Anti-soluble transferrin receptor monoclonal antibodies, methods of making and uses thereof

Abstract

This application discloses a monoclonal antibody against soluble transferrin receptor, its preparation method, and its application, belonging to the field of antibody detection technology. The antibody uses the L101-F760 amino acid sequence of human TfR protein as an immunogen. After specific screening by B cells, RT-PCR reverse RNA is converted to cDNA, followed by homologous recombination to construct heavy and light chain vectors. Finally, after multiple rounds of screening, a purified sTfR monoclonal antibody is obtained, which can specifically recognize sTfR protein, exhibiting high specificity and high sensitivity. It can recognize natural sTfR protein in human serum. It can be applied in detection and screening fields such as immunohistochemistry, immunofluorescence chemistry, Western blotting, immunoprecipitation, indirect ELISA, and flow cytometry. Especially in immunohistochemical detection, it has low background and no interference from non-specific binding proteins, enabling more accurate detection and evaluation results and avoiding false positives and false negatives. It has significant application value in basic scientific research and clinical pathology detection.

Description

Technical Field

[0001] This application belongs to the field of monoclonal antibody technology, specifically relating to anti-soluble transferrin receptor monoclonal antibodies, their preparation methods, and applications. Background Technology

[0002] Iron (Fe) is an essential trace element for the human body, participating in a variety of intracellular biological processes, such as DNA synthesis, cellular respiration, and energy metabolism. Therefore, maintaining iron balance is crucial for the body.

[0003] Transferrin (Tf) is a glycoprotein primarily synthesized by the liver and secreted into the bloodstream. Its main function is to transport iron in the blood. Transferrin has two similar domains, each capable of binding a ferric ion (Fe3+). 3+ In the bloodstream, transferrin exists in two forms: iron-bound and iron-unbound. Iron-bound transferrin is called iron-saturated transferrin, while iron-unbound transferrin is called deferrotransferrin.

[0004] Transferrin receptor (TfR) is a transmembrane glycoprotein that transports iron from the extracellular space to the intracellular space. It is widely expressed in tissues and organs such as the liver, kidneys, and lungs. It consists of two identical 95 kDa independent subunits linked by an intermolecular disulfide bond. Each subunit contains an N-terminal cytoplasmic domain (1-67 amino acid residues), a transmembrane domain (68-88 amino acid residues), and a C-terminal extracellular domain (89-760 amino acid residues). The extracellular domain contains a binding site for transferrin, specifically recognizing and binding to iron-saturated transferrin, thus transporting iron from the extracellular space to the intracellular space.

[0005] The soluble transferrin receptor (sTfR) is a hydrolysate of free plasma (101-760 amino acid residues) formed by protease cleavage of the extracellular domain R100-L101 of the transferrin receptor. It can bind to transferrin in plasma and plays a key role in regulating cellular iron metabolism and maintaining iron homeostasis.

[0006] Due to the crucial role of sTfR in iron metabolism, its abnormal expression or functional alterations are closely related to various diseases. Therefore, detecting the level of sTfR in the blood is of great clinical significance. For example, it can be used to diagnose iron deficiency anemia, as sTfR is one of the reliable biomarkers for this condition. When the body is iron deficient but hemoglobin levels are normal, the transferrin receptors on the cell surface will increase compensatorily, leading to elevated sTfR levels in the blood. Therefore, the detection of sTfR levels has high sensitivity and can help doctors diagnose iron deficiency anemia more quickly. It can also be used to differentiate iron deficiency anemia from anemia caused by other chronic diseases. Iron deficiency anemia and anemia caused by chronic diseases present similarly, but their treatments differ. sTfR detection can effectively distinguish between these two types of anemia. Furthermore, as a tumor marker, abnormal iron metabolism has been recognized as one of the specific markers of tumors. Studies have found that cell carcinogenesis is often accompanied by high iron intake and upregulation of TfR and sTfR expression. sTfR detection can serve as a potential biomarker for tumor diagnosis, prognostic assessment, and efficacy monitoring.

[0007] In related technologies, for example, Chinese invention patent CN112409487A describes anti-soluble transferrin receptor antibodies and their applications. It uses sTfR as an immunogen, and after immunizing mice, murine anti-soluble transferrin receptor antibodies are obtained through screening. Murine and rabbit antibodies are two commonly used types of antibodies in biomedical research, diagnosis, and treatment. Murine antibodies have advantages such as mature technology, good immune response, and low cost, but they also have disadvantages such as the need for humanization and limited affinity. Correspondingly, although rabbit antibodies have problems such as higher cost, they have advantages such as high affinity, good specificity, and relatively lower difficulty and potential risks in humanization. Summary of the Invention

[0008] 1. Purpose of the invention

[0009] The purpose of this application is to provide a rabbit-derived monoclonal antibody against soluble transferrin receptor, its preparation method and application. This monoclonal antibody against soluble transferrin receptor can specifically recognize soluble transferrin receptor and has the characteristics of high affinity and good specificity.

[0010] 2. Technical Solution

[0011] To achieve the aforementioned objectives, the technical solution adopted in this application is as follows:

[0012] As a first aspect of this application, this application provides an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, capable of specifically binding to the soluble transferrin receptor, including:

[0013] (i) the heavy chain variable region with the amino acid sequence as shown in SEQ ID NO.2, and the light chain variable region with the amino acid sequence as shown in SEQ ID NO.3; or

[0014] (ii) the heavy chain variable region with the amino acid sequence as shown in SEQ ID NO.4, and the light chain variable region with the amino acid sequence as shown in SEQ ID NO.5; or

[0015] (iii) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO.6 and the light chain variable region with the amino acid sequence shown in SEQ ID NO.7.

[0016] Furthermore, the aforementioned anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment includes: a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.8, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO.9.

[0017] Furthermore, the above-mentioned anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain Fv protein (scFv), or a disulfide-stabilized Fv protein (dsFv).

[0018] As a second aspect of this application, this application provides a nucleic acid encoding the above-mentioned anti-soluble transferrin receptor or its antigen-binding fragment, or its heavy chain variable region and / or light chain variable region.

[0019] Furthermore, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO.2 is shown in SEQ ID NO.10.

[0020] Furthermore, the nucleotide sequence of the nucleic acid encoding the light chain variable region as shown in SEQ ID NO.3 is shown in SEQ ID NO.11.

[0021] Furthermore, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO.4 is shown in SEQ ID NO.12.

[0022] Furthermore, the nucleotide sequence of the nucleic acid encoding the light chain variable region as shown in SEQ ID NO.5 is shown in SEQ ID NO.13.

[0023] Furthermore, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO.6 is shown in SEQ ID NO.14.

[0024] Furthermore, the nucleotide sequence of the nucleic acid encoding the light chain variable region as shown in SEQ ID NO.7 is shown in SEQ ID NO.15.

[0025] Furthermore, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region as shown in SEQ ID NO.8 is shown in SEQ ID NO.16.

[0026] Furthermore, the nucleotide sequence of the nucleic acid encoding the light chain constant region as shown in SEQ ID NO.9 is shown in SEQ ID NO.17.

[0027] As a third aspect of this application, this application provides a recombinant expression vector containing the aforementioned nucleic acid, which can express an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

[0028] Furthermore, the recombinant expression vector includes: a pcDNA3.4 plasmid containing nucleic acid encoding a monoclonal antibody against soluble transferrin receptor or its antigen-binding fragment.

[0029] As a fourth aspect of this application, this application provides a recombinant expression cell comprising the above-described recombinant expression vector or the above-described nucleic acid encoding an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, which can express an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

[0030] Furthermore, the recombinant expression cells mentioned above include the Expi293F cell line or the CHO cell line.

[0031] As a fifth aspect of this application, this application provides the use of the above-described nucleic acid, recombinant expression vector, or recombinant expression cell encoding the above-described anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment in the preparation of the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

[0032] Furthermore, the above applications include: transfecting cells with the above recombinant expression vector to obtain recombinant expression cells, and culturing the recombinant expression cells; or directly culturing the above recombinant expression cells; collecting the supernatant after culture and purifying it to obtain an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

[0033] As a sixth aspect of this application, this application provides a method for preparing the above-mentioned anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, comprising:

[0034] Recombinant expression cells were obtained by transfecting cells with the above-mentioned recombinant expression vector and then cultured; or the above-mentioned recombinant expression cells were cultured directly; after culture, the supernatant was collected and purified to obtain an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

[0035] As a seventh aspect of this application, this application also provides the use of the above-mentioned anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, nucleic acid encoding the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, recombinant expression vector or recombinant expression cell in detecting soluble transferrin receptor or preparing products for detecting soluble transferrin receptor.

[0036] Furthermore, the above-mentioned detection of soluble transferrin receptors refers to the detection of free soluble transferrin receptors in plasma.

[0037] Furthermore, the above-mentioned detection methods include any one or more of ELISA, immunoblotting, immunofluorescence, and immunohistochemistry.

[0038] As an eighth aspect of this application, this application also provides a kit for detecting soluble transferrin receptors, the kit comprising any one or more of the above-described anti-soluble transferrin receptor monoclonal antibodies or their antigen-binding fragments.

[0039] Furthermore, the above-mentioned kit includes any one of the above-mentioned anti-soluble transferrin receptor monoclonal antibodies or their antigen-binding fragments.

[0040] Furthermore, the above kit includes a monoclonal antibody against soluble transferrin receptor or an antigen-binding fragment thereof, with the heavy chain variable region of amino acid sequence as shown in SEQ ID NO.6 and the light chain variable region of amino acid sequence as shown in SEQ ID NO.7; and a monoclonal antibody against soluble transferrin receptor or an antigen-binding fragment thereof, with the heavy chain variable region of amino acid sequence as shown in SEQ ID NO.2 and the light chain variable region of amino acid sequence as shown in SEQ ID NO.3; the paired antibody can simultaneously bind to different epitopes of the sTfR protein.

[0041] Furthermore, the above kit includes a monoclonal antibody against the soluble transferrin receptor or an antigen-binding fragment thereof, with the heavy chain variable region as shown in SEQ ID NO.6 and the light chain variable region as shown in SEQ ID NO.7; and a monoclonal antibody against the soluble transferrin receptor or an antigen-binding fragment thereof, with the heavy chain variable region as shown in SEQ ID NO.4 and the light chain variable region as shown in SEQ ID NO.5; the paired antibody can simultaneously bind to different epitopes of the sTfR protein.

[0042] 3. Beneficial effects

[0043] Compared with the prior art, the advantages of this application are as follows:

[0044] The monoclonal antibody against soluble transferrin receptor or its antigen-binding fragment provided in this application exhibits high specificity and sensitivity in binding to soluble transferrin receptor molecules. It can specifically recognize and detect the expression of soluble transferrin receptor in blood, showing positive high expression when detecting soluble transferrin receptor. Therefore, this monoclonal antibody against soluble transferrin receptor or its antigen-binding fragment can be applied to the detection and screening of soluble transferrin receptor using techniques such as immunohistochemistry, indirect ELISA, antibody microarray, flow cytometry, immunoblotting, and immunofluorescence, which is beneficial for obtaining accurate assessment and detection results. Attached Figure Description

[0045] Figure 1 This is an SDS-PAGE result of the recombinant sTfR in this application, where lane 1 is the protein molecule Maker and lane 2 is the purified recombinant sTfR protein.

[0046] Figure 2 This study uses ELISA to detect the immunization effect of recombinant sTfR protein antigen in rabbits. K1672, K1673, and K1674 are serum samples obtained from three different rabbits after antigen immunization. The positive control (Control) is an antibody known to bind to a specific antigen, and the negative control (NC) is serum from unimmunized rabbits.

[0047] Figure 3 This is an electrophoresis image of the antibody heavy and light chain variable regions in a 1% agarose gel. The leftmost lane is the 2000 DNA maker band, the rightmost lane is the blank control, the top row of lanes is the heavy chain band, and the bottom row of lanes is the light chain band.

[0048] Figure 4 The image shows the SDS-PAGE results of the sTfR monoclonal antibody. The left side represents the non-reduced antibody sample, and the right side represents the reduced antibody sample. Lanes 1-3 are the purified antibodies H34L151 (RMB1201023), H45L156 (RMB1201024), and H83L193 (RMB1201025), respectively.

[0049] Figures 5-7 This is an ELISA diagram showing the binding of sTfR monoclonal antibody to commercially available sTfR recombinant protein.

[0050] Figure 8 This is an immunoblot assay showing the binding of sTfR monoclonal antibody to sTfR recombinant protein.

[0051] Figure 9 This is a graph showing the results of cellular immunofluorescence detection of the tumor cell line (HT-1080) that specifically binds to the sTfR monoclonal antibody.

[0052] Figure 10 This is an image of the immunohistochemical (IHC) staining results, showing, from left to right, human kidney, human liver, and human placenta. Detailed Implementation

[0053] The present application will be further described below with reference to specific embodiments.

[0054] In this application, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0055] In this application, unless otherwise defined, conditions not specifically specified in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] In this application, unless otherwise defined, the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items. The term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0057] In this application, unless otherwise defined, the term "about" is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility of a particular variable.

[0058] In this application, unless otherwise defined, concentration, amount, and other numerical data may be presented herein in a range format. It should be understood that such a range format is used only for convenience and brevity and should be flexibly interpreted to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly stated. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single numerical value, such as "less than about 4.5," which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or feature described.

[0059] In this application, unless otherwise defined, as used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabetic bodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0060] The monoclonal antibodies of this application can be prepared using various techniques, such as hybridoma techniques (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA techniques (see, for example, U.S. Patent Application 4,816,567), or phage antibody library techniques (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991). The antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography. For purification of immunoglobulins, please refer to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0061] In this application, unless otherwise defined, indirect ELISA detection involves first coating an antigen (sTfR) onto a solid-phase carrier, adding the serum sample to be tested, and if the sample contains a specific antibody against the antigen, the antibody will bind to the solid-phase antigen; after washing away unbound components, an enzyme-labeled anti-human immunoglobulin is added, which can bind to the human antibody already bound to the solid-phase antigen; after adding the substrate, the antibody content in the sample is determined based on the degree of colorimetric reaction.

[0062] In this embodiment, the soluble transferrin receptor (sTfR) used is the L101-F760 cleavage fragment of the human transferrin receptor (TfR). sTfR can be prepared by chemical synthesis or biological expression. To facilitate purification, a His tag can be added to its C-terminus. Recombinant sTfR is obtained by constructing an expression vector, introducing it into a eukaryotic expression system, inducing expression, and purifying it. The SDS-PAGE results are shown below. Figure 1 As shown, its amino acid sequence is shown in SEQ ID NO.1:

[0063] (SEQ ID NO.1).

[0064] Example 1

[0065] This embodiment provides the screening and preparation of monoclonal antibodies against soluble transferrin receptors, specifically including the following steps:

[0066] (1) Animal Immunization

[0067] Three healthy adult New Zealand white rabbits (numbered K1672, K1673, and K1674) were selected. Recombinant sTfR, with the amino acid sequence shown in SEQ ID NO.1, was used as the antigen and mixed with Freund's adjuvant to prepare an immunogen. Multiple immunizations were administered via subcutaneous injection at multiple sites on the back. Specifically, the immunization included:

[0068] a. First immunization: 1 mL of complete Freund's adjuvant was mixed with 600 μg of recombinant sTfR and emulsified as an immunogen;

[0069] b. Second immunization: Three weeks after the first immunization, 1 mL of incomplete Freund's adjuvant was mixed with 600 μg of recombinant sTfR and emulsified as an immunogen;

[0070] c. Third immunization: Five weeks after the second immunization, 300 μg of recombinant sTfR was mixed with the same volume of 0.01 M PBS (pH 7.4) and emulsified to serve as an immunogen;

[0071] d. Fourth immunization: Seven weeks after the third immunization, 300 μg of recombinant sTfR was mixed with the same volume of 0.01 M PBS (pH 7.4) and emulsified to serve as the immunogen;

[0072] f. Seven days after the fourth immunization, blood was collected from the marginal ear vein of the rabbits, and rabbit antiserum was obtained by centrifugation at 4000 rpm for 20 min. The serum antibody titer was detected by indirect ELISA to evaluate the antigen immunization effect.

[0073] The indirect ELISA detection of serum antibody titers includes: diluting recombinant sTfR with carbonate buffer (50mM, pH 9.6), coating 100μL / well of the protein onto the microplate, and incubating overnight at 4°C; blocking with 1% BSA the next day at 37°C for 1 hour, serially diluting rabbit antiserum as the primary antibody, incubating at 37°C for 1 hour, adding 1:5000 secondary antibody (goat anti-rabbit), incubating at 37°C for 1 hour, adding chromogenic solution for color development, and reading the absorbance value at OD450 using a microplate reader.

[0074] ELISA test results as follows Figure 2 As shown, the rabbit antiserum titer against recombinant sTfR is not less than 1:64000, indicating that recombinant sTfR has good immunogenicity as an antigen.

[0075] (2) Preparation of anti-sTfR monoclonal antibody

[0076] Under aseptic conditions, the spleens of rabbits numbered K1672 and K1673, which had been immunized with the antigen, were removed and ground in 10cm culture dishes, then passed through a cell sieve to obtain B lymphocytes. The obtained B lymphocytes were resuspended and diluted to a density of 102. 5 Cells / mL. Following the immunomagnetic bead operation instructions, recombinant sTfR was conjugated to the magnetic beads. The conjugated magnetic beads were incubated with the isolated B lymphocytes at room temperature for 50 min, followed by elution and resuspending. The resuspended B lymphocytes were cultured in 96-well cell plates for 7 days using a limiting dilution method. The supernatant was collected for indirect ELISA identification of antibodies. The detection method was the same as described above for indirect ELISA to detect serum antibody titers, ultimately yielding B lymphocytes that specifically secrete anti-sTfR monoclonal antibodies.

[0077] The selected specific B lymphocytes were lysed, and total RNA was obtained using the TurboCapture96mRNAPlate (QIAGEN, cat#72251) kit. cDNA was obtained using a reverse transcription kit (Shanghai Tongke Biotechnology Co., Ltd., cat#TD002B). The reverse transcription product was used as a template for PCR.

[0078] PCR involves designing primers to amplify nucleic acids encoding the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody.

[0079] The primer sequences are as follows:

[0080] VL-Primer-F: 5'-GCTCGTGATGACCCAGACTCCA-3' (SEQ ID NO.18),

[0081] VL-Primer-R: 5'-CCACCTCGGTCCCTCCG-3' (SEQ ID NO. 19),

[0082] VH-Primer-F: 5'-CCTGGTCGCTGTGCTCAAAGGTGTCCAG-3 (SEQ ID NO. 20),

[0083] VH-Primer-R: 5'-CGTTGGTCAGTGTGCCGCTA-3' (SEQ ID NO. 21);

[0084] The reaction procedure is as follows:

[0085] VH-DNA: Amplification was performed for 35 cycles: 95℃ for 5 min, 95℃ for 30 s, 70℃ for 30 s, 72℃ for 1 min, and 72℃ for 10 min.

[0086] VL-DNA: Amplify by performing 35 cycles of 95℃ for 5 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, and 72℃ for 10 min;

[0087] The amplification products were detected by 1% agarose gel electrophoresis (results are shown below). Figure 3 Then, a single target band was selected for gel recovery. The gel-recovered target band and a mammalian cell expression vector (pcDNA3.4) containing genes for the heavy and light chain constant regions were transformed into TOP10 competent cells (Shanghai Weidi Biotechnology Co., Ltd., cat#DF1010M) via homologous recombination. The cells were cultured at 37℃ for 12 hours, and single clones were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using Snapgene software to screen for nucleic acids encoding the heavy chain variable region (VH-DNA) and the light chain variable region (VL-DNA) that conformed to the sequence characteristics of rabbit-derived antibodies, along with their corresponding VH and VL amino acid sequences. Specifically:

[0088] a.H34L151 monoclonal antibody

[0089] The amino acid sequence (H34L151-VH) of the heavy chain variable region of the H34L151 monoclonal antibody:

[0090] QSVEESGGRLVTPGTPLTLTCTVFGFSLSNYNIQWVRQAPGKGLEYIGIIVAGGSAFYAS WAKGRFTISKTSTTVDLKITSPTIEDTATYFCAGSNSNLFKFNLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL(SEQ ID NO.2);

[0091] The amino acid sequence (H34L151-VL) of the light chain variable region of the H34L151 monoclonal antibody:

[0092] LTQTPSPVSAAVGGTVTINCQASQTVDNSNYLAWFQQKPGQPPKLLIYKASTLESGVPS RFSGSGSGTQFTLTISGVQCDDAATYYCQGTYWSSGWYIAFGGGTEVVVKGDPV(SEQ ID NO.3);

[0093] The nucleic acid sequence (H34L151-VH-DNA) encoding the heavy chain variable region against H34L151 is as follows:

[0094] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTTTTTGGATTCTCCCTCAGTAACTACAACATTCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGTTGCTGGTGGTAGCGCATTCTACGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAATCGAGGACACGGCCACCTATTTCTGTGCCGGTTCTAATAGTAATCTTTTTAAATTTAACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTC(SEQID NO.10);

[0095] Nucleic acid sequence of the light chain variable region encoding H34L151 (H34L151-VL-DNA):

[0096] CTGACCCAGACACCTTCGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGACTGTTGATAATAGCAACTACTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGAATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCACTTATTGGAGTAGTGGTTGGTATATTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTT(SEQ ID NO.11).

[0097] b. Monoclonal antibody H45L156

[0098] The amino acid sequence (H45L156-VH) of the variable region of the heavy chain of the H45L156 monoclonal antibody:

[0099] QSLVESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIINVSGRTWYA SWAKGRFSISKTSTTVDLKMTSPTTEDTATYFCARDAGRSYDIYFDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYL(SEQ ID NO.4);

[0100] The amino acid sequence of the variable region of the light chain of the H45L156 monoclonal antibody (H45L156-VL):

[0101] PTQTPSPVSAAVGGTVTISCQSSQSVYNDNDLAWYQQKPGQPPKLLIYQASSLASGVPS RFKGSGSGTQFTLTISDVQCDDAATYYCQGTYDSSGWYVAFGGGTEVVVKGDPV(SEQ ID NO.5);

[0102] The nucleic acid sequence (H45L156-VH-DNA) encoding the heavy chain variable region of H45L156 is as follows:

[0103] CAGTCGCTGGTGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGGGCTGGGTCCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAATGTTAGTGGTAGGACATGGTACGCGAGCTGGGCGAAAGGCCGATTCAGCATCTCCAAAACCTCGACCACGGTGGA TCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGGGATGCTGGTAGAAGTTATGATAATTTACTTTGACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTC(SEQ ID NO.12);

[0104] The nucleic acid sequence (H45L156-VL-DNA) encoding the light chain variable region of H45L156 is as follows:

[0105] CCAACCCAGACACCATCACCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAATGACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACCAGGCATCCAGTCTGGCATCTGGGGTC CCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCACTTATGATAGTAGTGGTTGGTACGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTT(SEQ ID NO.13).

[0106] c.H83L193 monoclonal antibody:

[0107] The amino acid sequence (H83L193-VH) of the heavy chain variable region of the H83L193 monoclonal antibody:

[0108] QSVEESGGRLVTPGTPLTLTCTVSGFSLSYYGVSWVRQAPGKGLEWIGIMATDGSAAY ASWAKGRFTISKTSTAVDLKISSPTTEDTATYFCARGGSLWGQGTLVTVSSGQPKAPSVFPLA PCCGDTPSSTVTLGCLVKGYL(SEQ ID NO.6);

[0109] The amino acid sequence (H83L193-VL) of the light chain variable region of the H83L193 monoclonal antibody:

[0110] LTQTASPVSAAVGGTVTINCQASQSVYTNNYLAWFQQKPGQPPKRLIYQASKLASGVS SRFSGSGSGTQFTLTISDVQCDDAASYYCLGTFDCSSTDCAAFGGGTEVVVKGDPV(SEQ ID NO.7);

[0111] The nucleic acid sequence (H83L193-VH-DNA) encoding the heavy chain variable region of H83L193:

[0112] CAGTCGGTGGAGGAGTCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTTACTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAATCATGGCTACTGATGGTAGCGCAGCCTACGCGAGCTGGGCGAAAGCCCGATTCACCATCTCCAAGACCTC GACCGCAGTGGATCTGAAAATCTCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGAGGTAGTTTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTC(SEQ ID NO.14);

[0113] The nucleic acid sequence encoding the light chain variable region of H83L193 (H83L193-VL-DNA) is as follows: CTGACCCAGACTGCTTCGCCCGTGTCTGCGGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATACTAACAACTACTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCGCCTGATCTACCAGGCATCCAAACTGGCATCTGGGGTCTCATCGCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACTATTAGCGACGTGCAGTGTGACGATGCTGCCTCTTACTACTGTCTAGGCACTTTTGATTGTAGTAGTACTGATTGTGCTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTT (SEQ ID NO.15); d. Heavy and light chain constant regions

[0114] The amino acid sequence of the heavy chain constant region (CH) of the above monoclonal antibody is as follows:

[0115] SGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPI AHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.8);

[0116] The amino acid sequence of the light chain constant region (CL) of the above monoclonal antibody is as follows:

[0117] FGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQT TGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCK(SEQ ID NO.9);

[0118] The nucleic acid sequence encoding the heavy chain constant region (CH-DNA) of a monoclonal antibody is as follows:

[0119] AGCGGCACACTGACCAACGGCGTGAGGACCTTTCCTAGCGTGAGACAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTGTCCGTGACAAGCAGCTCCCAGCCCGTGACATGCAACGTGGCCCACCCCGCCACAAACACAAAGGTGGATAAGACAGTGGCCCCTAGCACCTGCAGCAAGCCCACCTGTCCCCCTCCTGAGCTGCTGGGCGGCCCTTCTGTGTTTATCTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCAGGACGATCCTGAGGTGCAGTTCACCTGGTACATCAATAACGAGCAGGTGAGAACAGCCAGACCTCCTCTGAGAGAGCAGCAGTTCAACTCCACCATCAGAGTGGTGAGCACCCTGCCCATCGCCCACCAGGACTGGCTGAGGGGCAAGGAGTTTAAGTGTAAGGTGCACAACAAGGCCCTGCCCGCCCCTATCGAGAAGACCATCAGCAAGGCCAGGGGCCAGCCCCTGGAGCCTAAGGTGTACACCATGGGCCCCCCTAGGGAGGAGCTGAGCTCCAGGTCCGTGAGCCTGACCTGCATGATCAATGGCTTCTACCCTTCCGACATCTCCGTGGAGTGGGAGAAGAACGGCAAGGCCGAGGATAACTACAAGACCACACCTGCCGTGCTGGACTCCGACGGCAGCTACTTCCTGTACAGCAAGCTGAGCGTGCCCACAAGCGAGTGGCAGAGGGGCGATGTGTTCACCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCATCTCCAGGTCCCCCGGCAAG(SEQ ID NO.16);

[0120] The nucleic acid sequence encoding the constant region of the monoclonal antibody light chain (CL-DNA) is:

[0121] TTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCT GGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAG(SEQID NO.17).

[0122] Expression vectors containing the complete encoding of the rabbit monoclonal antibody light chain gene and heavy chain gene were paired 1:1 and co-transfected into cells at a density of 3 × 10⁶ cells / year. 6 In 100 mL of Expi 293F cells, the cell supernatant was collected after 5 days of culture and purified by affinity chromatography using Protein A resin to obtain the antibody.

[0123] The purity and molecular weight of the sTfR antibody were initially identified using SDS-PAGE. 2 μg of antibody was added to an appropriate amount of loading buffer to make a total volume of 20 μL. Reducing and non-reducing SDS-PAGE samples were prepared simultaneously. A 10% SDS-PAGE gel was prepared, and the sample was loaded. Electrophoresis was performed at 80V for 30 min, followed by 120V until the bands were clearly separated. The gel was removed after electrophoresis and stained with Coomassie brilliant blue. After 15 min, the staining solution was removed, and the gel was rinsed thoroughly with water. Destaining solution was then added until clear bands were observed. The results are as follows: Figure 4 As shown, the samples marked 1-8 on the left are non-reducing antibody samples, and the samples marked 1-8 on the right are reducing antibody samples. Lanes 1-3 are purified antibodies H34L151 (RMB1201023), H45L156 (RMB1201024), and H83L193 (RMB1201025), respectively.

[0124] Example 2

[0125] This embodiment provides the application of the anti-soluble transferrin receptor monoclonal antibody screened in this application in the detection of soluble transferrin receptor, including: ELISA method, Western blotting method, cell immunofluorescence method, and immunohistochemistry method.

[0126] (1) ELISA method

[0127] The specific binding of the purified antibody to the sTfR protein was detected using an ELISA method.

[0128] Using the sTfR recombinant protein from this application respectively ( Figure 5 ), and the sTfR recombinant protein purchased from Suzhou Nearshore Protein Technology Co., Ltd. Figure 6 (cat#CU75) and sTfR recombinant protein purchased from Nanjing Youai Biotechnology R&D Co., Ltd. Figure 7 The antibody (cat#UA010340) was used as the coating antigen. The detection method was the same as the ELISA detection method for rabbit antiserum in Example 1.

[0129] ELISA test results as follows Figures 5-7 As shown, H34L151, H45L156, and H83L193 all exhibited good binding effects with commercially available sTfR recombinant proteins.

[0130] (2) Immunoblotting method

[0131] The specific binding of the purified antibody to the sTfR protein was analyzed using immunoblotting.

[0132] HeLa, K562, HT-1080, and 293T cells in good culture condition were collected using SDS loading buffer to prepare cell lysis buffer. SDS-PAGE electrophoresis was performed according to standard methods, and the target protein gel region was transferred to a PVDF membrane (activated with methanol) at a constant current of 250 mA for 120 min. After transfer, the PVDF membrane was blocked in 5% skim milk powder on a shaker at room temperature for 1 h. H34L151 (RMB1201023) monoclonal antibody (0.5 mg / mL) was diluted 1:1000 as the primary antibody and incubated on a shaker at room temperature for 2 h. The membrane was washed 3 times with TBST for 10 min each time. HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson Immuno Research, GPRB051801H) was added as the secondary antibody (1:10000) and incubated on a shaker at room temperature for 1 h. The membrane was washed 5 times with TBST for 10 min each time. The ECL colorimetric solution is used to expose and develop the film for imaging analysis in a luminescent imaging system.

[0133] Immunoblotting results as follows Figure 8 As shown, the sTfR monoclonal antibody H34L151 can specifically bind to the sTfR protein.

[0134] (3) Cell immunofluorescence method

[0135] The specific binding of purified antibodies to sTfR-positive tumor cell lines was analyzed using cell immunofluorescence.

[0136] Cell spreaders were pre-seeded into 24-well cell culture plates at a density of 1 × 10⁻⁶ cells / well. 5 HT-1080 cells (sTfR positive cell material) were cultured at / mL for 12h. Cell status was observed afterward. HT-1080 cells were stimulated with BFA (5μg / mL) for 15min as a positive control, while HT-1080 cells without BFA (5μg / mL) treatment served as a negative control. The culture supernatant was then discarded, and the cells were washed three times with pre-frozen PBS. The cells were then fixed with anhydrous ethanol at room temperature for 15min, followed by three washes with PBS. 5% skim milk powder was added, and the cells were blocked at 37℃ for 2h. After blocking, the cells were washed three times with PBS and stored at 4℃ for later use. The purified H34L151 (RMB1201023) monoclonal antibody (0.5 mg / mL) was diluted 1:50 as the primary antibody and added to the prepared cell culture plate. After incubation at 37°C for 60 min, the plate was washed three times with TBST. Goat anti-rabbit fluorescent secondary antibody was added under dark conditions, and the plate was incubated again at 37°C for 45 min, followed by three washes with TBST. DAPI was added to stain the nuclei under dark conditions, and the plate was incubated at room temperature for 5 min, followed by three washes with TBST. A mounting medium was then placed on a glass slide and placed on top of the mounting medium from one side. Fluorescence was observed under an inverted fluorescence microscope.

[0137] Cell immunofluorescence detection results as follows Figure 9 As shown, the results on the left indicate that in HT-1080 cells treated with BFA (5 μg / mL), the sTfR protein was not only specifically recognized by the H34L151 monoclonal antibody on the cell membrane, but also showed punctate aggregation inside the cell; the results on the right indicate that in HT-1080 cells not treated with BFA (5 μg / mL), the sTfR protein was only specifically recognized on the cell membrane.

[0138] (4) Immunohistochemical methods

[0139] Paraffin-embedded tissue sections containing human kidney, liver, and placental extracts were placed in an oven at 65°C for approximately 1 hour for dewaxing and hydration. Afterward, the sections were rinsed three times with distilled water. The sections were then placed in a staining chamber containing EDTA retrieval solution and placed in an autoclave for antigen retrieval under high temperature and pressure. After heating, the sections were allowed to cool naturally. They were rinsed thoroughly with distilled water and washed three times with 1×PBS. The antigen-retrieved sections were then placed in 3% H2O2 solution for 10 minutes, followed by three washes with 1×PBS. Non-specific antigens were blocked with 5% BSA + 10% sheep serum (prepared with PBST) at room temperature for 30 minutes. After blocking, the blocking solution was discarded. Antibody H34L151 (RMB1201023) (0.5 mg / mL) was diluted 1:2000 as the primary antibody and incubated overnight at 4°C. The following morning, the sections were rinsed three times with 1×PBST and then HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoRes) was added. Using earch,GPRB051801H)(1:10000) as the secondary antibody, incubate at room temperature for 30 minutes, then wash 3 times with 1×PBST; develop DAB staining for 1-2 minutes, which can be observed with the naked eye until a brownish-yellow color appears. After staining, rinse directly with tap water to stop the staining process; counterstain with hematoxylin for 2-5 minutes; rinse with distilled water, then destain with PBS for 30 seconds, rinse with distilled water again, and dehydrate the sections; after sequentially dehydrating and drying with graded alcohols, add 50-100 μL of neutral resin to each section, then cover with a coverslip and mount; air dry the mounted sections overnight, and observe under a microscope the next day and scan the sections with a tissue sectioner to obtain immunohistochemical results.

[0140] Immunohistochemical test results as follows Figure 10 As shown, the sTfR monoclonal antibody H34L151 exhibited sTfR molecule-specific positive staining signals in human kidney, human liver, and human placental tissues.

[0141] Example 3

[0142] This embodiment provides a method for screening monoclonal antibody pairings to detect sTfR in biological samples using a double-antibody sandwich ELISA method.

[0143] One of the sTfR monoclonal antibodies from Example 1 will be used as the capture antibody, and the other will be used as the labeling antibody (Biotin labeling). The capture antibody will be coated into the wells of the ELISA plate. First, a biological sample (serum from a patient with iron deficiency anemia) will be added. After incubation, the unbound antigen will be washed away. Then, the labeling antibody will be added. After incubation, the unbound labeling antibody will be washed away. Finally, the colorimetric solution will be added for color development.

[0144] The specific steps are as follows: Dilute the capture antibody to 2 μg / mL with coating buffer, add 50 μL / well to each well of the ELISA plate, and coat overnight at 4°C. Pat the ELISA plate dry, add 200 μL of blocking buffer (PBS containing 1% BSA) to each well, block at 37°C for 2 hours, and discard the blocking buffer. Add 50 μL of diluted patient serum to each well (first dilute the blood sample to 20 ng / mL with 0.5% BSA / PBS). Incubate at 37°C for 1 hour, then wash the plate 3 times. Add 50 μL of biotinylated antibody to each well (first dilute the labeled antibody to 0.5 μg / mL with 0.5% BSA / PBS). Incubate at 37°C on a shaker for 1 hour, then wash the plate 3 times. Add 50 μL of SA-HRP to each well (first dilute 1:20000 with 0.5% BSA / PBS), incubate at 37°C on a shaker for 1 hour, then wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 min. Then, add 100 μL of 4.9% H3PO4 to each well to terminate the reaction. Read the OD value at 450 nm using a microplate reader.

[0145] The results of the pairing screening of monoclonal antibodies are shown in Table 1.

[0146] Table 1. Statistics of P / N values ​​for different paired sTfR monoclonal antibodies

[0147]

[0148]

[0149] As shown in Table 1, a positive result cannot be detected if the P / N ratio of the same monoclonal antibody is less than 2.1; however, when two different monoclonal antibodies are used in pairs, except for H45L156 and H34L151 which cannot be used together, the other paired antibodies with a P / N ratio greater than or equal to 2.1 can detect positive samples.

Claims

1. A monoclonal antibody against a soluble transferrin receptor or its antigen-binding fragment, characterized in that, include: The amino acid sequence is shown in the heavy chain variable region of SEQ ID NO.2, and the amino acid sequence is shown in the light chain variable region of SEQ ID NO.

3.

2. The anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody comprises a heavy chain constant region as shown in SEQ ID NO.8 and a light chain constant region as shown in SEQ ID NO.

9.

3. The anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain Fv protein, or a disulfide-stabilized Fv protein.

4. A nucleic acid, characterized in that, The nucleic acid encodes the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid encoding the heavy chain variable region as shown in SEQ ID NO.2 is shown in SEQ ID NO.10; The nucleotide sequence of the nucleic acid encoding the light chain variable region as shown in SEQ ID NO.3 is shown in SEQ ID NO.

11.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 4 or 5.

7. A recombinant expression cell, characterized in that, The recombinant expression cells comprise the recombinant expression vector of claim 6, or the nucleic acid encoding a monoclonal antibody against soluble transferrin receptor or its antigen-binding fragment as described in claim 4 or 5.

8. The use of the nucleic acid encoding an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment as described in claim 4 or 5, or the recombinant expression vector as described in claim 6, or the recombinant expression cell as described in claim 7 in the preparation of an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

9. A method for preparing an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment, comprising: Recombinant expression cells are obtained by transfecting cells with the recombinant expression vector described in claim 6 and culturing the recombinant expression cells; or by culturing the recombinant expression cells described in claim 7; the supernatant is collected and purified to obtain an anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment.

10. The use of the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, or the nucleic acid encoding the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment as described in claims 4 or 5, or the recombinant expression vector as described in claim 6, or the recombinant expression cell as described in claim 7, in the preparation of products for detecting soluble transferrin receptors.

11. The application according to claim 10, characterized in that, The detection methods include any one or more of ELISA, immunoblotting, immunofluorescence, and immunohistochemistry.

12. A kit for detecting soluble transferrin receptors, characterized in that, The kit comprises the anti-soluble transferrin receptor monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

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