A single-chain antibody specifically binding to thrombopoietin and use thereof

Abstract

The present disclosure belongs to the field of bioengineering and immune detection technology, and specifically provides a single-chain antibody specifically binding to human thrombopoietin (TPO) and applications thereof. The present disclosure also provides nucleic acid molecules, expression vectors, expression strains and kits, which can be used for detecting the content of TPO in a test substance. The present disclosure overcomes the shortcomings of high production cost and complex process of traditional antibodies, and meets the needs of high-sensitivity, high-affinity and high-specificity TPO immune detection, and is suitable for developing high-performance and low-cost TPO detection reagents and methods.

Description

Technical Field

[0001] This disclosure belongs to the field of bioengineering and immunoassay technology, specifically relating to a single-chain antibody that specifically binds to thrombopoietin and its application in thrombopoietin detection. Background Technology

[0002] Thrombopoietin (TPO) is a hematopoietic growth factor primarily secreted by liver parenchymal cells and kidneys, belonging to the type I cytokine family. As a key physiological factor regulating platelet production, TPO plays a crucial role in the blood system. It primarily activates the JAK-STAT signaling pathway by specifically binding to the c-Mpl receptor on the surface of megakaryocytes, thereby stimulating the proliferation, differentiation, and maturation of megakaryocytes in the bone marrow. Ultimately, this leads to the detachment of platelets from the megakaryocyte cytoplasm and their release into the bloodstream. Furthermore, TPO can synergistically work with erythropoietin (EPO) to promote the differentiation of hematopoietic progenitor cells and accelerate the recovery of hematopoietic function after bone marrow suppression.

[0003] Under physiological conditions, TPO maintains the dynamic balance of peripheral blood platelet count through a negative feedback mechanism. Clinical studies have shown a significant negative correlation between serum TPO concentration and platelet count. When platelets or megakaryocytes are reduced (e.g., in aplastic anemia, myelodysplastic syndrome), TPO levels significantly increase; while in patients with immune thrombocytopenic purpura (ITP) or cirrhosis, TPO levels are normal or slightly elevated. Therefore, accurate detection of TPO levels is of significant clinical value for the etiological identification, treatment optimization, and prognostic assessment of thrombocytopenia.

[0004] Currently, TPO detection mainly relies on enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay based on double-antibody sandwich methods. The antibodies used in these assays are often prepared using animal or mammalian cell expression systems, which suffers from long processing times, high costs, and poor batch stability. Furthermore, the large molecular weight of intact IgG molecules can cause steric hindrance in solid-phase coating, affecting the flexibility of antigen epitope recognition. This invention aims to overcome these limitations by developing a single-chain anti-human TPO antibody (scFv) that can be efficiently expressed in *E. coli* through genetic engineering recombination technology. This scFv not only has a small molecular weight and controllable production costs but also maintains good affinity and specific recognition ability for native TPO conformation. Therefore, it provides an ideal core raw material for constructing high-performance, low-cost TPO immunoassay methods, effectively meeting the urgent needs of clinical testing and basic research. The anti-human TPO single-chain antibody disclosed in this invention and its application in thrombopoietin detection can be used to detect thrombopoietin in human blood, contributing significantly to the differential diagnosis, efficacy prediction, and severity assessment of thrombocytopenia and related diseases. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this disclosure provides an anti-TPO single-chain antibody (scFv) with excellent binding activity and its applications. The scFv is composed of a heavy chain variable region and a light chain variable region linked by a short peptide. It not only retains the ability to bind to TPO with high affinity and high specificity, but also possesses significant advantages such as small molecular weight, good stability, and ease of large-scale expression in *E. coli*. The single-chain antibody provided in this disclosure solves the problems of high cost and difficult preparation of existing TPO detection reagents, providing a new technical path for developing high-performance, low-cost TPO detection reagents.

[0006] Therefore, this disclosure provides the following technical solution: As a first aspect of this disclosure, a single-chain antibody is provided.

[0007] As a second aspect of this disclosure, a nucleic acid molecule is provided.

[0008] As a third aspect of this disclosure, a means of expression is provided.

[0009] As a fourth aspect of this disclosure, an expression strain is provided.

[0010] As a fifth aspect of this disclosure, a thrombopoietin detection kit is provided.

[0011] As a sixth aspect of this disclosure, the use of the single-chain antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the expression strain described in the fourth aspect, or the kit described in the fifth aspect in detecting an analyte containing thrombopoietin is provided.

[0012] As a seventh aspect of this disclosure, a method for detecting thrombopoietin is provided.

[0013] According to a first aspect of this disclosure, a single-chain antibody that specifically binds to thrombopoietin is provided, the single-chain antibody comprising at least one light chain variable region and at least one heavy chain variable region; The light chain variable region has: LCDR1 as shown in SEQ ID NO: 5 or an active variant thereof, and / or LCDR2 as shown in SEQ ID NO: 6 or an active variant thereof, and / or LCDR3 as shown in SEQ ID NO: 7 or an active variant thereof; and / or The heavy chain variable region has: HCDR1 or an active variant thereof as shown in SEQ ID NO: 8, and / or HCDR2 or an active variant thereof as shown in SEQ ID NO: 9, and / or HCDR3 or an active variant thereof as shown in SEQ ID NO: 10.

[0014] In a preferred embodiment, the single-chain antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 1 or an active variant thereof obtained by adding, deleting, substituting, or modifying one or more amino acids; and / or It contains the light chain variable region sequence shown in SEQ ID NO:2 or an active variant thereof obtained by adding, deleting, substituting or modifying one or more amino acids.

[0015] In a preferred embodiment, the single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 3.

[0016] In a preferred embodiment, the heavy chain variable region and the light chain variable region are connected by a connector.

[0017] In a preferred embodiment, the connector has the formula (Gly4Ser). n Connector.

[0018] In a preferred embodiment, n is selected from 1, 2, 3, 4, and 5.

[0019] In a preferred embodiment, the connector is one or more of the following: (Gly4Ser) connector, (Gly4Ser)2 connector, (Gly4Ser)3 connector, (Gly4Ser)4 connector, or (Gly4Ser)5 connector.

[0020] According to another aspect of this disclosure, a method for preparing the scFv that provides the anti-TPO is provided, the method comprising: The recombinant expression vector was transformed into competent E. coli cells; expression was induced (e.g., by IPTG induction); and soluble scFv protein was obtained by purifying the protein through steps such as cell disruption, affinity chromatography (e.g., nickel column purification of His-tagged scFv), and ion exchange chromatography.

[0021] According to another aspect of this disclosure, a nucleic acid molecule is provided that encodes the single-chain antibody described in this disclosure.

[0022] In a preferred embodiment, the nucleic acid molecule includes DNA and / or RNA.

[0023] In a preferred embodiment, the nucleic acid molecule is selected from the group consisting of DNA, cDNA, RNA, and mRNA.

[0024] In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the single-chain antibody disclosed herein.

[0025] In a preferred embodiment, the nucleotide sequence encoding the single-chain antibody disclosed herein comprises the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0026] According to another aspect of this disclosure, an expression vector is provided that contains the nucleotide sequence of the nucleic acid molecule described in this disclosure.

[0027] In a preferred embodiment, the expression vector is a prokaryotic expression vector.

[0028] In a preferred embodiment, the expression vector includes, but is not limited to, pET series vectors, pBV220 vectors, etc.

[0029] According to another aspect of this disclosure, an expression strain is provided that comprises the expression vector described in this disclosure.

[0030] In a preferred embodiment, the expression strain is an engineered bacterium.

[0031] In a preferred embodiment, the host cells used to construct the expression strain include, but are not limited to, BL21(DE3), Rosetta, etc.

[0032] According to another aspect of this disclosure, a test kit is provided that comprises the single-chain antibody described in this disclosure.

[0033] In a preferred embodiment, the kit is based on the principle of double-antibody sandwich ELISA, using the scFv of this disclosure as a capture antibody and / or a detection antibody (labeled).

[0034] In a preferred embodiment, the kit uses the scFv of this disclosure as a capture antibody.

[0035] According to another aspect of this disclosure, the use of the single-chain antibody, the nucleic acid molecule, the expression vector, the expression strain, or the kit described herein is provided in detecting a analyte containing thrombopoietin; the analyte being serum and / or plasma.

[0036] According to another aspect of this disclosure, a method for detecting thrombopoietin is provided, which includes using the single-chain antibody described in this invention to detect thrombopoietin in samples such as serum and plasma.

[0037] In a preferred embodiment, the basic principle of the detection method (i.e., human thrombopoietin immunoassay method) or detection kit provided in this disclosure is as follows: Thrombopoietin in human serum or plasma is detected using a double-antibody sandwich combined with a biotin-streptavidin signal amplification system. First, anti-human thrombopoietin (TPO) antibody is coated onto a solid-phase carrier. During the experiment, after adding serially diluted standards or samples to be tested, the TPO (antigen) in the standards or samples to be tested binds to the coated antibody on the enzyme-linked immunosorbent assay (ELISA) plate. Then, [the following steps are added]... Biotin-labeled anti-human TPO antibody, coated on a solid-phase support, binds to the TPO antigen and the biotin-labeled anti-human TPO antibody to form a sandwich complex. Unbound compounds are then washed away, and streptavidin-labeled horseradish peroxidase (HRP) or alkaline phosphatase is added. Biotin binds to streptavidin to form an immune complex. After washing away free components, a chromogenic substrate is added for color development. The reaction is terminated with a stop solution, and the absorbance (OD) of the solution in the reaction wells is measured at 450 nm using a microplate reader. 450 TPO concentration and OD 450 The value is directly proportional, and the concentration of human TPO in the sample can be calculated by plotting a standard curve.

[0038] The beneficial effects of this invention are as follows: (1) An anti-TPO single-chain antibody (scFv) is provided: This invention successfully screened and obtained an anti-human TPO single-chain antibody containing a specific CDR sequence through genetic engineering recombination technology. This scFv not only has the ability to specifically recognize TPO in its natural conformation, but also overcomes the defects such as steric hindrance that are easily generated in the detection system by intact IgG molecules, providing a high-performance core recognition element for TPO immunoassay.

[0039] (2) The preparation process is simple and the production cost is significantly reduced: The scFv described in this invention can be efficiently expressed in Escherichia coli, avoiding the drawbacks of traditional animal immunization or mammalian cell expression systems, such as long cycle, high cost, and large batch-to-batch variability. High-purity recombinant protein can be obtained by combining conventional purification steps such as nickel column affinity chromatography. The process route is mature, the operation is simple, and it is suitable for large-scale production, which can significantly reduce the raw material cost of TPO detection reagents.

[0040] (3) Excellent binding performance and strong detection specificity: Through reasonable vector design and optimization of induction expression conditions, scFv with high soluble expression rate and activity was obtained. ELISA verified that it has high affinity and high specificity for TPO, which ensures the high specificity of the immunoassay process and helps to avoid the potential impact of interfering compounds in the sample on the detection results.

[0041] (4) Clear application prospects: The scFv of this invention exhibits better coating efficiency and antigen binding activity during solid-phase coating or label conjugation. Its molecular structure can be flexibly introduced with different tags or conjugated signal molecules through genetic engineering. It can be used as a capture antibody or a detection antibody. It can be directly used to develop a high-sensitivity and high-specificity TPO detection ELISA kit, providing a new, economical and reliable detection tool for clinical diagnosis and basic research. Attached Figure Description

[0042] Figure 1 The plasmid map containing the thrombopoietin single-chain antibody sequence constructed in Example 1 is shown.

[0043] Figure 2 The image shows the SDS-PAGE electrophoresis results from Example 2. Lane M: protein marker; Lane 1: purified recombinant TPO protein.

[0044] Figure 3 A flowchart of the human thrombopoietin immunoassay method in Example 4 is shown.

[0045] Figure 4 A standard curve of TPO constructed in Example 5 is shown.

[0046] Figure 5 The stability test results of the human thrombopoietin assay kit in Example 5 are shown in the figure. Detailed Implementation

[0047] Currently, the detection of thrombopoietin (TPO) levels is mainly based on immunological methods (such as ELISA and chemiluminescence immunoassay), with anti-TPO antibodies being one of the core reagents. However, existing antibodies have several drawbacks, including high preparation costs, reliance on immunized animal or mammalian cell expression systems, and a tendency to cross-react with related cytokines.

[0048] To address the aforementioned issues, this disclosure aims to provide a genetically engineered single-chain antibody (scFv) that is readily expressed on a large scale in *E. coli*. This scFv has a small molecular weight, good stability, and low production cost, and possesses the ability to bind with high affinity and high specificity to TPO, making it suitable for developing high-performance, low-cost TPO detection reagents and methods. This scFv can be directly used to develop highly sensitive and specific TPO detection ELISA kits, providing a novel, economical, and reliable detection tool for clinical diagnosis and basic research.

[0049] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this disclosure, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0050] Unless otherwise stated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the practice of this disclosure employs conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0051] It should be noted that all headings and subheadings used herein are for convenience only and should not be construed as limiting this disclosure in any way. Unless otherwise stated, the use of any and all examples or exemplary wording provided herein (e.g., “such”) is intended only to better illustrate this disclosure and does not constitute a limitation on the scope of this disclosure.

[0052] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0053] The terms “comprising” and “including” as used in the specification and claims of this application mean the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or combinations thereof.

[0054] It should be understood that the terminology used in this application specification is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Unless the context clearly indicates otherwise, the terms "a," "an," "the," or "the" as used in the specification and claims of this application may mean one or more. Unless the context clearly indicates otherwise, terms presented in the singular also include plural cases.

[0055] In this disclosure, "specific binding" means binding selectively to an antigen and distinguishable from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen.

[0056] In this disclosure, "% sequence identity" or "sequence identity" is used in the context of this disclosure to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percentage identity". The percentage homology of two sequences can be calculated as follows: after aligning the two sequences, divide the number of identical residue positions by the total length of the aligned sequence and then multiply by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST kit available on the NCBI website (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410). The phrase "at least 80% sequence identity" as used herein refers to having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence.

[0057] According to the technical solution of this disclosure, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below.

[0058] Table 1

[0059] Furthermore, due to the degeneracy of bases, bases in polynucleotide sequences can be substituted without altering the activity or function of the polynucleotide sequence, as shown in Table 2 below.

[0060] Table 2

[0061] It should also be understood that the term "and / or" as used in this specification and claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations. Furthermore, in some embodiments of this disclosure, features or combinations of features set forth herein may be excluded or omitted.

[0062] The various aspects and specific embodiments of this disclosure will now be described in more detail through non-limiting implementations and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0063] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0064] Example Example 1: Construction of a strain expressing the thrombopoietin single-chain antibody TPO-scFv The antibody disclosed herein is a recombinant single-chain antibody, obtained by expression in *E. coli*. The antibody consists of a heavy chain variable region and a light chain variable region, the specific sequence composition of which is shown in Table 3. The heavy chain variable region and the light chain variable region are linked by a (Gly4Ser)3 linker. To facilitate subsequent protein isolation and purification, a six-histidine tag (His×6) is designed at the C-terminus of the TPO single-chain antibody.

[0065] Table 3 Recombinant single-chain antibody sequences

[0066] The target gene was synthesized by the company and then inserted into the E. coli expression vector pET-22b, forming the TPO-scFv recombinant expression plasmid PET-22b-TPO-Ab. (The plasmid map is shown below.) Figure 1As shown. The recombinant expression plasmid was first transformed into *E. coli* DH5α to amplify the plasmid, and then transformed into *E. coli* BL21(DE3) competent cells. After induction of expression, purification, and identification, a high-performance protein form of the single-chain antibody TPO-scFv was obtained. The amino acid sequence of the thrombopoietin TPO single-chain antibody is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4. Details are as follows: DH5α glycerol bacteria containing a thrombopoietin single-chain antibody plasmid were inoculated onto LB agar plates containing ampicillin resistance and incubated overnight at 37°C with the plates inverted. Single colonies were then picked and inoculated onto ampicillin-resistant LB liquid medium and incubated overnight at 37°C with shaking at 200 rpm. The cultured bacterial suspension was centrifuged at 12,000 rpm for 2 min, the supernatant was removed, and the bacterial cells were retained for plasmid extraction.

[0067] Add 1 μg of purified plasmid to 100 μL of *E. coli* BL21 competent cells, mix gently, and incubate on ice for 30 min. Then, place the centrifuge tube containing the plasmid and *E. coli* competent cells in a 42°C water bath for 90 s heat shock, followed by an ice bath for 2-3 minutes. Add 800 μL of LB medium to the heat-shocked bacterial culture and incubate at 37°C with shaking at 150 rpm for 1 h. After incubation, spread 200 μL onto ampicillin-resistant LB agar plates and incubate overnight at 37°C. Pick positive transformant colonies and inoculate them into LB medium containing ampicillin, incubate at 37°C at 200 rpm for 12 h, then add 10%-15% sterile glycerol to a portion of the bacterial culture and store at -80°C for later use.

[0068] Example 2: Expression and purification of thrombopoietin single-chain antibody TPO-scFv Take 10 μL of glycerol bacteria and inoculate it into 10 mL of ampicillin-resistant LB medium. Incubate overnight at 37°C and 200 rpm for activation. Inoculate 1% to 10% of the culture into ampicillin-resistant medium and continue incubation at 37°C and 200 rpm. Observe the OD of the bacterial culture. 600 When the expression level reaches 0.6-1.0, add 0.1-1.0 mM IPTG to induce the expression of the target protein, and culture at 20℃ and 200 rpm for 15 h. After induction, remove the culture medium from the shaker and centrifuge at 8000 rpm and 4℃ for 15 min using a refrigerated centrifuge. Collect the bacterial cells for subsequent protein purification.

[0069] The collected bacterial cells were dissolved in 10 mM PBS buffer, placed on ice, and sonicated for 25 minutes. The cells were then centrifuged at 12,000 rpm for 40 minutes. The supernatant of the cell lysis was collected and purified using Ni-NTA agarose gel electrophoresis. The purified thrombopoietin single-chain antibody was dialyzed against PBS buffer (pH 7.4) at 4°C for 2 days, with the dialysate changed on both sides. Protein concentration was monitored, and if the concentration was low, it was concentrated using an ultrafiltration tube. The purified protein was stored at -80°C.

[0070] The medium was washed with 5 times its volume of equilibration buffer, and adsorption was performed three times with cell lysis buffer. Gradient washing with 10 mM imidazole, 50 mM imidazole, and 250 mM imidazole was then performed to remove non-specifically bound impurity proteins. The 250 mM eluent was collected (the high concentration of 250 mM imidazole occupies the binding sites of Ni ions, displacing the tightly bound target protein (TPO-scFv)). The results were analyzed by SDS-PAGE. Figure 2 As shown, the molecular weight of the actual recombinant protein is consistent with the theoretical size of 28 kDa, and the concentration of the purified TPO-scFv protein is approximately 1 mg / mL.

[0071] Example 3: Preparation of Human Thrombopoietin Detection Kit The detection kit includes: (1) Core reaction components: an enzyme-labeled plate coated with anti-human thrombopoietin antibody TPO-scFv, biotin-labeled thrombopoietin antibody, and streptavidin-labeled horseradish peroxidase solution; (2) Standard: TPO protein; (3) Auxiliary reagents: sample diluent, biotin-labeled antibody diluent, enzyme conjugate diluent, washing solution, blocking solution, TMB colorimetric solution and stop solution, etc.

[0072] The preparation methods for each reagent are as follows: Coating buffer: Weigh 8.00g sodium chloride, 0.20g potassium chloride, 0.24g dipotassium hydrogen phosphate, and 1.42g disodium hydrogen phosphate, and bring the volume to 1L. Adjust the pH to 8.0 with hydrochloric acid or sodium hydroxide.

[0073] Sample diluent: Weigh 8.00g sodium chloride, 0.20g potassium chloride, 0.24g dipotassium hydrogen phosphate, and 1.42g disodium hydrogen phosphate to a final volume of 1L. Adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide.

[0074] Biotin-labeled antibody dilution solution: a washing solution containing 2% newborn calf serum by volume.

[0075] Enzyme conjugate diluent: A washing solution containing 2% newborn calf serum by volume.

[0076] Cleaning solution: Weigh 8.00g sodium chloride, 0.20g potassium chloride, 0.24g dipotassium hydrogen phosphate, and 1.42g disodium hydrogen phosphate, add 5mL Tween-20 and bring the volume to 1L. Adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide.

[0077] Blocking solution: Sample diluent containing BSA and fetal bovine serum.

[0078] TMB developer: a commercially available product.

[0079] Termination solution: Add 1 mL of concentrated sulfuric acid dropwise to 17 mL of ultrapure water.

[0080] Example 4: Immunoassay Method and Procedure for Human Thrombopoietin This embodiment uses the double-antibody sandwich ELISA method as an example to further illustrate the immunoassay method for thrombopoietin in human serum or plasma. The method mainly includes the following steps ( Figure 3 ): (1) Coating human thrombopoietin antibody: Dilute anti-human thrombopoietin antibody TPO-scFv with 1×ELISA coating buffer, add 100 μL / well to ELISA microplate, incubate overnight in the refrigerator, and wash the plate 5 times with washing buffer after incubation; (2) Blocking: Add blocking solution, 300 μL / well, incubate in a constant temperature incubator at 37℃ for 1-2 h, and wash the plate 5 times; (3) Add standard and sample: According to the experimental needs, set up standard wells (add different concentrations of TPO protein), blank wells (add only sample diluent) and sample wells (human serum, plasma and other samples), 100 μL per well, cover the ELISA plate with a new sealing film, and incubate at 37°C for 2 h.

[0081] (4) Add biotinylated antibody: aspirate or shake off the liquid in the well, add 100 μL of biotinylated human thrombopoietin antibody working solution to each well, cover the plate with a membrane, incubate at 37°C for 1 hour, and wash the plate 3 times with washing buffer after incubation.

[0082] (5) Add streptavidin-labeled horseradish peroxidase conjugate: Add 100 μL of streptavidin-labeled horseradish peroxidase conjugate working solution to each well, cover the plate with a membrane, incubate at 37°C for 30 minutes, and wash the plate 6 times.

[0083] (6) Add substrate for color development: Add 90 μL of TMB color development solution to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for about 15 minutes.

[0084] (7) Add stop solution: Add 50 μL of stop solution to each well to terminate the reaction.

[0085] (8) Detection reading: Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0086] In step (4), the detection antibody is diluted with biotin-labeled antibody diluent, and the concentration of biotin-labeled antibody is 0.1-1.0 μg / mL.

[0087] Example 5 Performance Analysis of Human Thrombopoietin Detection Kit (1) Linear range Thrombopoietin (TPO protein) was diluted using the sample diluent from Example 3 to prepare standard solutions with final concentrations of 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL. The absorbance of different concentrations of thrombopoietin (3 replicates) was measured using the steps and methods from Example 4, as shown in Table 4.

[0088] Table 4 Linearity Test Results

[0089] Plotting concentration on the x-axis and average absorbance on the y-axis, a four-parameter logistic curve was fitted using ELISA Calc, as shown in the figure. Figure 4 As shown. The resulting linear equation is: y = (AD) / [1 + (x / C)^B] + D, Where A=6.101, B=-0.9989, C=2444.07, D=0.1219, r^2=0.9993; Within the reference range, the linear correlation coefficient r^2 ≥ 0.990. These results demonstrate that the detection values ​​are linear within the detection range of 31.25 pg / mL–2000 pg / mL, proving its quantitative ability and accuracy within this range.

[0090] (2) Evaluation of the stability of the reagent kit coated antibody ELISA plate The ELISA plates coated with human thrombopoietin antibodies were placed in a 4°C freezer and a 37°C incubator for 5 days, respectively. Detection was performed using different concentrations of standard diluents and the detection procedure shown in Example 4. The results are as follows: Figure 5As shown, the left-hand "color development" image shows the blue substance formed by the reaction of TMB and HRP after the addition of TMB. The right-hand "post-termination" image shows that the blue substance formed after the reaction of TMB and HRP is terminated with sulfuric acid or other substances, resulting in a more stable yellow substance. It can be seen that there is no significant difference in color between the two wells in each row after accelerated treatment at 37℃ for 5 days and after storage at 4℃, although the detection value is slightly lower. The CV% between the 4℃ storage and 37℃ accelerated treatment plates is less than 10%, indicating that the ELISA plate coated with human thrombopoietin antibody has high stability.

[0091] Furthermore, in the graph, from top to bottom (TPO concentration from 2000 pg / mL to NC), the color inside the well gradually lightens as the concentration decreases. This clear gradient change visually confirms the validity of the "linear correlation coefficient," indicating that the detection system is effective.

[0092] While some exemplary embodiments of this application have been described and illustrated, this application is not limited to the disclosed embodiments. Rather, those skilled in the art will recognize that modifications and changes may be made to the described embodiments without departing from the spirit and scope of this application as described in the appended claims.

Claims

1. A single-chain antibody that specifically binds to thrombopoietin, characterized in that, The light chain variable region of the single-chain antibody includes LCDR1 as shown in SEQ ID NO: 5, LCDR2 as shown in SEQ ID NO: 6, and LCDR3 as shown in SEQ ID NO: 7; the heavy chain variable region of the single-chain antibody includes HCDR1 as shown in SEQ ID NO: 8, HCDR2 as shown in SEQ ID NO: 9, and HCDR3 as shown in SEQ ID NO:

10.

2. The single-chain antibody according to claim 1, characterized in that, The single-chain antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 1; and the light chain variable region sequence shown in SEQ ID NO:

2.

3. The single-chain antibody according to claim 1, characterized in that, The single-chain antibody contains the amino acid sequence shown in SEQ ID NO:

3.

4. The single-chain antibody according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region are connected by connectors.

5. The single-chain antibody according to claim 4, characterized in that, The connector has the formula (Gly4Ser). n The connector is n, where n is selected from 1 to 5.

6. A nucleic acid molecule, characterized in that, It encodes the single-chain antibody as described in any one of claims 1-5.

7. An expression carrier, characterized in that, It comprises the nucleotide sequence of the nucleic acid molecule of claim 6.

8. An expression strain, characterized in that, It comprises the expression vector as described in claim 7.

9. A test kit, characterized in that, It contains any one of the single-chain antibodies described in 1-5.

10. The application of the single-chain antibody according to any one of claims 1-5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7, the expression strain according to claim 8, or the kit according to claim 9, characterized in that, The application is in the preparation of reagents for detecting thrombopoietin in a analyte; the analyte is serum and / or plasma.

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