Atf7b antibodies and uses thereof

Abstract

This invention provides an ATP7B antibody and its application. The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of H-CDR1 in the heavy chain variable region is shown in SEQ ID NO:2, the amino acid sequence of H-CDR2 in the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of H-CDR3 in the heavy chain variable region is shown in SEQ ID NO:4; the amino acid sequence of L-CDR1 in the light chain variable region is shown in SEQ ID NO:6, the amino acid sequence of L-CDR1 in the light chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of L-CDR1 in the light chain variable region is shown in SEQ ID NO:8. The antibody of this invention exhibits high affinity and specificity for ATP7B.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an ATP7B antibody and its applications. Background Technology

[0002] ATP7B protein belongs to the P-type ATPase superfamily and is a copper ion transporter primarily responsible for the transport and regulation of intracellular copper ions. Mutations in the ATP7B gene lead to Wilson's disease, an autosomal recessive inherited disorder of copper metabolism. In Wilson's disease patients, impaired ATP7B protein function leads to abnormal accumulation of copper in the liver, brain, and other organs, causing liver disease, neurological symptoms, and mental health issues. Gene therapy based on adeno-associated virus (AAV) vectors, which deliver the ATP7B gene into the patient's body to enable the expression of functional ATP7B protein in the liver, promises to provide a "one-time treatment, lifelong benefit" approach for Wilson's disease patients.

[0003] In drug development, the detection of ATP7B protein concentration and anti-ATP7B binding antibodies are crucial indicators of drug efficacy and safety, both requiring the use of antibodies with high affinity and high specificity. ATP7B is an eight-transmembrane protein, and its structural characteristics significantly influence the preparation of purified protein antigens and antibody recognition sites, making it difficult to obtain antibodies that meet the detection requirements.

[0004] Therefore, there is an urgent need in this field to develop an antibody with high affinity and specificity for ATP7B protein that can be stably prepared to meet the needs of preclinical and clinical drug research testing. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an ATP7B antibody and its application, in order to solve the problem of the lack of effective means for detecting ATP7B protein in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides an antibody comprising a heavy chain variable region and a light chain variable region. The amino acid sequence of H-CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of H-CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of H-CDR3 of the heavy chain variable region is shown in SEQ ID NO: 4; the amino acid sequence of L-CDR1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of L-CDR1 of the light chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of L-CDR1 of the light chain variable region is shown in SEQ ID NO: 8.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1. Inconsistencies may occur in the backbone region.

[0008] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 17. Inconsistencies may occur in the backbone region.

[0009] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5. Inconsistencies may occur in the backbone region.

[0010] Furthermore, the amino acid sequence of the light chain is shown in SEQ ID NO: 18. Inconsistencies may occur in the backbone region.

[0011] In some embodiments, the antibody described in this invention is an IgG antibody. Further, in some embodiments, the IgG antibody is an IgG1 antibody. In some embodiments, the light chain type of the antibody is Kappa type. The antibody may be mouse, human, chimeric, or humanized.

[0012] Another aspect of the present invention provides a gene fragment encoding the antibody described above.

[0013] Another aspect of the present invention provides a vector containing the above-mentioned gene fragment.

[0014] In some embodiments, the nucleotide sequence encoding H-CDR1 in the gene fragment is shown in SEQ ID NO:10, the nucleotide sequence encoding H-CDR2 is shown in SEQ ID NO:11, the nucleotide sequence encoding H-CDR3 is shown in SEQ ID NO:12, the nucleotide sequence encoding L-CDR1 is shown in SEQ ID NO:14, the nucleotide sequence encoding L-CDR1 is shown in SEQ ID NO:15, and the nucleotide sequence encoding L-CDR1 is shown in SEQ ID NO:16.

[0015] Another aspect of the present invention provides a host cell containing or integrating the above-mentioned vector.

[0016] Another aspect of the present invention provides an antibody-drug conjugate comprising an antibody portion and a conjugating portion, wherein the antibody portion comprises any one of the antibodies described above. The selection of the conjugating portion must match the antibody's targeting and application scenario. For example, for therapeutic applications, the conjugating portion may be selected from cytotoxic drugs, immunomodulators, radionuclides, etc.; for diagnostic / imaging applications, the conjugating portion may be selected from fluorescent dyes, radioactive contrast agents, etc.; for research applications, the conjugating portion may be selected from affinity tags, enzymes, and other auxiliary molecules. In some embodiments, the conjugating portion is HRP.

[0017] Another aspect of the present invention provides a pharmaceutical composition comprising the above-described antibody and a pharmaceutically acceptable excipient. The excipient may be an excipient, diluent, carrier, etc.

[0018] Another aspect of the present invention provides the use of the above-mentioned antibody in the preparation of products for detecting ATP7B.

[0019] Another aspect of the present invention provides the use of the above-mentioned gene fragment, vector or host cell for the preparation of antibodies to detect ATP7B.

[0020] In practical applications, the main uses of the antibody in this application include, but are not limited to: 1) clinical detection of ATP7B protein expressed in the liver. A portion of liver tissue can be obtained during a liver biopsy to measure the level of the expressed protein. 2) Detection of ATP7B protein concentration as a target in laboratories or other settings. 3) Use as a positive control when clinically detecting serum ATP7B antibodies after drug administration.

[0021] As described above, the ATP7B antibody of the present invention has the following beneficial effects:

[0022] The ATP7B antibody prepared in this application has high affinity and specificity for ATP7B. Attached Figure Description

[0023] Figure 1 It showed affinity for ATP7B hybridoma supernatant;

[0024] Figure 2 The purity of the anti-ATP7B recombinant antibody was shown.

[0025] Figure 3 The affinity of the anti-ATP7B recombinant antibody was demonstrated;

[0026] Figure 4 This demonstrates the use of anti-ATP7B recombinant antibody for Western blot (WB) detection;

[0027] Figure 5 This demonstrates the use of anti-ATP7B recombinant antibody for ELISA detection;

[0028] Figure 6 The use of anti-ATP7B recombinant antibody for ADA detection is shown. Detailed Implementation

[0029] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0030] As used herein, the term "antigen" refers to a molecule that elicits an immune response, which may involve antibody production or activation of specific immune-active cells. Those skilled in the art will understand that any macromolecule, including all proteins or peptides, can be used as an antigen. Antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, encoding what is referred to herein as an "antigen." Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all; antigens may be generated, synthesized, or derived from a biological sample. Such a biological sample may include, but is not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0031] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of this invention can exist in a variety of forms, including polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies; the antibody forms of this invention also include full-length antibodies, antibody fragments such as Fab, Fab', F(ab')2, Fv, scFv, di-scFv, tri-scFv, Fd, and other antibody fragments that retain antigen-binding function; they can also be dimeric (diabody) or trimeric (triabody) structures. Typically, the fragment should include an antigen-binding fragment, which usually includes VL and VH; however, it does not necessarily have to include both. For example, a so-called Fd antibody fragment consists only of VH and CH1 domains, yet still retains some of the antigen-binding functions of a complete antibody. The term "antibody," as used for immunoglobulins or fragments thereof or derivatives thereof, includes any polypeptide containing an antigen-binding site, regardless of whether it is produced in vitro or in vivo. The VH or VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions interspersed throughout, called framework regions (FWRs). The variable regions of the heavy and light chains contain binding domains that interact with the antigen. CDRs in the heavy chain are abbreviated as VH-CDRs, such as VH-CDR1, VH-CDR2, and VH-CDR3, while CDRs in the light chain are abbreviated as VL-CDRs, such as VL-CDR1, VL-CDR2, and VL-CDR3.

[0032] As used herein, the terms "specific binding" or "specifically binding" refer to a non-random binding reaction between two molecules, such as the binding reaction between an antibody and an antigen.

[0033] As used herein, the term "vector" refers to a vector comprising a recombinant polynucleotide, which includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. Expression vectors include sufficient cis-acting elements for expression; other elements for expression may be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0034] As used herein, the term "host cell" can refer to any prokaryotic or eukaryotic cell containing a cloning or expression vector, including those that have been genetically engineered to contain cloned genes in the host cell's chromosome or genome. Suitable mammalian host cells include myeloma cells, such as SP2 / 0 and NSO cells, as well as Chinese hamster ovary (CHO) cells, hybridoma cell lines, and other mammalian host cells that can be used to express antibodies. Special transgenic animals with modified immune systems can also be used to produce antibodies.

[0035] As used herein, “identity” refers to sequence similarity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence aligned for comparison purposes. When positions in the compared sequences are occupied by the same bases, then the molecules at that position are identical. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or procedures can be used to calculate identity between two sequences, including those available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wis.) and which can be used, for example, by default, as FASTA or BLAST. For example, those skilled in the art can anticipate polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding the aforementioned polypeptides.

[0036] Unless otherwise specified, the nucleotide sequence of a nucleic acid molecule or amino acid sequence of a protein as used herein includes all nucleotide sequences that are degenerate from each other and encode the same amino acid sequence. The nucleotide sequence may also include one or more introns.

[0037] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0039] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0040] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0041] Unless otherwise specified, experimental methods in the following examples were performed using conventional methods and conditions, or according to the product instructions. All raw materials and equipment used in the examples are well-known to those skilled in the art and are commercially available or readily obtained or prepared. Key experimental material information is shown in Table 1.

[0042] Table 1. Experimental Materials

[0043] Experimental materials Manufacturer's part number ELISA reader Thermo Varioskan LUX HRP-labeled anti-mouse IgG secondary antibody CST 7076S Protein A affinity chromatography medium Bio-link 1024-1821 Immuno Transparent Standard Version (96 holes) Thermo 468667 Coating buffer (20×) Biotech E661004 Rinse solution (PBST) PBS + 0.1% Tween 20 Sealing liquid PBST + 3% BSA Analysis buffer PBST + 1% BSA Streptavidin-HRP (SA-HRP) CST 3999S 1-Step Ultra TMB-ELISA Thermo 34029 .

[0044] Example 1: Hybridoma Monoclonal Preparation

[0045] Anti-ATP7B mouse monoclonal hybridoma cells were prepared by Nanjing Genscript Biotech Co., Ltd.: the immunogen was a recombinant N-terminal fragment of the ATP7B protein. Five BALB / c mice were initially immunized with 50 μg of antigen each; subsequently, immunizations were performed on days 14, 28, and 50, with each mouse receiving 25 μg of antigen. Four days after the final immunization, two mice with the highest serum immune response were selected for hybridoma cell fusion. The fused cells were diluted and seeded into 96-well plates. After expansion, the supernatant was analyzed using indirect ELISA to select clones positive for the ATP7B protein. The selected clones were subcloned using limiting dilution and expanded to obtain monoclonal hybridoma cells.

[0046] Affinity of hybridoma monoclonal 13A10F4 culture supernatant was detected by indirect ELISA: 1 μg / ml or 100 μl / well of ATP7B protein N-terminal fragment or irrelevant protein (negative control) was added to each well of a 96-well plate, and incubated at 2–8°C for 18–72 hours. The liquid in the wells was discarded, and 200 μL of wash buffer was added to each well, then discarded again, and the plate was dried on paper. This washing process was repeated 3 times. 200 μL of blocking buffer was added to each well, and the plate was incubated with shaking at 450 rpm at room temperature for 1–2 hours. The plate was washed 3 times. The hybridoma supernatant was diluted 10, 30, 90, 270, 810, and 2430 times with analysis buffer, and 100 μL was added to each well. The plate was incubated with shaking at 50 rpm at room temperature for 1 hour, and washed 3 times. The labeled anti-mouse IgG secondary antibody was diluted to 100 ng / mL with analysis buffer, and 100 μL was added to each well. The plate was incubated with shaking at 450 rpm at room temperature for 1 hour. Rinse 3 times. Add 100 μL SA-HRP to each well and incubate at 450 rpm with shaking at room temperature for 45 minutes. Rinse 3 times. Add 100 μL TMB substrate to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL stop solution (2 M H2SO4) to each well and read the OD450 using a microplate reader within 30 minutes.

[0047] The results are as follows Figure 1 As shown, the culture supernatant of hybridoma monoclonal 13A10F4 has a very strong affinity for ATP7B protein (titer > 1:2430) and does not recognize irrelevant proteins (titer < 1:10).

[0048] Sequencing of the heavy chain and light chain variable regions of hybridoma cells expressing monoclonal antibodies was commissioned to Shanghai Bio-Tech Co., Ltd., as shown in Table 2.

[0049] Table 2. Sequence Information

[0050] .

[0051] Example 2: Expression and purification of recombinant antibodies

[0052] RNA was extracted from hybridoma cells, and 1 μg of RNA was added to a reverse transcription system (Sangon Biotech, B639251-0100) to synthesize cDNA. Specific primers were designed based on sequencing results, and cDNA was used as a template for PCR amplification and purification of the heavy chain variable region and light chain variable region fragments of the antibody. The backbone plasmid containing the mouse IgG1 heavy chain constant region (GenBank: BCQ06376.1) (MouseIgG1 H-chain backbone) and the backbone plasmid containing the mouse IgG kappa light chain constant region (EntrezID 16071) (Mouse IgG.k L-chain backbone) were linearized with NotI and purified. 100-200 ng of the variable region fragment and 70 ng of the linearized backbone plasmid were added to a seamless cloning reaction system (NEB, 10238675) for ligation. The ligation product was transformed into stbl3 competent bacteria, plated, and single clones were picked. After successful sequencing, the plasmid was extracted after culture.

[0053] CHO cells were used at a rate of 1×10 6 After culturing at a density of 1 / mL for 18-24 hours, plasmid transfection was performed: Add 0.2 μg / mL (cell culture volume, the same below) of heavy chain plasmid and 0.3 μg / mL of light chain plasmid to Dynamis medium, and vortex to mix. Add 1 μL / mL of FectoPRO (Polyplus, 101000007) transfection reagent to another clean container; add the diluted plasmid to the transfection reagent, mix immediately, and incubate at room temperature for 10 minutes before adding cells. Continue culturing for 72 hours, then centrifuge at 3000 rpm for 10 minutes to collect the supernatant for later use. Use a 2 mL Protein A affinity chromatography column, equilibrate with 40 mL of PBS, and then pass the culture supernatant through the column. After loading, wash with 40 mL of PBS, then elute with 10 mL of glycine (100 mM, pH 3.5). The affinity eluent was replaced with PBS using a 30 kD ultrafiltration tube, and the purified recombinant antibody was quantified using BCA (Thermo, 23227).

[0054] Add 1 μg of purified recombinant antibody to the wells of a SurePAGE precast gel (GenScript, M00654) and electrophoresis at 120 V for 80 minutes. After electrophoresis, add approximately 30 mL of BeyoBlue Coomassie Brilliant Blue Ultrafast Staining Solution (Beyotime, P0017F) and stain on a shaker at room temperature for 30 minutes. After staining, add an appropriate amount of deionized water and destain on a shaker. Replace the deionized water every 10-30 minutes and continue destaining on a shaker until clear protein bands are obtained, then photograph using a gel imaging system. The purity of the purified recombinant antibody is as follows: Figure 2 As shown, there is a single band at the theoretical molecular weight of 55 kDa (heavy chain) and 25 kDa (light chain).

[0055] Example 3: Affinity Detection of Anti-ATP7B Recombinant Antibody

[0056] Dilute the N-terminal protein of ATP7B to 100 ng / mL with coating buffer, add 100 μL to each well of a 96-well plate, and incubate at 2–8°C for 18–72 hours. Discard the liquid in the wells, add 200 μL of wash buffer to each well, then discard the buffer, blot dry on paper, and repeat the washing process 3 times. Add 200 μL of blocking buffer to each well and incubate with shaking at 450 rpm at room temperature for 1–2 hours. Wash 3 times. Dilute the recombinant antibody to seven concentrations between 0.3 and 20 ng / mL with analysis buffer, add 100 μL to each well of a pre-coated 96-well plate. Incubate with shaking at 450 rpm at room temperature for 1 hour, and wash 3 times. Dilute the labeled anti-mouse IgG secondary antibody to 100 ng / mL with analysis buffer, add 100 μL to each well, and incubate with shaking at 450 rpm at room temperature for 1 hour. Wash 3 times. Add 100 μL of SA-HRP to each well and incubate at 450 rpm with shaking for 45 minutes at room temperature. Rinse three times. Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL of stop solution (2 M H2SO4) to each well and read the OD450 using a microplate reader within 30 minutes. Results are as follows. Figure 3 As shown, the recombinant antibody can bind to the ATP7B protein, and the reaction signal is positively correlated with the antibody concentration.

[0057] Example 4: Method for Western Blot Detection Using Anti-ATP7B Recombinant Antibody

[0058] Two weeks after AAV-ATP7B infection in mice, samples were collected. Four grinding steel balls were added to 20 mg of mouse liver tissue, along with 1 mL of urea lysis buffer containing a protease inhibitor. The sample was then ground in an automated cryogenic homogenizer (run time 30 s, interruption time 30 s, set number of times 5, set frequency 70 Hz). After homogenization, the sample was placed on ice for 20 min, centrifuged at 16000 ×g for 10 min at 4 ℃, and the supernatant was collected and denatured at 95 ℃ for 10 min. 10 μL of the supernatant was added to the sample wells of a SurePAGE pre-cast gel, and electrophoresis was performed at 120 V for 80 min. After electrophoresis, the gel was placed on a sponge paper on a black transfer plate, covered with a membrane soaked in methanol, and all air bubbles were removed. The electrophoresis apparatus was assembled, and the membrane was transferred at 400 mA for 1 hour. After transfer, the membrane was soaked in 5% BSA / TBST solution and blocked on a shaker at room temperature for 2 hours. The blocking solution was discarded, and 1 μg / mL of recombinant antibody solution was added and incubated overnight at 4 ℃ with shaking. Discard the recombinant antibody solution, wash three times with 1× TBST (10 min / wash), add 1000-fold diluted Anti-mouse IgG solution (Cell Signaling technology, 7076S), and incubate at room temperature with shaking for 1 h. Discard the secondary antibody solution, wash three times with 1× TBST (10 min / wash), add Chemiluminescent Substrate (Thermo, 34577) detection reagent onto the membrane, and develop using a chemiluminescence analyzer. Results are as follows. Figure 4 As shown, the recombinant antibody can specifically bind to human ATP7B protein expressed by AAV infection in mouse liver lysate, and can be used for Western blotting detection of ATP7B protein.

[0059] Example 5: Method for detecting ATP7B protein concentration using recombinant anti-ATP7B antibody via enzyme-linked immunosorbent assay (ELISA).

[0060] Dilute anti-ATP7B rabbit polyclonal antibody (GenScript) to 1 μg / mL with coating buffer, add 100 μL to each well of a 96-well plate, and incubate at 2–8°C for 18–72 hours. Discard the liquid in the wells, add 200 μL of wash buffer to each well, then discard the buffer, blot dry on paper, and repeat the wash 3 times. Add 200 μL of blocking buffer to each well and incubate with shaking at 450 rpm at room temperature for 1–2 hours. Wash 3 times. Add 100 μL of serially diluted ATP7B N-terminal protein or full-length recombinant human ATP7B protein (abmart, RG323635S) to each well and incubate with shaking at 450 rpm at room temperature for 1 hour. Wash 3 times. Dilute biotin-labeled monoclonal antibodies (13A10F4-bio, 14B7D3-bio) to 1 μg / mL with analysis buffer, add 100 μL to each well, and incubate with shaking at 450 rpm at room temperature for 1 hour. Wash 3 times. Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL of stop solution to each well and read the OD value using a microplate reader within 30 minutes. 450 The result is as follows Figure 5 As shown, the two standard curves exhibit good linearity, and the parameter fit R0 is satisfactory. 2 >0.99; This combination of recombinant antibody and polyclonal antibody can simultaneously detect recombinant N-terminal protein and full-length protein, and can be used for ELISA detection of ATP7B protein concentration, with a limit of quantitation <10 ng / mL.

[0061] Example 6: Method for using anti-ATP7B recombinant antibody for antibody detection

[0062] Dilute the N-terminal fragment of ATP7B protein to 1 μg / mL with coating buffer, add 100 μL to each well of a 96-well plate, and incubate at 2–8°C for 18–72 hours. Discard the liquid in the wells, add 200 μL of wash buffer to each well, then discard the buffer, blot dry on paper, and repeat the rinsing process 3 times. Add 200 μL of blocking buffer to each well and incubate at 450 rpm at room temperature for 1–2 hours. Rinse 3 times. Dilute the recombinant antibodies (13A10F4, 14B7D3) to 100, 200, and 500 ng / mL with analysis buffer, respectively, and add 100 μL to each well of a pre-coated 96-well plate. Incubate at 450 rpm at room temperature for 1 hour, and rinse 3 times. Dilute the biotin-labeled N-terminal fragment of ATP7B protein to 500 ng / mL with analysis buffer, add 100 μL to each well, and incubate at 450 rpm at room temperature for 1 hour. Rinse 3 times. Add 100 μL of SA-HRP to each well and incubate at 450 rpm with shaking for 45 minutes at room temperature. Rinse three times. Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL of stop solution to each well and read the OD450 using a microplate reader within 30 minutes. Results are as follows. Figure 6As shown, there is a dose-response relationship between the signal value and the antibody concentration; this recombinant antibody can be used as a positive antibody in the detection of anti-ATP7B binding antibodies.

[0063] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An ATP7B antibody, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of H-CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of H-CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of H-CDR3 of the heavy chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of L-CDR1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of L-CDR2 of the light chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of L-CDR3 of the light chain variable region is shown in SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

3. The ATP7B antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 17; and / or the amino acid sequence of the light chain is shown in SEQ ID NO:

18.

4. A gene fragment, characterized by: The gene fragment encodes the antibody as described in any one of claims 1-3.

5. The gene fragment according to claim 4, characterized in that: The nucleotide sequence encoding H-CDR1 in the gene fragment is shown in SEQ ID NO:10, the nucleotide sequence encoding H-CDR2 is shown in SEQ ID NO:11, the nucleotide sequence encoding H-CDR3 is shown in SEQ ID NO:12, the nucleotide sequence encoding L-CDR1 is shown in SEQ ID NO:14, the nucleotide sequence encoding L-CDR2 is shown in SEQ ID NO:15, and the nucleotide sequence encoding L-CDR3 is shown in SEQ ID NO:

16.

6. A carrier, characterized in that: The vector contains the gene fragment as described in any one of claims 4-5.

7. A host cell, characterized by: The host cell contains or integrates the vector as described in claim 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as described in any one of claims 1-3 and a pharmaceutically acceptable excipient.

9. Use of the antibody as described in any one of claims 1-3 in the preparation of ATP7B detection products.

10. The use of the gene fragment as described in any one of claims 4-5 in the preparation of the ATP7B antibody as described in claim 1.

11. Use of the carrier as described in claim 6 in the preparation of the ATP7B antibody as described in claim 1.

12. The use of the host cell as described in claim 7 in the preparation of the ATP7B antibody as described in claim 1.

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