Antibody combined with anti-fumaric acid hydratase or antigen-binding fragment thereof and application of antibody or antigen-binding fragment

Abstract

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

Description

TECHNICAL FIELD

[0001] The application relates to an antibody or antigen-binding fragment thereof binding to fumarate hydratase and application thereof, and belongs to the technical field of biological medicines. BACKGROUND

[0002] Fumarate hydratase (FH), also known as fumarase, is a highly conserved key protein involved in energy metabolism in prokaryotes and eukaryotes, which has multiple biological functions such as regulating mitochondrial tricarboxylic acid cycle, urea cycle, purine nucleotide cycle, DNA damage repair and tumor suppression. As an important metabolism-related enzyme, the tumor related to the functional defect of FH has relatively special characteristics in pathogenesis, clinical symptoms, pathological morphology, immunophenotype, treatment outcome and prognosis. The role of abnormal metabolism of such organisms in tumor occurrence and development has attracted widespread attention in recent years. The FH antibody is an antibody capable of specifically recognizing and binding to the FH protein.

[0003] The FH gene is located in the q43 region of chromosome 1 and contains 10 exons. The transcription and translation product of the FH gene can be located in mitochondria and cytoplasm and exerts biological functions in the form of a homotetramer. The FH monomer is composed of 510 amino acids and contains three key functional domains, namely, an N-terminal fumarate lyase-1 domain, a C-terminal fumarase-C domain and a central domain for binding to other monomers. The N-terminal extended form exists in mitochondria and participates in the tricarboxylic acid cycle to catalyze the generation of L-malic acid from fumaric acid. The cytoplasmic type of FH participates in the metabolism of amino acids and fumaric acid and can be transferred to the nucleus to participate in the repair of DNA damage.

[0004] The defect of the FH gene can cause the cell to lose the ability to metabolize fumaric acid, resulting in a series of compensatory metabolic changes such as pseudo-hypoxic drive, oxidative stress, reduced AMPK level and inhibited DNA repair, thereby triggering carcinogenic changes, such as causing hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, paraganglioma, pheochromocytoma, testicular interstitial cell tumor, ovarian cystadenoma and other diseases. Among them, HLRCC has a dangerous course and poor prognosis. The clinical manifestations are FH-deficient skin and / or uterine leiomyoma and renal cell carcinoma. FH-deficient leiomyoma accounts for 0.4% to 1.6% of uterine leiomyoma, and the onset is 10 years earlier than other leiomyomas. Up to 62% of HLRCC syndrome patients develop renal cell carcinoma at a young age, which is the main cause of death.

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

[0006] The main detection method for FH deficiency in clinical practice is immunohistochemistry (immunohistochemistry), which is a traditional detection means for detecting the expression amount of target protein in tissues by using the principle of specific binding of antigen-antibody. Immunohistochemical detection mainly relies on antibodies that specifically bind to target antigens for detection, and there are few FH antibodies available, and the affinity for FH needs to be improved. SUMMARY

[0007] In order to solve the above problems, an antibody or antigen-binding fragment thereof that binds to fumarate hydratase and its application are provided. The fumarate hydratase antibody or antigen-binding fragment thereof prepared in the present application is superior to commercially available products in terms of sensitivity, affinity and specificity, and has better market competitiveness compared to the prior art.

[0008] The present application provides an antibody or antigen-binding fragment thereof that binds to fumarate hydratase, which 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; 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.

[0009] Optionally, the antibody or antigen-binding fragment thereof can bind to fumarate hydratase with a K D ≤10nM affinity.

[0010] Optionally, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 17; The light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 18.

[0011] Optionally, the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 8; The light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 16.

[0012] Optionally, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; or, The antibody or antigen-binding fragment thereof is a half antibody or an antigen-binding fragment of a half antibody.

[0013] The present application provides a biological material, which is any one of the following: A) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described above; B) a vector comprising the nucleic acid molecule described in A); C) a host cell comprising the nucleic acid molecule described in A) or the vector described in B); D) a composition comprising the antibody or antigen-binding fragment thereof described above, or the nucleic acid molecule described in A), or the vector described in B), or the host cell described in C).

[0014] The present application provides the use of the antibody or antigen-binding fragment thereof described above in the preparation of a fumarate hydratase detection product.

[0015] Optionally, the detection product is an immunohistochemical detection product, an ELISA detection product, or a chemiluminescence detection product.

[0016] The present application provides a detection reagent for detecting fumarate hydratase, comprising the antibody or antigen-binding fragment thereof described above.

[0017] The present application provides a kit for detecting fumarate hydratase, comprising the antibody or antigen-binding fragment thereof described above or comprising the detection reagent described above.

[0018] The beneficial effects of the present application include but are not limited to: The fumarate hydratase antibody or antigen-binding fragment thereof prepared by the present application is superior to commercially available products in terms of sensitivity, affinity, and specificity, and the affinity is significantly improved, which has better market competitiveness compared to the prior art.

[0019] The fumarate hydratase antibody or antigen-binding fragment thereof prepared by the present application has high specificity and sensitivity, can specifically recognize cells expressing fumarate hydratase (FH) protein, is not only suitable for immunological detection, but also can effectively prevent the occurrence of false positive and false negative phenomena, and can significantly improve the accuracy of detection and diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which are included to provide a further understanding of the present application and constitute a part of this application, illustrate certain illustrative embodiments of the present application and together with the description serve to explain the present application. In the drawings: Figure 1 Figure 2 shows the comparison of immunohistochemical staining results of one case of renal clear cell carcinoma (A: LBP2-FH prepared in the present application; B: commercially available fumarate hydratase FH (Elabscience, E-AB-22031)); Figure 2 Figure 3 shows the kinetics curve of the LBP2-FH monoclonal antibody and the antigen; Figure 3 Figure 4 shows the kinetics curve of the commercially available fumarate hydratase FH (Elabscience, E-AB-22031) antibody and the antigen; Figure 4 Figure 5 shows the comparison of immunoblotting detection results (A: commercially available fumarate hydratase FH (Elabscience, E-AB-22031); B: LBP2-FH prepared in the present application). DETAILED DESCRIPTION

[0021] The present application will be described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0022] Example 1 1) Preparation of antigen The antigen is the N-terminal amino acid sequence AA 1-200 of recombinant human fumarate hydratase (FH) (Uniport code: P07954, in the UniProt database, detailed information such as amino acid sequence, domain, functional annotation can be queried according to the number). The antigen sequence (SEQ ID NO: 19) is: MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARMASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMPTPVIKAFGILKRAAAEVNQDYGLDPKIANAIMKAADEVAEGKLNDHFPLVVWQTGSGTQTNMNVNEVISNRAIEMLGGELGSKIPVHPNDHVNKSQSSNDTFPTAMHIAAA For ease of description, the recombinant human fumarate hydratase (FH) antigen is labeled as hu-FH.

[0023] 2) Immunization 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.

[0024] 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.

[0025] 3) Single B cell screening and sequencing 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).

[0026] 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.

[0027] 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.

[0028] Using single B cell sequencing technology, the efficiency of screening high affinity antibodies and the success rate of cloning are improved. Because B cells cannot survive for a long time in vitro, even if the ideal antibody is produced, it will be lost due to the death of B cells. The traditional antibody screening method mostly uses hybridoma technology to fuse B cells with myeloma cells to obtain the ability of immortality. However, the defect of this technology is that the success rate of fusion is generally only about 5%, and hybridoma cells also have the possibility of two cell rejection and ultimately losing antibody information. Single B cell sequencing technology is to use B cell separation equipment to sort single B cells into culture dishes, and use cell culture medium rich in nutrients and rich in growth factors that can stimulate B cell growth, which can screen B cells that can express high affinity antibodies with very low loss rate, and DNA sequencing. DNA sequences can be permanently preserved or regenerated through vector cloning technology.

[0029] 4) Preparation and purification of cell supernatant monoclonal antibody After confirming the positive expression vector, a large number of cell transfection was carried out, and after 3-5 days of continuous culture, the cell suspension was collected, centrifuged, and the supernatant was taken. Using affinity chromatography, the supernatant was purified using Protein A to obtain high-purity antibodies. Finally, the purified anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody was subjected to concentration determination and sub-packaging, labeled as LBP2-FH (anti-fumarate hydratase (FH) protein monoclonal antibody), and stored in a 4°C refrigerator.

[0030] The obtained anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody was sequenced, which included heavy chain and light chain. The heavy chain included heavy chain variable region and heavy chain constant region; the light chain included light chain variable region and light chain constant region.

[0031] The amino acid sequences of CDR1, CDR2, and CDR3 of 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 of 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, as shown in Table 1.

[0032] The amino acid sequences of CDR1, CDR2 and CDR3 of 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 of 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, for details, see Table 1.

[0033] Table 1 Sequences of anti-fumarate hydratase (FH) protein monoclonal antibody

[0034] Table 1 Table 1

[0035] Table 1 Table 2

[0036] Example 2 Immunohistochemical detection The anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody was used as a primary antibody for immunohistochemical detection, and the method was as follows: 1) Sample section preparation: The kidney clear cell carcinoma fixed with formalin and embedded in paraffin was baked in a constant temperature oven at 60°C for 1-2h, and stored for standby use.

[0037] 2) Use an automatic repair instrument to deparaffinize, hydrate and repair the section; in this process, the temperature is preferentially controlled at 95°C, and the time is preferentially controlled at 30min.

[0038] 3) Block by soaking in a hydrogen peroxide jar for 10min, then take out and rinse with pure water for 3 times, then circle the tissue to be tested with an immunohistochemical pen, rinse with pure water for 2 times, and soak in phosphate buffered saline (PBST) for 2-3min.

[0039] 4) Primary antibody incubation: add 100μL of anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody to completely cover the tissue, incubate at room temperature for 1h, and then rinse with PBST for 3 times, each for 3min.

[0040] 5) Secondary antibody incubation: incubate the secondary antibody according to the instructions of the DAB staining system secondary antibody staining kit for 30min, and then rinse with PBST for 3 times, each for 3min.

[0041] 6) DAB color development: configure the DAB color developing solution according to the instructions of the kit, drop an appropriate amount of the prepared DAB color developing solution to completely cover the tissue, incubate for 10min, and then rinse with pure water for 3 times, each for 3min.

[0042] 7) Hematoxylin counterstaining: according to the operation steps and suggestions of the hematoxylin manufacturer, the slice is counterstained for 2 min, and PBST is returned to blue for 5 min.

[0043] 8) Dehydration and transparency: soak in alcohol for 2-3 min, and air dry.

[0044] 9) Mounting: the sample is mounted with neutral gum.

[0045] 10) Slice scanning.

[0046] 11) Result analysis and statistics.

[0047] The immunohistochemical staining results are usually divided into positive and negative. Positive expression must be at the specific antigen site of cells and tissues to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results can be further divided according to the difference in staining intensity, as follows: 1. The sample is weakly positive, marked as "+"; 2. The sample is moderately positive, marked as "++"; 3. The sample is highly positive, marked as "+++"; 4. The sample is negative, marked as "-".

[0048] The obtained fumarate hydratase (FH) monoclonal antibody and the commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031) were used to synchronously detect 40 cases of renal clear cell carcinoma and compare the detection results. The whole test process adopted a double-blind design, and the results of the immunohistochemistry of fumarate hydratase (FH) were statistically analyzed, and the statistical results are shown in Table 2. Figure 1 The following is a comparison chart of the immunohistochemical staining results of one case of renal clear cell carcinoma, wherein, Figure 1 LBP2-FH of the present application was used in middle A; Figure 1 Commercial fumarate hydratase (FH) (E-AB-22031) was used in middle B.

[0049] Table 2. Immunohistochemical results of fumarate hydratase (FH)

[0050] From Figure 1 it can be seen that for the same renal clear cell carcinoma tissue, the staining intensity of the LBP2-FH monoclonal antibody provided by the present application is obviously stronger than that of the commercially available fumarate hydratase (FH) (E-AB-22031), and the LBP2-FH monoclonal antibody provided by the present application has accurate staining localization, clear staining, no non-specific staining, and clean background.

[0051] As can be seen from Table 2, in the immunohistochemical detection, the positive rate of the antibody of the application is comparable to that of the commercially available antibody, and the detection of 20 known negative samples (FH deletion mutant samples) is all negative, indicating that the immunohistochemical staining detection specificity of LBP2-FH is comparable to that of the commercially available antibody, and the positive intensity of 7 LBP2-FH monoclonal antibodies is higher than that of the commercially available antibody, indicating that the sensitivity of the LBP2-FH monoclonal antibody provided by the application is higher than that of the commercially available antibody.

[0052] Example 3 Affinity determination The affinity determination of the anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody was performed as follows: The ability of the antibody to bind to the antigen was characterized by Biacore SPR. Specifically, a protein A chip (1 channel as a reference channel and 2 channel as a ligand channel) was used, and the antibody was immobilized to the 2nd channel of the protein A chip as a ligand at a concentration of 10 μg / ml, the binding time was 30 s, and the flow rate was 10 ul / min.

[0053] The human fumarate hydratase (FH) antigen was used, and the antigen was used as an analyte, and the antigen sample ID was hu-FH; the LBP2-FH monoclonal antibody in the application was used as an antibody, and the antibody was diluted to 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM and 0 nM using running buffer.

[0054] The analyte flowed through the 1st channel and the 2nd channel, the binding time was 60 s, the dissociation time was 90 s, and the flow rate was 30 ul / min. After the analyte was loaded, the chip surface was regenerated using a glycine buffer with pH = 1.5. Before each analyte loading, the ligand was first immobilized, and after the analyte loading was completed, the chip surface was regenerated with the regeneration solution.

[0055] The affinity of the fumarate hydratase (FH) antibody to the human fumarate hydratase (FH) antigen at different concentrations was determined by Biacore SPR, and the corresponding analysis software was used to analyze the binding and separation by using a 1:1 kinetic model; at the same time, a control group was set, and the antibody in the control group was a commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031), and the rest of the test parameters and conditions were the same as above.

[0056] The results are shown in Table 3 and Figure 2 , Figure 3 , Figure 2 and Figure 3 The raw data of the determination of the data in Table 3 are shown in the original graph.

[0057] In Table 3, KD represents the dissociation constant, reflecting the comprehensive index of affinity, the lower the KD value, the higher the affinity between molecules, that is, a lower concentration is required to achieve a certain degree of binding, which means that the two molecules are more likely to bind to form a complex and more stable. kon represents the association rate constant, reflecting the speed of antibody and antigen binding, the larger the value, the more efficient the binding process. Kdis represents the dissociation rate constant, reflecting the speed of complex dissociation, which is the core parameter for measuring the stability of antibody-antigen complex, the smaller the value, the more stable the complex, the higher the affinity.

[0058] Table 3 Comparison of affinity determination results (NA indicates that the affinity is too low to accurately fit)

[0059] According to the comparison of data in Table 3, the affinity of the LBP2-FH monoclonal antibody provided by the application to human fumarate hydratase (FH) antigen is much higher than that of the commercially available fumarate hydratase (FH) (Elabscience, E-AB-22031) to human fumarate hydratase (FH) antigen. It can be known that the LBP2-FH monoclonal antibody of the application has higher sensitivity.

[0060] Example 4 Western blotting detection The anti-fumarate hydratase (FH) recombinant rabbit monoclonal antibody LBP2-FH was used as a primary antibody for Western blotting detection, and the method was as follows: 1) Select polyvinylidene fluoride (PVDF) membrane of Hela / HEK293T / HepG2 / MCF7 / NIH / 3T3 / PC-12 cell lysate for activation, methanol activation for 1 min, and TBST washing for 3 times; 5% skim milk blocking for 1 h; TBST is a commonly used washing buffer suitable for experiments such as Western blotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and surfactant Tween-20.

[0061] 2) Primary antibody incubation: place the blocked PVDF membrane in a solution containing diluted LBP2-FH antibody solution, incubate at 4°C overnight, and then take out the membrane and shake wash with TBST for 5 times, each time for 5 min.

[0062] 3) Secondary antibody incubation: place the washed PVDF membrane in a diluted HRP-anti-rabbit IgG (1:10000) solution, shake at room temperature for 1 h, incubate, then take out the membrane and shake wash with TBST for 5 times, each time for 5 min.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] according to Figure 4 It 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.

[0067] 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.

[0068] 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, a single-chain antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; or, The antibody or its antigen-binding fragment is a half antibody or an antigen-binding fragment of a half antibody.

6. 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 5; 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 5, or a nucleic acid molecule as described in A), or a vector as described in B), or a host cell as described in C).

7. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5 in the preparation of fumarate hydratase detection products.

8. The application according to claim 7, characterized in that, The detection products are immunohistochemical detection products, ELISA detection products, or chemiluminescence detection products.

9. A detection reagent for detecting fumarate hydratase, characterized in that, It contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5.

10. A kit for detecting fumarate hydratase, characterized in that, It contains an antibody or its antigen-binding fragment as described in any one of claims 1 to 5, or a detection reagent as described in claim 9.

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