An antibody or antigen-binding fragment thereof that binds to fumarylhydrazine hydratase and uses thereof

Abstract

The application relates to an antibody or antigen binding fragment thereof binding to fumarase hydratase and application thereof, and belongs to the technical field of biological medicine. The application provides an antibody or antigen binding fragment thereof binding to fumarase hydratase, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; and the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 respectively. The fumarase hydratase antibody or antigen binding fragment thereof prepared in the application is superior to a commercially available product in sensitivity, affinity and specificity, and has better market competitiveness compared with prior art.

Description

Technical Field

[0001] This application relates to an antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase and its application, belonging to the field of biomedical technology. Background Technology

[0002] Fumarate hydratase (FH), also known as fumarate hydratase, is a highly conserved key protease involved in energy metabolism in prokaryotes and eukaryotes. It plays a variety of biological functions, including regulating the mitochondrial tricarboxylic acid cycle, urea cycle, purine nucleotide cycle, DNA damage repair, and tumor suppression. As an important metabolic enzyme, FH dysfunction in tumors exhibits relatively unique characteristics in terms of pathogenesis, clinical symptoms, pathological morphology, immunophenotype, treatment outcomes, and prognosis. The role of such metabolic abnormalities in tumor development and progression has attracted widespread attention in recent years. FH antibodies are antibodies that specifically recognize and bind to the FH protein.

[0003] The FH gene is located in the q43 region of chromosome 1 and contains 10 exons. The FH gene transcription and translation products can be localized in mitochondria and cytoplasm, exerting their biological functions as a homotetramer. The FH monomer consists of 510 amino acids and contains three key functional domains: the N-terminal fumarate lyase-1 domain, the C-terminal fumarate-C domain, and the central domain that binds to other monomers. The N-terminal extended form exists in mitochondria and participates in the tricarboxylic acid cycle, catalyzing the conversion of fumarate to L-malate. The cytoplasmic FH participates in the metabolism of amino acids and fumarate and can translocate into the nucleus to participate in DNA damage repair.

[0004] Defects in the FH gene can cause cells to lose their ability to metabolize fumarate, leading to a series of compensatory metabolic changes such as pseudohypoxia-driven processes, oxidative stress, decreased AMPK levels, and DNA repair inhibition. These changes can trigger carcinogenic alterations, such as hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, paraganglioma, pheochromocytoma, testicular interstitial cell tumor, and ovarian cystadenoma. HHLRCC is particularly aggressive with a poor prognosis, clinically manifesting as FH-deficient skin and / or uterine leiomyomas and renal cell carcinoma. FH-deficient leiomyomas account for 0.4%–1.6% of uterine leiomyomas and develop 10 years earlier than other leiomyomas; up to 62% of HHLRCC patients develop renal cell carcinoma at a young age, which is a leading cause of death.

[0005] In pathological diagnosis, preliminary morphological screening lacks sensitivity and specificity for FH-deficient tumors, and the commonly used IHC test is an FH-negative screening. FH deletion and 2SC overexpression can serve as immunohistochemical markers for diagnosing and screening tumors caused by FH deletion or mutation, and the combined detection of the two can enhance the potential of IHC in detecting FH-deficient diseases.

[0006] The primary clinical method for detecting FH deficiency is immunohistochemistry, a traditional method that uses the principle of specific antigen-antibody binding to detect the expression level of the target protein in tissues. Immunohistochemical detection relies mainly on antibodies that specifically bind to the target antigen; however, currently available FH antibodies are limited, and their affinity for FH needs improvement. Summary of the Invention

[0007] To address the aforementioned issues, this application provides an antibody or antigen-binding fragment of an anti-fumarate hydratase, and its application. The fumarate hydratase antibody or antigen-binding fragment prepared in this application is superior to commercially available products in terms of sensitivity, affinity, and specificity, and has better market competitiveness compared to existing technologies.

[0008] This application provides an antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL;

[0009] The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0010] The light chain variable regions VL respectively include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0011] Optionally, the antibody or its antigen-binding fragment can be K D It binds to fumarate hydratase with an affinity of ≤10 nM.

[0012] Optionally, the heavy chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:17;

[0013] The variable region of the light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:18.

[0014] Optionally, the heavy chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:8;

[0015] The light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:16.

[0016] Optionally, the antibody or its antigen-binding fragment is a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; or,

[0017] The antibody or its antigen-binding fragment is a half antibody or an antigen-binding fragment of a half antibody.

[0018] This application provides a biomaterial, which is any one of the following:

[0019] A) Nucleic acid molecules encoding the antibodies or their antigen-binding fragments described above;

[0020] B) A carrier containing the nucleic acid molecules described in A);

[0021] C) A host cell containing the nucleic acid molecule described in A) or the vector described in B);

[0022] D) A composition comprising the antibody or its antigen-binding fragment described above, or the nucleic acid molecule described in A), or the vector described in B), or the host cell described in C).

[0023] This application provides the application of the above-mentioned antibody or its antigen-binding fragment in the preparation of fumarate hydratase detection products.

[0024] Optionally, the detection product is an immunohistochemical detection product, an ELISA detection product, or a chemiluminescence detection product.

[0025] This application provides a detection reagent for detecting fumarate hydratase, comprising the aforementioned antibody or its antigen-binding fragment.

[0026] This application provides a kit for detecting fumarate hydratase, comprising the antibody or its antigen-binding fragment described above, or comprising the detection reagent described above.

[0027] The beneficial effects of this application include, but are not limited to:

[0028] The fumarate hydratase antibody or its antigen-binding fragment prepared in this application is superior to commercially available products in terms of sensitivity, affinity, and specificity, with a significant improvement in affinity, giving it better market competitiveness compared to existing technologies.

[0029] The fumarate hydratase antibody or its antigen-binding fragment prepared in this application has high specificity and sensitivity, and can specifically recognize cells expressing fumarate hydratase (FH) protein. It is not only suitable for immunological detection, but can also effectively prevent false positives and false negatives, and can significantly improve the accuracy of detection and diagnosis. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0031] Figure 1 This is a comparison of immunohistochemical staining results of a case of clear cell renal cell carcinoma involved in Example 2 of this application (A is LBP2-FH prepared in this application; B is commercially available fumarate hydratase FH (Elabscience, E-AB-22031)).

[0032] Figure 2 This is the kinetic curve of the interaction between the LBP2-FH monoclonal antibody and the antigen involved in Example 3 of this application;

[0033] Figure 3 The image shows the kinetic curve of the interaction between the commercially available fumarate hydratase FH (Elabscience, E-AB-22031) antibody and antigen involved in Example 3 of this application.

[0034] Figure 4 The image shows a comparison of the immunoblotting results in Example 4 of this application (A is commercially available fumarate hydratase FH (Elabscience, E-AB-22031); B is LBP2-FH prepared in this application). Detailed Implementation

[0035] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0036] Example 1

[0037] 1) Preparation of antigen

[0038] The antigen is recombinant human fumarate hydratase (FH) (Uniport code: P07954, detailed information such as its amino acid sequence, domains, and functional annotations can be found in the UniProt database based on this number). The N-terminal amino acid sequence is AA1-200, and the antigen sequence (SEQ ID NO:19) is as follows:

[0039] MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARMASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMPTPVIKAFGILKRAAAEVNQDYGLDPKIANAIMKAADEVAEGKLNDHFPLVVWQTGSGTQTNMNVNEVISNRAIEMLGGELGSKIPVHPNDHVNKSQSSNDTFPTAMHIAAA

[0040] For ease of description, the recombinant human fumarate hydratase (FH) antigen is labeled as hu-FH.

[0041] 2) Immunotherapy

[0042] The fumarate hydratase (FH) antigen obtained in the previous step was mixed with and emulsified with complete Freund's adjuvant, and multiple rabbits were immunized subcutaneously three times. After the three immunizations, blood was collected and serum titers were determined by ELISA and immunohistochemistry. The rabbit with the highest antibody titer against the fumarate hydratase (FH) antigen was selected as the target animal for the next step of single B cell screening.

[0043] By comparing the hu-FH antigen with similar sequences from the same family, a highly specific region was selected as the antigen and conjugated with a highly immunogenic hapten to enhance the immune response in experimental rabbits, thus increasing the likelihood of obtaining specific antibodies.

[0044] 3) Single B cell screening and sequencing

[0045] Spleens of the target animal were harvested, and B cells secreting specific antibodies were isolated by antigen coating adsorption. After culture, the supernatant of the B cells was collected, and B cells capable of secreting antibodies binding to fumarate hydratase (FH) were identified by ELISA. These B cells were lysed to obtain total RNA (ribonucleic acid). cDNA (complementary DNA) was obtained using a reverse transcription kit, and the reverse transcription product was used as a template for PCR (polymerase chain reaction).

[0046] The corresponding primers were designed to amplify the antibody encoding the heavy chain variable region (VH) and light chain variable region (VL) sequences. The reaction program was as follows: VH: 95℃, 5 min; 95℃, 30 s; 70℃, 30 s; 72℃, 1 min; 72℃, 10 min for 35 cycles; VL: 95℃, 5 min; 95℃, 30 s; 55℃, 30 s; 72℃, 1 min; 72℃, 10 min for 35 cycles. The amplification products were detected by 1% agarose gel electrophoresis, and then a single target band was selected for gel recovery.

[0047] The target band recovered from the gel and a mammalian cell expression vector containing the constant region gene were transformed into TOP10 competent cells via homologous recombination. The cells were cultured at 37°C for 12 hours, and single clones were selected for sequencing. The sequencing results were analyzed using Snapgene software to screen expression plasmids with VH and VL sequences that match the characteristics of rabbit-derived antibody sequences.

[0048] Single-cell B-cell sequencing technology improves the efficiency and cloning success rate of screening high-affinity antibodies. Since B cells cannot survive in vitro for extended periods, even if ideal antibodies are produced, they will be lost due to B cell death. Traditional antibody screening methods often employ hybridoma technology, fusing B cells with myeloma cells to achieve immortality. However, this technology has drawbacks: the fusion success rate is generally only around 5%, and hybridoma cells may experience rejection between the two cell types, ultimately resulting in the loss of antibody information. Single-cell B-cell sequencing technology uses B-cell isolation equipment to sort individual B cells into culture dishes, using a nutrient-rich cell culture medium rich in growth factors that stimulate B cell growth. This allows for the screening of B cells expressing high-affinity antibodies with a very low loss rate, followed by DNA sequencing. The DNA sequence can then be permanently preserved or regenerated using vector cloning technology.

[0049] 4) Preparation and purification of monoclonal antibodies on cells

[0050] The confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography with Protein A to obtain high-purity antibody. Finally, the purified anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody was analyzed for concentration and aliquoted, labeled as LBP2-FH (anti-fumarate hydratase (FH) protein monoclonal antibody), and stored at 4°C.

[0051] The obtained recombinant rabbit monoclonal antibody against fumarate hydratase (FH) was sequenced, which included a heavy chain and a light chain. The heavy chain included a heavy chain variable region and a heavy chain constant region; the light chain included a light chain variable region and a light chain constant region.

[0052] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of FR1, FR2, FR3, and FR4 in the heavy chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and the amino acid sequence of the complete heavy chain is shown in SEQ ID NO:8. See Table 1 for details.

[0053] The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; the amino acid sequences of FR1, FR2, FR3, and FR4 in the light chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; and the amino acid sequence of the complete light chain is shown in SEQ ID NO:16. See Table 1 for details.

[0054] Table 1. Sequences of monoclonal antibodies against fumarate hydratase (FH) protein

[0055]

[0056] Table 1 (Continued from Table 1)

[0057]

[0058] Table 1 (Continued from Table 2)

[0059]

[0060] Example 2 Immunohistochemical detection

[0061] Immunohistochemical detection was performed using a recombinant rabbit monoclonal antibody against fumarate hydratase (FH) as the primary antibody, as follows:

[0062] 1) Sample section preparation: The formalin-fixed paraffin-embedded clear cell renal cell carcinoma was baked in a 60℃ constant temperature oven for 1-2 hours and stored for later use.

[0063] 2) Use an automated repair instrument to dewax, hydrate, and repair the sections; during this process, the temperature is preferably controlled at 95℃ and the time is preferably controlled at 30 minutes.

[0064] 3) Immerse in hydrogen peroxide for 10 minutes to block the reaction, then rinse with pure water 3 times. Use an immunohistochemical pen to circle the tissue to be tested, rinse with pure water 2 times, and immerse in phosphate buffered saline (PBST) for 2-3 minutes.

[0065] 4) Primary antibody incubation: Add 100 μL of recombinant rabbit monoclonal antibody against fumarate hydratase (FH) to completely cover the tissue, incubate at room temperature for 1 h, and then wash with PBST 3 times for 3 min each time.

[0066] 5) Secondary antibody incubation: Incubate with secondary antibody for 30 min according to the instructions of the DAB staining solution kit for the secondary antibody staining system used, and rinse with PBST 3 times, 3 min each time.

[0067] 6) DAB color development: Prepare the DAB color development solution according to the kit instructions. Add an appropriate amount of the prepared DAB color development solution to completely cover the tissue, incubate for 10 min, and rinse with pure water 3 times, 3 min each time.

[0068] 7) Hematoxylin counterstaining: Follow the instructions and recommendations of the hematoxylin manufacturer to counterstain the sections for 2 minutes, then perform PBST blueing for 5 minutes.

[0069] 8) Dehydrate and become transparent: Soak in alcohol for 2-3 minutes, then air dry.

[0070] 9) Mounting: Mount the sample with neutral resin.

[0071] 10) Perform slice scanning.

[0072] 11) Results analysis and statistics.

[0073] Immunohistochemical staining results are generally classified as positive or negative. Positive expression must occur at a specific antigenic site on the cell or tissue to be considered positive. When tissue staining distribution is clear and cell localization is accurate, staining results can be further subdivided based on differences in staining intensity, as follows:

[0074] 1. The sample is weakly positive; it is marked as "+";

[0075] 2. The sample is moderately positive; marked as "++";

[0076] 3. The sample is highly positive; marked as "+++";

[0077] 4. If the sample is negative, mark it as "-".

[0078] The obtained monoclonal antibody against fumarate hydratase (FH) and the commercially available antibody fumarate hydratase (FH) (Elabscience, E-AB-22031) were used to simultaneously detect and compare the results in 40 cases of clear cell renal cell carcinoma. The entire experiment was conducted using a double-blind design, and the immunohistochemical results of fumarate hydratase (FH) were statistically analyzed. The results are shown in Table 2 below. Figure 1 This is a comparison of immunohistochemical staining results for a case of clear cell renal cell carcinoma. Figure 1 In section A, LBP2-FH of this application is adopted; Figure 1 The enzyme used in the Chinese sample (B) is commercially available fumarate hydratase (FH) (E-AB-22031).

[0079] Table 2. Statistical analysis of immunohistochemical results of fumarate hydratase (FH)

[0080]

[0081] from Figure 1As can be seen, for the same clear cell renal cell carcinoma tissue, the staining intensity of the LBP2-FH monoclonal antibody provided in this application is significantly stronger than that of commercially available fumarate hydratase (FH) (E-AB-22031). Furthermore, the staining localization of the LBP2-FH monoclonal antibody provided in this application is accurate, the staining is clear and there is no nonspecific staining, and the background is clean.

[0082] As shown in Table 2, in immunohistochemical detection, the positive rate of the antibody in this application is comparable to that of commercially available antibodies. All 20 known negative samples (FH deletion mutation samples) were negative, indicating that the specificity of immunohistochemical staining for LBP2-FH is comparable to that of commercially available antibodies. Among them, the positive intensity of the LBP2-FH monoclonal antibody in 7 cases was higher than that of commercially available antibodies, indicating that the sensitivity of the LBP2-FH monoclonal antibody provided in this application is higher than that of commercially available antibodies.

[0083] Example 3 Affinity Measurement

[0084] The affinity of the recombinant rabbit monoclonal antibody against fumarate hydratase (FH) was determined using the following method:

[0085] The ability of antibodies to bind to antigens was characterized using Biacore SPR. Specifically, a proteinA chip (channel 1 is the reference channel, and channel 2 is the ligand channel) was used. The antibody, as a ligand, was immobilized in channel 2 of the proteinA chip at a concentration of 10 μg / ml. The binding time was 30 s, and the flow rate was 10 μl / min.

[0086] Human fumarate hydratase (FH) antigen was used as the analyte, and the antigen sample ID was hu-FH. The LBP2-FH monoclonal antibody in this application was used as the antibody, and the antibody was diluted with running buffer to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM and 0 nM respectively.

[0087] The analytical stream was fed through channels 1 and 2, with a binding time of 60 s and a dissociation time of 90 s, at a flow rate of 30 μL / min. After analyte loading, the chip surface was regenerated using glycine buffer at pH 1.5. Ligand immobilization was required before each analyte loading, and the chip surface was regenerated with regeneration buffer after analyte loading.

[0088] The affinity of the fumarate hydratase (FH) antibody for human fumarate hydratase (FH) antigen at different concentrations was determined using Biacore SPR. The binding and separation were analyzed using a 1:1 kinetic model with corresponding analytical software. A control group was set up, in which the antibody was commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031), and the other test parameters and conditions were the same as above.

[0089] The results are shown in Table 3 and Figure 2 , Figure 3 As shown, Figure 2 and Figure 3 The original spectra were obtained from the data in Table 3.

[0090] In Table 3, KD represents the dissociation constant, a comprehensive indicator of affinity. A lower KD value indicates higher intermolecular affinity, meaning a lower concentration is needed to achieve a certain degree of binding, implying that the two molecules bind more easily to form a more stable complex. Kon represents the binding rate constant, reflecting the speed at which the antibody binds to the antigen; a higher value indicates a more efficient binding process. Kdis represents the dissociation rate constant, reflecting the speed of complex dissociation and is a core parameter for measuring the stability of antibody-antigen complexes; a lower value indicates a more stable complex and higher affinity.

[0091] Table 3 Comparison of affinity test results (NA indicates affinity too low to fit accurately)

[0092]

[0093] According to the data comparison in Table 3, the affinity of the LBP2-FH monoclonal antibody provided in this application to human fumarate hydratase (FH) antigen is much higher than that of commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031) to human fumarate hydratase (FH) antigen. Therefore, the LBP2-FH monoclonal antibody of this application has higher sensitivity.

[0094] Example 4: Western blotting detection

[0095] The recombinant rabbit monoclonal antibody LBP2-FH against fumarate hydratase (FH) was used as the primary antibody for Western blotting detection, as follows:

[0096] 1) Activate the membrane using polyvinylidene fluoride (PVDF) in HeLa / HEK293T / HepG2 / MCF7 / NIH / 3T3 / PC-12 cell lysate. Activate with methanol for 1 min, wash three times with TBST, and block with 5% skim milk for 1 h. TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and the surfactant Tween-20.

[0097] 2) Primary antibody incubation: Place the blocked PVDF membrane in a solution containing diluted LBP2-FH antibody and incubate overnight at 4°C. After incubation, remove the membrane and wash it 5 times with TBST for 5 minutes each time.

[0098] 3) Secondary antibody incubation: Place the cleaned PVDF membrane in a diluted HRP-anti-rabbit IgG (1:10000) solution and shake at room temperature for 1 hour. After incubation, remove the membrane and wash it 5 times with TBST for 5 minutes each time.

[0099] 4) Prepare the developing solution according to the Immobilon Western Developing Kit instructions, and evenly drop it onto the above membrane. Then, develop the membrane using a GelView+6000ProⅡ multi-functional image workstation according to the instructions.

[0100] The theoretical molecular weight of fumarate hydratase (FH) protein is around 54 kDa. There is also an N-terminal deleted isoform with a theoretical molecular weight of around 50 kDa. According to literature reports, the actual measured molecular weight is smaller than the theoretical value.

[0101] Western blotting results are as follows Figure 4 As shown, where, Figure 4 In section B, LBP2-FH prepared according to the present invention is used. Figure 4 In the study, fumarate hydratase (FH) (Elabscience, E-AB-22031) was used. Figure 4 The HeLa / HEK293T / HepG2 / MCF7 cell lines were positive, while the NIH / 3T3 / PC-12 cell lines were negative.

[0102] according to Figure 4It is evident that the fumarate hydratase (FH) monoclonal antibody prepared in this application and the commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031) antibody showed no bands at NIH / 3T3 / PC-12 in the Western blotting results. However, in the HeLa / HEK293T / HepG2 / MCF7 bands, the fumarate hydratase (FH) monoclonal antibody prepared in this application showed only one band at the theoretical molecular weight, while the commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031) antibody showed two bands. This indicates that the recombinant rabbit monoclonal antibody against fumarate hydratase (FH) in this application can specifically recognize the full-length fumarate hydratase (FH) protein, demonstrating superior specificity compared to commercially available antibodies.

[0103] In summary, the fumarate hydratase (FH) monoclonal antibody prepared in this application is superior to commercially available products in terms of sensitivity, affinity, and specificity, and has significant technological advancements compared to existing technologies.

[0104] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. An antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The light chain variable regions VL respectively include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

2. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 1, characterized in that, The antibody or its antigen-binding fragment can be K D It binds to fumarate hydratase with an affinity of ≤10 nM.

3. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 1 or 2, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:17; The variable region of the light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:

18.

4. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 3, characterized in that, The heavy chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:8; The light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:

16.

5. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 4, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 4, characterized in that, The antibody or its antigen-binding fragment is a half antibody or an antigen-binding fragment of a half antibody.

7. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 5, characterized in that, The monoclonal antibody is a single-chain antibody or a chimeric antibody.

8. The antibody or antigen-binding fragment thereof that binds to anti-fumarate hydratase according to claim 5, characterized in that, The monoclonal antibody is a fully or partially humanized antibody.

9. A biomaterial, characterized in that, The biomaterial is any one of the following: A) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8; B) A carrier containing the nucleic acid molecules described in A); C) A host cell containing the nucleic acid molecule described in A) or the vector described in B); D) A composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or a nucleic acid molecule as described in A), or a vector as described in B), or a host cell as described in C).

10. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8 in the preparation of fumarate hydratase detection products.

11. The application according to claim 10, characterized in that, The detection products are immunohistochemical detection products, ELISA detection products, or chemiluminescence detection products.

12. A detection reagent for detecting fumarate hydratase, characterized in that, It contains an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8.

13. A kit for detecting fumarate hydratase, characterized in that, It contains an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or a detection reagent as described in claim 12.

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