incubator for future use.

[0112] 4.2 Preparation of SP2 / 0 cells

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

[0114] 4.3 Preparation of splenocytes

[0115] 3 to 5 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 spleen cells and SP2 / 0 cells at a ratio of (2-5) / 1, centrifuge at 1500rpm for 5min, discard the supernatant, fully disperse, slowly add 1mL fusion agent (SIGMA), let stand for 1min, then slowly add 30-50mL basal culture medium incubated at 37℃, centrifuge at 1200rpm for 8min, and discard the supernatant. Add an appropriate amount of HAT culture medium and mix carefully. Add the fused cell suspension to a 96-well cell culture plate with feeder cells, 0.1mL / well, 37℃, 5% CO 2 Culture in an incubator and observe the cells for 4 to 7 days before changing to HT medium.

[0118] 4.5 Cell supernatant detection and limiting dilution

[0119] Perform cell supernatant detection in the same manner as tail blood titer detection. When the fusion cells are cultured for 8 to 14 days, add 0.1 mL of cell supernatant to each well for enzyme immunoassay. 450nm The positive cell wells with OD values ​​greater than 2.0 were selected for limiting dilution. The monoclonal wells were selected and the positive cell wells with OD values ​​greater than 2.0 in the cell supernatant were tested for expansion culture, cell freezing and ascites preparation.

[0120] 4.6 Ascites preparation

[0121] Male Bacb / C mice aged 6 to 8 weeks were intraperitoneally injected with Freund's complete adjuvant (sigma catalog number: F5506-10X10ml) at 0.5 mL / mouse. Cell immunization was performed 7 days later, with 500,000 to 1,000,000 cells / mouse. The growth of ascites in the mice was observed and the cells were collected after 9 to 15 days.

[0122] 4.7 Antibody Preparation

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

[0124] 4.8 Reproducibility experiments of synthetic peptides as immunogens

[0125] According to the method described in the present invention, the synthetic polypeptide 2 of the present invention was used as an immunogen to immunize 60 mice, and a total of 38 monoclonal strains were prepared, with concentrations as shown in Table 3.

[0126] Table 3. Monoclonal clones prepared

[0127] Number Clone Number Antibody Concentration (mg / ml) Number Clone Number Antibody Concentration (mg / ml) Number Clone Number 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] As can be seen from Table 3, the concentration of the prepared antibodies is good, and the titers of the 38 antibody enzyme immunoassay developed by the present invention are all good. The 38 antibodies are individually coated on NC membranes or labeled with colloidal gold and then arranged in combination. There are a total of 1444 combinations. The colloidal gold test strips prepared from these 1444 combinations are used for the detection of quality control products and clinical samples. Among them, 20 combinations have good test results, among which the best effect is obtained when labeled with antibody No. 38 and coated with antibody No. 9. Figure 5 , only the best pair is listed). This shows that the HCBP6 immunogen has good reproducibility in preparing hybridoma cells and monoclonal antibodies and can be used in industry.

[0129] 5. Application of Monoclonal Antibodies

[0130] 5.1 Preparation of colloidal gold test strips

[0131] Coating: dilute the antibody (antibody No. 9) to 0.5-2 mg / ml in 1×PBS containing 2% sucrose, streak-coat on the NC membrane, and dry at 37°C overnight (antibody performance test, no quality control line).

[0132] Labeling: Take an appropriate amount of 0.2% colloidal gold, add 0.1M potassium carbonate 10μL / mL and mix well, react at room temperature for 10min, add antibody (antibody number 38) 10μg / mL and mix well, react at room temperature for 10min, add 10% BSA (w / v) 50μl / ml, react at room temperature for 10min, centrifuge (12000rpm, 10min), discard the supernatant. Resuspend with 0.01M PB (pH=7.4) containing 3%~5% (w / v) sucrose, 0.5%~3% (w / v) inositol, 1%~2% (w / v) BSA, 0.1%~0.5% (w / v) Tween-20 to 1 / 3 of the original volume.

[0133] Preparation of gold pad: Evenly apply 750 μL of colloidal gold suspension to each 0.6 cm × 30 cm gold pad and dry at 37°C overnight.

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

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

[0136] According to Table 4 and Figure 5 (Antibody No. 38 labeled, antibody No. 9 coated) The colloidal gold test results show that the anti-HCBP6 antibody detection quality control product of the present invention has a more and more obvious color from low value to high value, and the detection of clinical fatty liver indicators is also very clear.

[0137] Table 4. Test results

[0138]

[0139] At present, there is no kit developed for HCBP6 protein in my country. The present invention provides high value for developing HCBP6 detection kit and provides good assistance for the diagnosis of fatty liver.

[0140] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. HCBP6 monoclonal antibody, It is characterized in that comprising antibody 1 and / or antibody 2, The heavy chain variable region of the antibody 1 has at least one of the amino acid sequences shown in SEQ ID NO: 1, 2 or 3; The light chain variable region of the antibody 1 has at least one of the amino acid sequences shown in SEQ ID NO: 4, 5 or 6; 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; 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.

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

16.

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

4. Immunization to obtain the antigenic polypeptide of the HCBP6 monoclonal antibody according to any one of claims 1 to 3, It is characterized in that The antigen polypeptide has at least one of the amino acid sequences shown in the following I) to III): 1), the amino acid sequence shown in SEQ ID NO: 2; or II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or III), an amino acid sequence having at least 90% sequence identity with the amino acid sequence described in (I) or (II).

5. Recombinant antigens, It is characterized in that It comprises the antigen polypeptide as claimed in claim 4 and a carrier protein.

6. Biomaterials, It is characterized in that Including at least one of the following i) to iii): i) a nucleic acid encoding the HCBP6 monoclonal antibody according to any one of claims 1 to 3, the antigenic polypeptide according to claim 4, or the recombinant antigen according to claim 5; ii), a recombinant vector comprising a vector backbone and the nucleic acid as shown in i); iii), transfecting or transforming a host cell with the recombinant vector as shown in ii); iv) culturing the host cell as shown in iii) to obtain a culture product.

7. A method for preparing the HCBP6 monoclonal antibody according to any one of claims 1 to 3, the antigen polypeptide according to claim 4, or the recombinant antigen according to claim 5, It is characterized in that The HCBP6 monoclonal antibody or the antigenic polypeptide or the recombinant antigen is obtained by culturing the host cells in the biological material according to claim 4.

8. Labeling antibodies, It is characterized in that The method comprises the HCBP6 monoclonal antibody according to any one of claims 1 to 3 and a marker.

9. The labeled antibody according to claim 8, It is characterized in that 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.

10. Conjugates, It is characterized in that The invention comprises the HCBP6 monoclonal antibody according to any one of claims 1 to 3 and a coupling medium.

11. Use of at least one of the following A) to F) in the preparation of a product for HCBP6 detection or fatty liver diagnosis: A), the HCBP6 monoclonal antibody according to any one of claims 1 to 3; B), the antigen polypeptide according to claim 4; C) the recombinant antigen according to claim 5; D) the biomaterial according to claim 6; E), the labeled antibody according to claim 8 or 9; F) The conjugate according to claim 10.

12. Products for fatty liver testing, It is characterized in that The raw materials include at least one of the following a) to d): a), the HCBP6 monoclonal antibody according to any one of claims 1 to 3; b) The biomaterial according to claim 6; c) The labeled antibody according to claim 8 or 9; d) The conjugate according to claim 10.

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