Anti-human thrombopoietin antibody AF687, kit and application thereof

Abstract

The invention provides an anti-human thrombopoietin antibody AF687, a kit and application thereof. The anti-human thrombopoietin antibody disclosed by the invention can specifically recognize epitopes without glycosylation modification of natural TPO protein, and has relatively high affinity for recombinant TPO protein and specific epitopes of the recombinant TPO protein. Meanwhile, the anti-human thrombopoietin antibody disclosed by the invention can accurately detect the content of human thrombopoietin in serum, is not influenced by heterogeneity of natural protein glycosylation sites, can be used for accurately identifying and diagnosing early aplastic anemia and primary immune thrombocytopenia, and has the advantages that compared with an existing detection reagent, the anti-human thrombopoietin antibody is high in sensitivity and high in sensitivity. The false positive rate can be reduced from 13.9% to 2.7%, and a more accurate detection method is provided for differential diagnosis of the primary immune thrombocytopenia.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to an anti-human thrombopoietin antibody AF687, a kit and applications thereof. Background Art

[0002] Thrombopoietin (TPO) is a cytokine that promotes platelet production. It is primarily synthesized by liver cells and released into the blood. Mature TPO contains 332 amino acids and is composed of two distinct domains: an EPO-like domain (amino acid residues 22-153) and an unstructured domain (amino acid residues 154-332). It also contains eight O-glycosylation sites and six N-glycosylation sites (Uniprot ID: P40225). After binding to its receptor, TPO exerts diverse effects depending on the cell type, such as maintaining the survival of hematopoietic stem cells, stimulating the mitosis of megakaryocyte colony-forming cells and megakaryocytes, stimulating megakaryocyte maturation, and promoting platelet differentiation and maturation.

[0003] Under normal physiological conditions, TPO is synthesized and released in the liver at a constant rate and is cleared primarily by binding to TPO receptors on the platelet surface, resulting in low concentrations in the blood. In conditions of decreased platelet production, such as those caused by chemotherapy, aplastic anemia, and myelodysplastic syndrome, the ability of platelets and megakaryocytes to clear TPO is impaired, leading to a significant increase in serum TPO levels to promote megakaryocyte proliferation and platelet production. However, in patients with primary immune thrombocytopenia (ITP), rapid immune clearance of platelets accelerates the clearance of platelet-bound TPO, and a certain number of megakaryocytes remain in the blood, maintaining plasma TPO levels at normal to slightly elevated levels. Therefore, blood TPO levels can serve as an effective marker for the differential diagnosis of ITP and are recommended in ITP diagnosis and treatment guidelines. The Chinese Guidelines for the Diagnosis and Treatment of Primary Immune Thrombocytopenia in Adults point out that TPO is a special laboratory test for diagnosing ITP. The determination of serum TPO levels is helpful in the differential diagnosis of ITP (normal TPO levels) and bone marrow failure diseases (elevated TPO levels).

[0004] Mass spectrometry analysis showed that the glycosylation modifications at multiple sites of the TPO protein were not uniform. On the one hand, some glycosylation sites were incompletely modified, and on the other hand, the sugar chains at most glycosylation sites had multiple sugar combinations, which means that the TPO protein does not exist in a uniform form in the blood. This protein heterogeneity caused by post-translational modification may affect immunoassays, causing some types of proteins to be unable to be effectively identified by immunological tests, resulting in low test values and thus deviations in clinical diagnosis. Previous research literature also showed that in large-scale clinical sample testing, among patients with thrombocytopenia caused by aplastic anemia, chemotherapy, etc., the serum TPO levels of some patients were no different from those of healthy people or ITP patients.

[0005] A major problem with existing detection methods is that the heterogeneity of TPO protein glycosylation in serum can lead to lower-than-actual levels in some samples, resulting in low specificity for distinguishing ITP from thrombocytopenia caused by chemotherapy or aplastic anemia. Recent studies have developed antibodies for sandwich assays of serum TPO levels, but one of these antibodies targets a glycosylation site, potentially leading to this problem. Therefore, there is a need to develop new monoclonal antibodies and immunoassays for serum TPO levels that are not significantly affected by the heterogeneity of native TPO protein glycosylation modifications and that exhibit high sensitivity and specificity for the differential diagnosis of ITP from thrombocytopenia caused by chemotherapy or aplastic anemia. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide an anti-human thrombopoietin (TPO) antibody, a kit and applications thereof.

[0007] In the first aspect of the present invention, an anti-human thrombopoietin (TPO) antibody or an antigen-binding fragment thereof is provided, characterized in that it includes a light chain variable region and a heavy chain variable region, and the amino acid sequences of the complementary determining regions HCDR1, HCDR2 and HCDR3 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 the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0009] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0010] Furthermore, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.9.

[0011] Furthermore, the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.10.

[0012] Furthermore, the antibody or antigen-binding fragment thereof is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment, a sc(Fv)2 fragment, a F(ab')2 fragment, a Fab or Fab' fragment, a full-length antibody, a bivalent antibody, a trivalent antibody, and a tetravalent antibody.

[0013] Furthermore, the antibody is a rabbit-derived antibody.

[0014] In a second aspect of the present invention, a biomaterial related to the antibody or antigen-binding fragment thereof according to the first aspect of the present invention is provided, wherein the biomaterial comprises at least one of the following 1) to 5):

[0015] 1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0016] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0017] 3) a recombinant vector containing the nucleic acid molecule described in 1);

[0018] 4) a recombinant microorganism or recombinant cell containing the nucleic acid molecule described in 1);

[0019] 5) A recombinant microorganism or recombinant cell containing the recombinant vector described in 3).

[0020] Furthermore, in 4) or 5), the recombinant microorganism is a recombinant Escherichia coli.

[0021] Furthermore, in 4) or 5), the recombinant cell is an animal cell or a human cell.

[0022] In the third aspect of the present invention, an antibody conjugate is provided, characterized in that it comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and a detection label connected to the antibody or antigen-binding fragment thereof.

[0023] Furthermore, the detection label is selected from one or more of biotin, fluorescein, chemiluminescent group, chemifluorescent group, fluorescent protein, horseradish peroxidase, acid phosphatase, colloidal gold, colored magnetic beads, magnetic microspheres, latex particles, radioactive nuclides and detection antibodies.

[0024] In a fourth aspect of the present invention, a kit is provided, characterized in that it comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the antibody conjugate according to the third aspect of the present invention.

[0025] Furthermore, the kit is a chemiluminescence kit, an immunoblotting kit or an immunohistochemistry kit.

[0026] Furthermore, the kit is used to detect TPO protein or its epitope.

[0027] Furthermore, the epitope is an epitope without a glycosylation site.

[0028] Furthermore, the kit is used to diagnose thrombocytopenia caused by ITP or chemotherapy or aplastic anemia.

[0029] In the fifth aspect of the present invention, there is provided the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention in the preparation of a product for detecting TPO protein or its epitope.

[0030] Furthermore, the epitope is an epitope without a glycosylation site.

[0031] Furthermore, the product is a reagent or a kit.

[0032] Furthermore, the kit is a chemiluminescence kit, an immunoblotting kit or an immunohistochemistry kit.

[0033] In the sixth aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention in the preparation of a product for diagnosing thrombocytopenia caused by ITP or chemotherapy or aplastic anemia.

[0034] Furthermore, the product is a reagent or a kit.

[0035] Furthermore, the kit is a chemiluminescence kit, an immunoblotting kit or an immunohistochemistry kit.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0037] Compared with the prior art, the present invention has the following advantages and improvements:

[0038] The anti-human thrombopoietin antibody of the present invention can specifically recognize an epitope of the natural TPO protein without glycosylation modification and has a high affinity for both the recombinant TPO protein and its specific epitope. The EC50 value for binding to the recombinant TPO protein can reach below 8 ng / mL, and the EC50 value for binding to the specific epitope can reach below 10 ng / mL. Furthermore, the anti-human thrombopoietin antibody of the present invention can accurately detect the level of human thrombopoietin in serum, unaffected by the heterogeneity of glycosylation sites of the natural protein. It can be used to accurately differentiate between early-stage aplastic anemia and primary immune thrombocytopenia. Compared with existing detection reagents, the false positive rate can be reduced from 13.9% to 2.7%, providing a more accurate test method for the differential diagnosis of primary immune thrombocytopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0040] Figure 1 Figure 2: SDS-PAGE electrophoresis results of recombinant full-length anti-human TPO antibody AF687. The purified AF687 antibody and a bovine serum albumin (BSA) control sample were resolved by 10% polyacrylamide gel electrophoresis under 50 mM dithiothreitol reducing conditions. The results showed that the AF687 antibody exhibited two bands with molecular weights of approximately 50 kDa and 25 kDa, representing the heavy and light chains of the antibody, respectively. BSA exhibited a single band with a molecular weight of approximately 70 kDa.

[0041] Figure 2 : Calibration curve of TPO chemiluminescence kit. DETAILED DESCRIPTION

[0042] The present invention provides an anti-human thrombopoietin antibody, a kit and its application, and its mutants and applications. The present invention is described in detail below in conjunction with the examples to facilitate further understanding of the present invention by those skilled in the art. However, the examples described below are only part of the examples of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. Specific technical operation steps and operators are not specified in the examples, and are carried out in accordance with the general technical conditions described in the literature of this field or the relevant product instructions.

[0043] Example 1 Expression of TPO recombinant protein and antibody preparation

[0044] A full-length TPO protein expression plasmid with a 6×His tag at the C-terminus was prepared by full gene synthesis (ordered from BGI). The vector was pcDNA-3.4, and the gene sequence used was NM_000460.3 (nucleotides 280 to 1341) in the NCBI database. The plasmid was transformed into the Escherichia coli DH5α strain for plasmid amplification, and the plasmid for cell transfection was extracted using an endotoxin-free plasmid extraction kit. 293T cells were cultured adherently in a T25 culture flask, and when the density reached about 80%, lipofactamine 3000 transfection reagent (Thermo Fisher, L3000075) was used for plasmid transfection (refer to the transfection reagent instructions). Three days after transfection, the cells were digested with trypsin, and the cell culture supernatant was collected by centrifugation at 1000 rpm for protein purification. The recombinant TPO protein was purified using Ni-NTA affinity chromatography medium (GenScript, L00250) according to the instructions. The recombinant TPO protein was replaced into PBS solution using an ultrafiltration tube and the final concentration was adjusted to 1 mg / mL.

[0045] Two New Zealand white rabbits were immunized with recombinant TPO protein as an immunogen. Each rabbit received 200 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant (Sigma, F5881) to form an emulsion, which was then injected subcutaneously at multiple sites on the rabbit's back. Three weeks thereafter, 100 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant (Sigma, F5506) to form an emulsion, which was then injected subcutaneously at multiple sites on the rabbit's back, for a total of four immunizations. One week after the final immunization, blood was collected from the ear vein, and serum was isolated for antibody titer determination. 1 μg / mL of recombinant TPO protein was immobilized on an ELISA plate, and rabbit serum was diluted with PBS to prepare a 1 / 100–1 / 125,000 dilution series. Standard enzyme-linked immunosorbent assay (ELISA) was used to obtain absorbance at OD 450 nm. Rabbits with a titer of 1 / 62,500 or higher were selected and sacrificed, and their spleens were harvested. B lymphocytes in the spleen are isolated by conventional methods, and then antigen-specific B lymphocytes are sorted. For related methods, please refer to the patent "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and the patent "A B lymphocyte in vitro culture system and application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0046] Example 2 Antibody Screening

[0047] The isolated single B cells were cultured using conventional methods, and the cell culture supernatant was assayed for titer using recombinant TPO protein. Nine single cell clones with higher titers were selected, lysed with cell lysis buffer, and total RNA was extracted and reverse transcribed into cDNA. The following primers were used to amplify the light and heavy chain variable regions of the rabbit monoclonal antibody:

[0048] VL-Primer-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC;

[0049] VL-Primer-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG;

[0050] VH-Primer-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG;

[0051] VH-Primer-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGAGCTG.

[0052] High-fidelity PCR (Yisheng Bio, 10153) was used for the amplification system. A 25 μL reaction volume was used, and 2 μL of cDNA was added. The remaining component amounts and reaction conditions were determined according to the reagent instructions. The amplified cDNA was sequenced by BGI, and the variable region nucleic acid sequences were obtained.

[0053] pcDNA-3.4 expression vectors containing the light chain variable region, light chain constant region, and heavy chain variable region, heavy chain constant region were prepared using whole-gene synthesis. A signal peptide was attached to the nitrogen terminus of the antibody sequence to ensure secretion. Signal peptides used commonly used in the art for antibody expression signal peptide sequences, such as the light chain variable region upstream signal peptide "MDTRAPTQLLGLLLLWLPGARC" from the patents "Anti-human interferon α2 rabbit monoclonal antibodies and their applications (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and "High-affinity human IL-5 rabbit monoclonal antibodies and their applications (Publication No.: CN115819578A, Publication Date: 2023-03-21)," and the heavy chain variable region upstream signal peptide "METGLRWLLLVAVLKGVQC."

[0054] The expression vectors containing the light chain gene and the heavy chain gene were transfected into 293F cells together with PEI (Polyscience, 24765-1). The transfection method was carried out according to the manufacturer's instructions. After transfection, the cells were cultured for 72-96 hours and the cell culture supernatant was collected by centrifugation. The recombinant rabbit monoclonal antibody was purified from the supernatant of the culture medium after transfection using a protein A affinity column (Yisheng Biological, 36410ES08). The purification conditions were according to the manufacturer's instructions. After purification, 9 anti-human TPO antibodies (AF684, AF685, AF686, AF687, AF688, AF689, AF691, AF692, and AF693) corresponding to the 9 single cell clones with higher titers were obtained. Among them, the purified AF687 antibody was subjected to 10% polyacrylamide gel electrophoresis under dithiothreitol reducing conditions with a final concentration of 50mM to detect its purity and molecular weight. Figure 1 As shown, the AF687 antibody presents two bands with molecular weights of 50 KDa and 25 KDa, which are the heavy chain and light chain bands of the antibody, respectively. This shows that the AF687 antibody prepared in the above example has a correct structure and its molecular weight is consistent with the theoretical value.

[0055] The specific amino acid sequence and encoding nucleic acid sequence of antibody AF687 are as follows:

[0056] Anti-human TPO antibody AF687 heavy chain variable region HCDR1 amino acid sequence:

[0057] CKASGFS (SEQ ID NO. 1).

[0058] Anti-human TPO antibody AF687 heavy chain variable region HCDR2 amino acid sequence:

[0059] WIGCIN (SEQ ID NO. 2).

[0060] Anti-human TPO antibody AF687 heavy chain variable region HCDR3 amino acid sequence:

[0061] CATQPGGAALW (SEQ ID NO. 3).

[0062] Anti-human TPO antibody AF687 light chain variable region LCDR1 amino acid sequence:

[0063] QASQSISSYLS (SEQ ID NO. 4).

[0064] Anti-human TPO antibody AF687 light chain variable region LCDR2 amino acid sequence:

[0065] DASTLAS (SEQ ID NO. 5).

[0066] Anti-human TPO antibody AF687 light chain variable region LCDR3 amino acid sequence:

[0067] CQNYYFSSSSSYGNVF (SEQ ID NO. 6).

[0068] Anti-human TPO antibody AF687 heavy chain variable region (VH) amino acid sequence:

[0069] QQLEQSGGGAEGGLVKPGGSLELCCKASGFSLSSTVWIFWVRQAPGKGLEWIGCI NGGSSGGTFYATWVNGRFTLSRDIDQSTGCLQLNGLTVADTGMYYCATQPGGAALW GPGTLVTVSS (SEQ ID NO. 7).

[0070] Anti-human TPO antibody AF687 light chain variable region (VL) amino acid sequence:

[0071] DIVMTQTPASVEAAVGGTVTIKCQASQSISSYLSWYQQKPGQPPNLLIYDASTLAS GVSSRFKGSGSGTEFTLTISDLECADAATYYCQNYYFSSSSSYGNVFGGGTEVVVK (SEQ ID NO. 8).

[0072] Anti-human TPO antibody AF687 heavy chain amino acid sequence:

[0073] QQLEQSGGGAEGGLVKPGGSLELCCKASGFSLSSTVWIFWVRQAPGKGLEWIGCINGGSSGGTFYATWVNGRFTLSRDIDQSTGCLQLNGLTVADTGMYYCATQPGGAALWG PGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPT CPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQID NO.9).

[0074] Anti-human TPO antibody AF687 light chain amino acid sequence:

[0075] DIVMTQTPASVEAAVGGTVTIKCQASQSISSYLSWYQQKPGQPPNLLIYDASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQNYYFSSSSSYGNVFGGGTEVVV KRDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO.10).

[0076] Anti-human TPO antibody AF687 heavy chain variable region HCDR1 encoding nucleic acid sequence:

[0077] TGCAAGGCCTCCGGCTTCTCC (SEQ ID NO. 11).

[0078] Anti-human TPO antibody AF687 heavy chain variable region HCDR2 encoding nucleic acid sequence:

[0079] TGGATCGGCTGCATCAAC (SEQ ID NO. 12).

[0080] Anti-human TPO antibody AF687 heavy chain variable region HCDR3 encoding nucleic acid sequence:

[0081] TGCGCCACCCAGCCCGGCGGCGCCGCCCTGTGG (SEQ ID NO. 13).

[0082] Anti-human TPO antibody AF687 light chain variable region LCDR1 encoding nucleic acid sequence:

[0083] CAGGCCTCCCAGTCCATCTCCTCCTACCTGTCC (SEQ ID NO. 14).

[0084] Anti-human TPO antibody AF687 light chain variable region LCDR2 encoding nucleic acid sequence:

[0085] GACGCCTCCACCCTGGCCTCC (SEQ ID NO. 15).

[0086] Anti-human TPO antibody AF687 light chain variable region LCDR3 encoding nucleic acid sequence:

[0087] TGCCAGAACTACTACTTCTCCTCCTCCTCCTCCTACGGCAACGTGTTC (SEQ ID NO. 16).

[0088] Anti-human TPO antibody AF687 heavy chain variable region (VH) encoding nucleic acid sequence:

[0089] CAGCAGCTGGAGCAGTCCGGCGGCGGCGCCGAGGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGGAGCTGTGCTGCAAGGCCTCCGGCTTCTCCCTGTCCTCCACCGTGTGGATCTTCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCTGCATCAACGGCGGCTCCTCCGGC GGCACCTTTCTACGCCACCTGGGTGAACGGCCGCTTCACCCTGTCCCGCGACATCGACCAGTCCACCGGCTGCCTGCAGCTGAACGGCCTGACCGTGGCCGACACCGGCATGTACTACTGCGCCACCCAGCCCGGCGGCGCCGCCCTGTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC(SEQ IDNO.17).

[0090] Anti-human TPO antibody AF687 light chain variable region (VL) encoding nucleic acid sequence:

[0091] GACATCGTGATGACCCAGACCCCCGCCTCCGTGGAGGCCGCCGTGGGCGGCACCGTGACCATCAAGTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAACCTGCTGATCTACGACGCCTCCACCCTGGCCTCCGG CGTGTCCTCCCGCTTCAAGGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCGACCTGGAGTGCGCCGACGCCGCCACCTACTACTGCCAGAACTACTTCTCCTCCTCCTCCTCCTACGGCAACGTGTTCGGCGGCGGCACCGAGGTGGTGGTGAAG(SEQ ID NO.18).

[0092] Anti-human TPO antibody AF687 heavy chain encoding nucleic acid sequence:

[0093]

[0094] Nucleic acid sequence encoding the light chain of anti-human TPO antibody AF687:

[0095] GACATCGTGATGACCCAGACCCCCGCCTCCGTGGAGGCCGCCGTGGGCGGCACCGTGACCATCAAGTGCCAGGCCTCCCAGTCCATCTCCTCCTACCTGTCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAACCTGCTGATCTACGACGCCTCCACCCTGGCCTCCGGCGTGTCCTCCCGCTTCAAGGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCGACCTGGAGTGCGCCGACGCCGCCACCTACTACTGCCAGAACTACTACTTCTCCTCCTCCTCCTCCTACGGCAACGTGTTCGGCGGCGGCACCGAGGTGGTGGTGAAGCGCGACCCCGTGGCCCCCACCGTGCTGATCTTCCCCCCCGCCGCCGACCAGGTGGCCACCGGCACCGTGACCATCGTGTGCGTGGCCAACAAGTACTTCCCCGACGTGACCGTGACCTGGGAGGTGGACGGCACCACCCAGACCACCGGCATCGAGAACTCCAAGACCCCCCAGAACTCCGCCGACTGCACCTACAACCTGTCCTCCACCCTGACCCTGACCTCCACCCAGTACAACTCCCACAAGGAGTACACCTGCAAGGTGACCCAGGGCACCACCTCCGTGGTGCAGTCCTTCAACCGCGGCGACTGC(SEQ ID NO.20).

[0096] Experiment on antibody binding ability in Example 3

[0097] The binding ability of the prepared antibodies to recombinant TPO protein was evaluated using an indirect ELISA. 1 μg / mL of recombinant TPO protein was coated onto an ELISA plate using carbonate buffer (pH 9.6). The plate was incubated at 2-8°C for 16 hours. After coating, the plate was washed twice with PBST buffer and blocked with PBST containing 5% BSA for 1 hour at 37°C. 100 μL of 5 ng / mL recombinant antibody was added to the first well, and the antibody was serially diluted two-fold to each subsequent well. The plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, followed by the addition of a goat anti-rabbit Fc antibody conjugated to horseradish peroxidase and incubated at 37°C for 1 hour. The plate was washed three times with PBST, followed by the addition of TMB colorimetric solution for 15 minutes in the dark. Stop solution was then added, and the absorbance at OD450 nm was measured on a microplate reader. The EC50 values were calculated based on the OD450 values. The results are shown in Table 1.

[0098] Indirect ELISA was used to evaluate the binding ability of the prepared antibodies to various non-glycosylated epitope sequences on the TPO protein (Table 2). Each epitope sequence was obtained through contract synthesis (GenScript) with a purity of 95%. ELISA plates were coated with 0.5 μg / mL of the epitope sequence using carbonate buffer (pH 9.6) and incubated at 2-8°C for 16 hours. After coating, the plate was washed twice with PBST buffer and blocked with PBST containing 5% BSA for 1 hour at 37°C. Then, 100 μL of 2 μg / mL recombinant antibody was added to the first well, and the antibody was added to subsequent wells in a three-fold serial dilution. The plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, and then a goat anti-rabbit Fc antibody conjugated to horseradish peroxidase was added and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and then TMB colorimetric solution was added for 15 minutes in the dark. Stop solution was then added, and the absorbance at OD450 nm was measured on a microplate reader. The curve was drawn based on the OD450 value to obtain the EC50 value. The results are shown in Table 3.

[0099] Among them, the two antibodies AF687 and AF692 target two different antigen-binding epitopes respectively, and have low EC50 values, which can be used for subsequent development of detection kits.

[0100] Table 1 EC50 values of each antibody binding to recombinant TPO protein (ng / mL)

[0101]

[0102] Table 2 Epitope sequences without glycosylation sites used:

[0103] Serial number Amino acid position sequence 1 K35-L43 KLLRDSHVL 2 V60-L69 VLLPAVDFSL 3 T89-A98 TLLLEGVMAA 4 G94-L102 GVMAARGQL 5 Q101-L110 QLGPTCLSSL 6 S109-V118 SLLGQLSGQV 7 A147-L156 AIFLSFQHLL 8 F162-L171 FLMLVGGSTL 9 M164-V173 MLVGGSTLCV 10 S187-L195 SLVLTLNEL 11 Y246-L254 YLNRIHELL 12 T312-L320 TLPTPVVQL 13 L323-T331 LLPDPSAPT

[0104] Table 3 EC50 values of antibodies binding to various antigen epitopes (ng / mL)

[0105]

[0106]

[0107] Example 4 Preparation of Antibody Chemiluminescence Kit

[0108] The anti-TPO rabbit monoclonal antibody pair obtained above was used to construct a chemiluminescent immunoassay system for immunoassay of serum TPO levels. First, a magnetic microsphere-antibody conjugate was prepared. The AF687 antibody was chemically coupled to carboxylated magnetic microspheres (JSR, MS160 / Carboxyl) using the EDC method. Take 10 mg of magnetic beads, wash them twice with coupling buffer (MES buffer, pH 5.0), and remove the supernatant by centrifugation. Resuspend them with coupling buffer, add EDC (Sigma, E1769) and NHS (Sigma, 130672) solutions to final concentrations of 10 mg / mL and 50 mg / mL, respectively, and mix them on a mixer at room temperature for 30 minutes. Repeat the washing with coupling buffer twice, add 50 ug of antibody, and mix at room temperature for 2 hours. Centrifuge and remove the supernatant, add coupling buffer containing 5% BSA as a blocking agent, and block at room temperature for 30 minutes. The coupled magnetic microsphere-antibody conjugate was suspended in a solution containing 1% BSA, 100mM MES, 0.6M NaCl, and 0.02% Tween20 (pH 6.5). Next, an alkaline phosphatase-labeled antibody was prepared. The anti-TPO rabbit monoclonal antibody AF692 was coupled to alkaline phosphatase using the SMCC method. 3mg of alkaline phosphatase (Wuxi Aorui Dongyuan, DP0008) was dissolved in PBS, and 0.5mg of sulfo-SMCC (Thermo Fisher, A39268) was also dissolved in PBS. The dissolved sulfo-SMCC solution was slowly dripped into the alkaline phosphatase solution and reacted at room temperature for 2 hours. The solution was added to a dialysis bag and dialyzed with PBS solution overnight to remove excess SMCC. Dissolve 1 mg of antibody in PBS and 0.1 mg of Traut's Reagent (Thermo Fisher Scientific, 26101) in PBS. Slowly add the dissolved Traut's Reagent solution dropwise to the antibody solution, let it react at room temperature for 2 hours, then add the solution to a dialysis bag and dialyze overnight with PBS to remove excess reagent. Thoroughly mix the enzyme activator and antibody activator at a mass ratio of 1:1, then let it react at room temperature for 1 hour. Block the reaction by adding sodium 2-mercaptoethanesulfonate and N-ethylmaleimide to a final concentration of 1 mM, let it react at room temperature for 1 hour, then add the solution to a dialysis bag and dialyze overnight with PBS to remove excess reagent. The conjugated enzyme-labeled antibody was then replaced with a solution of 10 mM MES, 0.5% BSA, 150 mM NaCl, 0.1 mM ZnCl₂, and 10 mM MgCl₂ (pH 6.5) using an ultrafiltration tube. These solutions, as well as sample diluent (PBS solution), cleaning solution (PBST solution), and substrate solution (containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt)) were then filled into reagent strips compatible with the lumilite8 chemiluminescence analyzer to prepare a chemiluminescence detection kit.

[0109] The test procedure for the kit is as follows: 100 μL of sample diluent, 100 μL of magnetic microsphere-antibody conjugate, and 100 μL of sample are mixed and incubated at 37°C for 10 minutes. The reaction solution is adsorbed onto a magnetic rod and magnetic rod cover to immobilize the magnetic microsphere-antibody-antigen conjugate, which is then washed three times with 300 μL of washing solution. After washing, 150 μL of sample diluent is added to resuspend the magnetic microsphere conjugate, followed by 50 μL of alkaline phosphatase-labeled antibody and incubation at 37°C for 10 minutes. The reaction solution is adsorbed onto a magnetic rod and magnetic rod cover to immobilize the magnetic microsphere-antibody-antigen-enzyme-labeled antibody conjugate, which is then washed three times with 300 μL of washing solution. After washing, 150 μL of sample diluent is added to resuspend the magnetic microsphere conjugate, followed by 50 μL of substrate solution. The luminescence is then measured after incubation at 37°C for 5 minutes.

[0110] Example 5 Clinical Evaluation of Antibody Chemiluminescence Kit

[0111] The TPO chemiluminescence detection kit prepared above was compared with the ELISA TPO detection reagent produced by R&D systems (Human Thrombopoietin Quantikine ELISA Kit DTP00B, referred to as "R&D-ELISA").

[0112] The TPO chemiluminescence kit was calibrated using PBS solutions containing 0 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL, and 2000 pg / mL of TPO recombinant protein as standards. The calibration curve is shown in Figure 1 .

[0113] Serum samples from healthy individuals, ITP patients, and patients with aplastic anemia were tested using a TPO chemiluminescence kit and an R&D-ELISA kit, respectively. Using clinical diagnosis as the gold standard, the diagnostic accuracy of the R&D-ELISA and TPO chemiluminescence kits was statistically analyzed, as shown in Tables 4 and 5. Compared to the results of the R&D-ELISA kit, the TPO chemiluminescence detection kit of the present invention was able to better differentiate patients with aplastic anemia. Among the 36 patients with aplastic anemia, only one falsely low result was observed, accounting for 2.7%, while the control kit had five falsely low results, accounting for 13.9%.

[0114] Table 4 Statistics of clinical sample data detected by R&D-ELISA kit

[0115]

[0116] Table 5 Statistics of clinical sample data detected by R&D-ELISA kit

[0117]

[0118]

[0119] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modifications and changes made to the above embodiment based on the technical essence of the present invention should fall within the scope of protection of the technical solution of the present invention.

Claims

1. An anti-human thrombopoietin (TPO) antibody or an antigen-binding fragment thereof, characterized in that: It includes a light chain variable region and a heavy chain variable region, and the amino acid sequences of the complementary determining regions HCDR1, HCDR2 and HCDR3 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 the complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.9; the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.

10.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment, a sc(Fv)2 fragment, a F(ab')2 fragment, a Fab' fragment, a full-length antibody, a bivalent antibody, a trivalent antibody, and a tetravalent antibody.

5. A biomaterial related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising at least one of the following 1) to 5): 1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; 2) an expression cassette containing the nucleic acid molecule described in 1); 3) a recombinant vector containing the nucleic acid molecule described in 1); 4) a recombinant microorganism or recombinant cell containing the nucleic acid molecule described in 1); 5) A recombinant microorganism or recombinant cell containing the recombinant vector described in 3).

6. An antibody conjugate, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a detection label connected to the antibody or antigen-binding fragment thereof.

7. The antibody conjugate according to claim 6, characterized in that The detection label is selected from one or more of biotin, fluorescein, chemiluminescent group, chemifluorescent group, fluorescent protein, horseradish peroxidase, acid phosphatase, colloidal gold, colored magnetic beads, magnetic microspheres, latex particles, radioactive nuclides and detection antibodies.

8. A kit, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the antibody conjugate according to any one of claims 6 to 7.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the biomaterial according to claim 5, or the antibody conjugate according to any one of claims 6 to 7 in the preparation of a product for detecting TPO protein or an epitope thereof.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the biomaterial according to claim 5, or the antibody conjugate according to any one of claims 6 to 7 in the preparation of a product for diagnosing thrombocytopenia caused by ITP or chemotherapy or aplastic anemia.

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