Monoclonal Antibody Specific for Binding 25-Hydroxyvitamin D and Its Application

Abstract

The present invention provides a monoclonal antibody with high specificity and high affinity for 25-hydroxyvitamin D (25-OH-VD), and its application in immunoassay. The monoclonal antibody was successfully prepared through an innovative immunization procedure and cell fusion technology, breaking through the limitations of traditional detection methods and significantly improving the sensitivity and accuracy of detection. Based on the screened monoclonal antibody combination, an immunoassay kit using the sandwich ELISA method was developed. Experimental results confirmed that the kit has an extremely high coincidence rate compared with traditional detection methods, demonstrating good clinical relevance and consistency, providing a new technical means for the quantitative analysis of 25-OH-VD, and having important clinical application value and market potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody preparation and immunological detection, and particularly relates to a monoclonal antibody capable of specifically binding to 25-hydroxyvitamin D. In addition, the present invention also provides a method for immunological detection using the monoclonal antibody, as well as a detection kit containing the monoclonal antibody, providing a new technical means for the quantitative analysis of 25-hydroxyvitamin D. Background Art

[0002] 25-Hydroxyvitamin D (25(OH)D) is the main circulating form of vitamin D in the body, and vitamin D plays an important role in maintaining human health. Lack of vitamin D can lead to skeletal health, respiratory diseases, cardiovascular diseases, digestive diseases, endocrine and metabolic diseases, neuropsychiatric diseases, and urinary system diseases. 25-Hydroxyvitamin D is formed by hydroxylation of vitamin D in the liver. By detecting the level of 25-hydroxyvitamin D, early diseases such as rickets, osteomalacia, and osteoporosis can be detected and treated in a timely manner. Detection of 25(OH)D levels helps to evaluate an individual's skeletal health status and detect and prevent skeletal diseases in a timely manner.

[0003] Detecting the concentration of 25-hydroxyvitamin D (25(OH)D) is crucial for the diagnosis and treatment of related diseases. Currently, the methods for detecting 25(OH)D mainly include liquid chromatography-tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), etc. Among them, LC-MS / MS is considered the "gold standard" for detecting 25(OH)D due to its high sensitivity, high specificity, and wide linear range. However, due to the small molecular weight of 25(OH)D, it is difficult to develop antibodies that can recognize different epitopes, which limits the possibility of using the sandwich immunoassay method. Therefore, developing monoclonal antibodies with high specificity and high affinity is of great significance for the detection of 25(OH)D.

[0004] Existing detection methods have their own advantages and limitations. Although LC-MS / MS has high accuracy, it has high costs, complex operations, and the detection results between different laboratories may deviate, which requires standardization of the detection method to ensure the consistency and comparability of the results. In contrast, ELISA and CLIA are easy to operate and can be automated, but their specificity and linear range may not be as good as LC-MS / MS, and there is a problem of poor comparability of detection results.

[0005] Competitive protein binding assay (CPBA) may lead to inaccurate test results due to its insufficient specificity. Therefore, the development of monoclonal antibodies with high specificity and affinity is crucial for improving the accuracy and reliability of detection. This can not only promote the development of 25(OH)D detection technology but also provide stronger support for the diagnosis and treatment of related diseases. Summary of the Invention

[0006] To solve the above problems, the present invention provides a monoclonal antibody with high specificity and high affinity, which can specifically bind to 25(OH)D and can be used to develop an immunoassay kit based on the sandwich method with two antibodies. The development of this monoclonal antibody breaks through the limitations of traditional immunoassay techniques and improves the sensitivity and accuracy of detection.

[0007] The first aspect of the present invention is to provide a monoclonal antibody against 25-hydroxyvitamin D or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment has 3 light-chain complementary determining regions and 3 heavy-chain complementary determining regions. The 3 light-chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:1, LCDR2 shown in SEQ ID NO:2, and LCDR3 shown in SEQ ID NO:3, and the 3 heavy-chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, and HCDR3 shown in SEQ ID NO:6; or the 3 light-chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:10, and LCDR3 shown in SEQ ID NO:11, and the 3 heavy-chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, and HCDR3 shown in SEQ ID NO:15.

[0008] Furthermore, for the monoclonal antibody or its antigen-binding fragment, the monoclonal antibody or its antigen-binding fragment includes a light-chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:7, and a heavy-chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:8;

[0009] The monoclonal antibody or its antigen-binding fragment comprises a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 12, and a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 16.

[0010] Furthermore, the monoclonal antibody or its antigen-binding fragment comprises a heavy chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 17, and / or comprises a light chain constant having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 18; or

[0011] The monoclonal antibody or its antigen-binding fragment comprises a heavy chain constant region having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 19, and / or comprises a light chain constant having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 20.

[0012] The second aspect of the present invention provides a polynucleotide encoding the monoclonal antibody or its antigen-binding fragment of the present invention.

[0013] The third aspect of the present invention provides a nucleic acid construct comprising the polynucleotide of the present invention, and optionally, at least one expression regulatory element operably linked to the polynucleotide.

[0014] The fourth aspect of the present invention provides a recombinant vector comprising the polynucleotide of the present invention or the nucleic acid construct of the present invention.

[0015] The kit comprises:

[0016] (a) A monoclonal antibody specifically binding to 25-hydroxyvitamin D for coating, and

[0017] (b) A monoclonal antibody specifically binding to 25-hydroxyvitamin D for labeling;

[0018] The monoclonal antibody for coating comprises 3 light chain complementarity determining regions and 3 heavy chain complementarity determining regions, wherein

[0019] The three light chain complementary determining regions of the monoclonal antibody for coating comprise LCDR1 shown in SEQ ID NO:1, LCDR2 shown in SEQ ID NO:2, and LCDR3 shown in SEQ ID NO:3, and the three heavy chain complementary determining regions of the monoclonal antibody for coating comprise HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, and HCDR3 shown in SEQ ID NO:6;

[0020] The monoclonal antibody for labeling comprises three light chain complementary determining regions and three heavy chain complementary determining regions. Among them, the three light chain complementary determining regions of the monoclonal antibody for labeling comprise LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:10, and LCDR3 shown in SEQ ID NO:11, and the three heavy chain complementary determining regions of the monoclonal antibody for labeling comprise HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, and HCDR3 shown in SEQ ID NO:15.

[0021] Furthermore, in the double antibody sandwich immunoassay kit,

[0022] The monoclonal antibody (a) for coating comprises a light chain variable region of the amino acid shown in SEQ ID NO:7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:8;

[0023] The monoclonal antibody (b) for labeling comprises a light chain variable region of the amino acid shown in SEQ ID NO:12 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:16;

[0024] Optionally, the monoclonal antibody (a) for coating and the monoclonal antibody (b) for labeling comprise i) a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:17 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:18 or ii) a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:19 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:20.

[0025] Furthermore, the double antibody sandwich immunoassay kit includes a double antibody sandwich immunochromatographic test strip. In the double antibody sandwich immunochromatographic test strip, the monoclonal antibody (a) specifically binding to 25-hydroxyvitamin D is used for coating, and the monoclonal antibody (b) specifically binding to 25-hydroxyvitamin D is used for labeling.

[0026] The sixth aspect of the present invention provides an application, which is the application of the double-antibody sandwich immunoassay kit of the present invention in the preparation of a detection kit for diseases caused by abnormal vitamin D.

[0027] Further, the vitamin D abnormal diseases include osteoporosis and osteomalacia.

[0028] Further, the abnormal vitamin D is vitamin D deficiency.

[0029] More preferably, the vitamin D deficiency includes diseases related to diabetes, different types of cancers, cardiovascular and cerebrovascular diseases, and autoimmune diseases.

[0030] The seventh aspect of the present invention provides a method for preparing an immunoassay test strip. First, a coating pad is bonded on a PVC plate, then a blotting paper is lapped at one end of the quality control line c line close to the coating pad, and a conjugate pad is lapped at one end of the T line close to the coating pad. The yellow part of the conjugate pad contains fluorescent microspheres - ab and fluorescent microspheres - goat anti-rabbit secondary antibody (see Figure 4 ). The T line and the c line are sequentially coated on the coating pad; among them, the labeled antibody numbered MAb2135 of the present invention is used to prepare the fluorescent microsphere - antibody complex and placed on the conjugate pad, and the antibody coating antibody numbered MAb1024 is used for T line coating.

[0031] The eighth aspect of the present invention provides an immunoassay test strip prepared by the method of the seventh aspect.

[0032] Advantages of the present invention:

[0033] 1. High specificity and affinity: The monoclonal antibodies of the present invention have extremely high specificity and affinity, and can tightly bind to specific epitopes of 25-OH-VD. This high affinity makes the detection method have excellent sensitivity and accuracy, and helps to improve the reliability of disease diagnosis.

[0034] 2. Breaking through the limitations of traditional technologies: The invention overcomes the limitations existing in traditional immunoassay technologies, such as low sensitivity and low specificity. By developing new monoclonal antibodies, the double-antibody sandwich method becomes possible, thereby improving the efficiency and accuracy of 25-OH-VD detection.

[0035] 3. Simple operation and high cost-effectiveness: Compared with high-cost and complex-operation detection methods such as liquid chromatography - tandem mass spectrometry (LC-MS / MS), the immunoassay method of the present invention has simple operation, is easy to automate, and has low cost, and is suitable for large-scale screening and clinical applications.

[0036] 4. Good clinical relevance and consistency: The experimental results show that the detection method of the present invention has a very high coincidence rate compared with the traditional Roche competitive method, indicating good clinical relevance. This means that this method can provide more accurate detection results for doctors and patients, helping to guide clinical decision-making and disease management.

[0037] In summary, by providing a novel monoclonal antibody and corresponding detection method, the present invention not only improves the performance of 25-OH-VD detection but also reduces the detection cost, having important clinical application value and market potential.

[0038] Term definition:

[0039] Antibody: An antibody is a protein produced by the immune system, whose main function is to recognize and neutralize foreign pathogens. They are produced by B cells after being stimulated by specific antigens and play a key role in humoral immunity. Antibodies have a high degree of specificity and can bind to specific antigen epitopes, protecting the body from infection through various mechanisms (such as neutralization, opsonization, activation of the complement system). The antibodies of the present invention include but are not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies.

[0040] Monoclonal antibody: A monoclonal antibody (mAb) is an antibody produced by a single cloned B cell line. These antibodies are homogeneous in molecular structure and only target specific antigen epitopes. The production of monoclonal antibodies usually involves fusing B cells that produce specific antibodies with myeloma cells to form hybridoma cells. These hybridoma cells can proliferate infinitely in vitro and produce a large amount of the same antibody. Monoclonal antibodies are very useful in medical research, diagnosis, and treatment due to their high specificity and homogeneity.

[0041] Immunoassay: Immunoassay is a method that uses antigen-antibody reactions to detect specific substances (usually proteins or pathogens). This kind of detection can be used for disease diagnosis, monitoring treatment response, or evaluating immune status. There are various immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), immunofluorescence, immunoblot (Western Blot), flow cytometry, etc. These techniques can detect the presence of antibodies, quantify specific proteins, or identify specific cell types. The sensitivity and specificity of immunoassay make it an indispensable tool in medical research and clinical diagnosis.

[0042] Affinity, Binding Affinity: In immunology, the binding affinity or avidity of an antibody generally refers to the tightness of its binding to a specific antigenic epitope. High affinity means that the antibody can bind to the antigen with high specificity and strength, while low affinity means weaker binding and susceptibility to competitive molecules. Affinity can be measured by various experimental methods, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and enzyme-linked immunosorbent assay (ELISA). These methods can provide kinetic parameters regarding the binding of the antibody to the antigen, such as the binding constant (Kd) or the dissociation constant (Koff), which can be used to evaluate the magnitude of the affinity. The binding constant Kd is an inverse indicator reflecting affinity, i.e., the smaller the Kd value, the stronger the affinity. Brief Description of the Drawings

[0043] Figure 1 It is the gel diagram of the SDS-PAGE detection of the anti-25-OH-VD monoclonal antibody expressed and purified in Example 3; wherein, M represents the electrophoretic band of the molecular weight Marker, lane 1 shows the electrophoretic bands of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoretic bands of each monoclonal antibody under non-reducing conditions.

[0044] Figure 2 It is the HPLC chromatogram of the MAb1024 monoclonal antibody in Example 3.

[0045] Figure 3 It is the HPLC chromatogram of the MAb2135 monoclonal antibody in Example 3.

[0046] Figure 4 It is the schematic diagram of the structure of the colloidal gold immunochromatographic test strip prepared in the embodiment of the present invention.

[0047] Figure 5 It is the actual photo of the colloidal gold immunochromatographic test strip prepared in the embodiment of the present invention. Specific Embodiments

[0048] The present invention will be further described below in conjunction with specific embodiments. The described embodiments are some, but not all, of the embodiments of the present invention. It should be understood that the following embodiments are provided to give a complete disclosure and explanation to those of ordinary skill in the technical field to which the present invention belongs on how to use the methods and compositions of the present invention, and are not intended to limit the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0049] Example 1 Preparation of Monoclonal Antibody Specifically Binding to 25-OH-VD

[0050] Experimental Method:

[0051] (1) Immunization protocol design:

[0052] - Using conjugated protein 25-OHVD-BSA (purchased from Nanjing Kaitai'er Biotechnology Co., Ltd., catalog number AG0124) as an antigen, after fully emulsifying it with an equal volume of Freund's complete adjuvant, it was subcutaneously injected to immunize goats.

[0053] - Using an immune complex conjugated with 25-OH-VD antigen and its labeled antibody (obtained by mixing 25-OH-VD antigen with the labeled antibody screened in this project) as an antigen, after fully emulsifying it with an equal volume of Freund's complete adjuvant, it was subcutaneously injected to immunize rabbits (breed: New Zealand white rabbit, female, 6 - 8 weeks old).

[0054] - In the following two weeks, two booster immunizations were carried out according to the same immunization protocol, for a total of three immunizations.

[0055] - Five to eight days after the last booster immunization, blood was collected by intravenous puncture, and the enzyme-linked immunosorbent assay (ELISA) method was used to evaluate the antibody titer in the serum.

[0056] - Set 128 as the standard line. When the detected serum titer exceeded 128K, the preparation of peripheral blood mononuclear cell (PBMC) suspension began.

[0057] (2) Preparation of PBMC suspension:

[0058] - Add an appropriate amount of Ficoll lymphocyte separation solution to a centrifuge tube, and mix anticoagulated blood with sterile phosphate buffer solution (PBS) at a ratio of 1:1 to 1:5.

[0059] - Slowly pour the mixed solution along the tube wall onto the Ficoll layering solution, and perform centrifugal separation at a speed of 400g for 30 minutes.

[0060] - After centrifugation, remove most of the upper liquid, retain about 1 mL of the precipitate, and use a pipette to aspirate the cloudy layer containing mononuclear cells.

[0061] - Transfer the collected cells to a new centrifuge tube, add 5 volumes of PBS, and centrifuge at a speed of 300g for 10 minutes, repeating the washing twice.

[0062] - After washing, remove the supernatant, add red blood cell lysate, incubate at room temperature for 2 minutes to lyse red blood cells, then add 10 mL of PBS, and centrifuge at a speed of 300g for 10 minutes again to wash the cells twice.

[0063] - Discard the supernatant, and finally add RPMI1640 medium containing 10% fetal bovine serum (FBS) to resuspend the cells and perform cell counting.

[0064] (3) B cell screening:

[0065] - Using PBMC suspension, add FITC-labeled antigen (25-OHVD-BSA, or 25-OH-VD antigen and the labeled antibody obtained by screening in this project), and use flow sorting technology to screen out B cells that have a specific reaction to 25-OH-VD on a flow cytometer (BD Influx).

[0066] Experimental results:

[0067] Through the above steps (1)-(3), Example 1 successfully obtained a monoclonal antibody library against 25-OH-VD and obtained multiple monoclonal antibodies, all of which were of the IgG type after testing.

[0068] Table 1. Source table of monoclonal antibodies against 25-OH-VD

[0069] Clone Name Screening Method MAb1024 (Coated) Flow Cytometry Screening, Sequencing MAb2135 (Labeled) Flow Cytometry Screening, Sequencing

[0070] Example 2 Molecular construction and production of full antibodies

[0071] Sequence the monoclonal antibodies constructed for coating and labeling in Table 1. The amino acid sequences of the variable regions of 2 lead antibodies are as shown in Table 2 below (the CDR regions are marked with underlines, and the analysis system is the IMGT system). The constant region sequences of the heavy and light chains are shown in Table 3.

[0072] Table 2

[0073]

[0074]

[0075] Table 3 Amino acid sequences of the constant regions of the heavy and light chains of the coating antibody Mab1024

[0076]

[0077] Table 4 Amino acid sequences of the constant regions of the heavy and light chains of the labeling antibody Mab2135

[0078]

[0079] Example 3: Expression and purification of monoclonal antibodies against 25-OH-VD

[0080] Experimental method:

[0081] (1) Cell culture:

[0082] - Use suspension culture technology to culture N293 cells until the cell density reaches (1-3)×10^6 cells / mL.

[0083] - Before transfection, ensure that the cell viability is greater than 80 - 90%, which can be evaluated by the trypan blue exclusion test or similar cell viability assays.

[0084] - During cell culture, regularly monitor cell density and viability to ensure that the cells are in a healthy state before transfection.

[0085] (2) Cell transfection:

[0086] - After the cell density and viability meet the requirements, perform cell transfection. Before transfection, calculate the amounts of plasmid DNA and transfection reagent required based on the cell number and transfection efficiency.

[0087] - According to the light chain and heavy chain gene sequences of the monoclonal antibody measured in Example 2, chemically synthesize these gene fragments.

[0088] - Clone the synthesized light chain and heavy chain genes into the pcDNA3.4 plasmid vector respectively to construct recombinant expression vectors.

[0089] - Use polyethyleneimine (PEI) as the transfection reagent and prepare the transfection mixture according to the mass ratio of PEI to plasmid DNA of 3:1.

[0090] - During transfection, add the transfection mixture to the cell culture system according to the ratio of 2 μg plasmid DNA / 1 mL N293 cells.

[0091] - Gently mix the culture system to ensure that the transfection mixture is in full contact with the cells, and then incubate in a cell culture incubator to promote the formation of transfection complexes and intracellular delivery of DNA.

[0092] (3) Protein expression and purification:

[0093] - 5 - 7 days after transfection, collect the cell culture supernatant, and this step can remove cell debris by centrifugation.

[0094] - Purify the monoclonal antibody using Protein A affinity chromatography resin. Pass the cell supernatant through a chromatography column pre - loaded with Protein A resin and collect the flow - through.

[0095] - Elute the antibody bound to the Protein A resin with an appropriate eluent and collect the eluate.

[0096] - Replace the antibody buffer in the eluate with phosphate - buffered saline (PBS) by dialysis, and the PBS should be changed regularly during dialysis to ensure complete replacement.

[0097] - Determine the concentration of the antibody using a Nanodrop or similar spectrophotometer to ensure accurate usage of the antibody in subsequent experiments.

[0098] (4) Antibody purity detection:

[0099] (4.1) SDS-PAGE detection:

[0100] - Take 2 μg of the antibody to be tested and add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20 μL.

[0101] - Prepare a reducing SDS-PAGE sample: Add β-mercaptoethanol or DTT to the loading buffer to perform SDS-PAGE on the sample under reducing conditions.

[0102] - Prepare a non-reducing SDS-PAGE sample: Do not add a reducing agent to maintain the native disulfide bond state of the antibody.

[0103] - Perform pre-electrophoresis at 80 V for 30 minutes, and then perform electrophoresis at 120 V until the bands are completely separated.

[0104] - After electrophoresis is completed, take out the PAGE gel, perform Coomassie Brilliant Blue staining for 15 minutes.

[0105] - After staining, rinse the gel with deionized water to remove the staining solution, and then add decolorizing solution for decolorization until the bands are clearly visible.

[0106] (4.2) HPLC detection:

[0107] - Use a high-performance liquid chromatography (HPLC) system equipped with an appropriate chromatographic column and detector to analyze the antibody sample.

[0108] - Optimize the composition and flow rate of the HPLC mobile phase according to the characteristics of the antibody such as molecular size, charge, and hydrophobicity to achieve the best separation effect.

[0109] - Analyze the HPLC chromatogram to evaluate the purity of the antibody and possible impurities.

[0110] Experimental results:

[0111] The results of electrophoresis detection are as Figure 1Shown as follows: Under reducing conditions, both monoclonal antibodies numbered MAb1024 and MAb2135 have two electrophoretic bands, and the corresponding molecular weights of these two electrophoretic bands are consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, the monoclonal antibodies all have a single band, and the corresponding molecular weight of this electrophoretic band is consistent with the size of their respective intact antibodies; these results indicate that the expression of the monoclonal antibodies is accurate. In addition, all electrophoretic bands have clear edges and no impurity bands, indicating that the purity of the monoclonal antibodies obtained by the above expression and purification steps is relatively high.

[0112] HPLC chromatogram: The HPLC results show that the purity of the monoclonal antibodies is greater than 99%.

[0113] Example 5: Preparation of a double-antibody sandwich fluorescence immunochromatographic test strip and detection of its sensitivity

[0114] In this example, the antibody numbered MAb1024 in Example 3 was used as the coating antibody for the T line, and the antibody numbered MAb2135 in Example 3 was used as the labeled antibody to prepare a colloidal gold immunochromatographic test strip for detecting 25-OH-VD based on the principle of the double-antibody sandwich method. The schematic diagram of the structure of the test strip is as Figure 3 shown, in which the MAb2135-labeled antibody was used to prepare a fluorescence microsphere-antibody complex and placed on the conjugate pad, and the MAb1024 coating antibody was used for coating the T line.

[0115] The preparation method of the test strip is as follows:

[0116] (1) Preparation of the fluorescence microsphere label:

[0117] - Weigh 0.02 grams of latex fluorescence microspheres and centrifuge them at a speed of 10,000 revolutions per minute for 30 minutes using a high-speed centrifuge to remove the excess liquid.

[0118] - After discarding the supernatant, resuspend the microspheres with 2 ml of labeling buffer (0.05 M carbonate buffer).

[0119] - Add 0.4 mg of the labeled antibody 1 against 25-OH-VD and mix well.

[0120] - Simultaneously add 2 mg of carbodiimide and stir and react at room temperature for 1 hour to promote the binding of the antibody to the microspheres.

[0121] - After the reaction, centrifuge again at a speed of 10,000 revolutions per minute for 15 minutes and discard the supernatant.

[0122] - Dilute with 4 ml of label diluent (0.02 M phosphate buffer), mix well and set aside.

[0123] (2) Preparation of the conjugate pad:

[0124] - Use a glass cellulose membrane cut to an appropriate size.

[0125] - According to the operation method of the three-dimensional plane dot membrane gold spraying instrument, spray the labeled mixed working solution onto the glass cellulose membrane, with a spraying volume of 5 microliters per centimeter.

[0126] - After spraying, move the membrane to an environment with a humidity lower than 35% and dry it for 2 hours to ensure the stable fixation of the label. (3) Coating the quality control line C and the test line T:

[0127] - Dissolve the 25-OH-VD labeled antibody-coated antibody-2 in 0.02M phosphate buffer to prepare a coating working solution of 1mg / mL for the preparation of the T line.

[0128] - Similarly dissolve the goat anti-rabbit secondary antibody in 0.02M phosphate buffer to prepare a coating working solution of 1mg / mL for the preparation of the C line.

[0129] - Both the coated antibody and the goat anti-rabbit working solution are coated at a volume of 1 microliter per centimeter.

[0130] - After coating, place the test strip in a dry environment with a humidity lower than 35% and dry it for 2 hours.

[0131] (4) Assembly of the test strip:

[0132] - Cover one end near the test line T with the labeled pad and cover one end near the quality control line C with the absorbent paper.

[0133] - Cut the bottom plate with the strips attached into test strips 4 ± 0.2 mm wide.

[0134] - The antibody pairing method of the test strip is: MAb2135 labeled antibody (for preparing the fluorescent microsphere-antibody complex) + MAb1024 coated antibody (for coating the T line).

[0135] (5) Testing the detection performance of the test strip:

[0136] - For the fluorescent immunochromatographic test strip prepared in this example, dilute the clinical serum sample with the sample diluent (its composition is: 10 mM PBS + 0.5% Triton-100 + 0.5% Tween-20 + 0.5% sodium salicylate + 0.1% dithiothreitol + 0.1% proclin300, pH 7.4) and react at room temperature for 10 minutes.

[0137] - Add 75 microliters of the diluted sample to the test strip and detect it with a fluorescent immunoassay analyzer after 15 minutes.

[0138] - Read the detection values, process the data, and obtain the 25-OH-VD concentrations in samples of each dilution.

[0139] - For the Roche detection kit, operate according to the instructions in the manual. Dilute the serum samples and then perform the detection to obtain the 25-OH-VD concentration data.

[0140] Through the above steps, we successfully prepared a monoclonal antibody against 25-OH-VD and developed a double-antibody sandwich colloidal gold immunochromatographic test strip based on this antibody. The detection performance of this test strip in clinical serum samples was verified and compared with that of the traditional Roche competitive method [25-Hydroxyvitamin D Detection Kit (Electrochemiluminescence Method) REF05894913190].

[0141] The experimental results are shown in Table 4;

[0142] Table 4

[0143]

[0144]

[0145] The above results show that the 25-OH-D fluorescence immunochromatographic test strip prepared based on the paired antibodies of the present invention has a very high coincidence rate (r = 0.9579) compared with the traditional Roche competitive method, and thus has better clinical relevance.

Claims

1. A monoclonal antibody against 25-hydroxyvitamin D or an antigen-binding fragment thereof, characterized in that, The monoclonal antibody or antigen-binding fragment thereof comprises 3 light-chain complementarity-determining regions and 3 heavy-chain complementarity-determining regions, wherein the 3 heavy-chain complementarity-determining regions of the monoclonal antibody or antigen-binding fragment thereof are HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11, and the 3 light-chain complementarity-determining regions of the monoclonal antibody or antigen-binding fragment thereof are LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO:

15.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy-chain variable region having 100% identity with the amino acid sequence shown in SEQ ID NO: 12, and a light-chain variable region having 100% identity with the amino acid sequence shown in SEQ ID NO:

16.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy-chain constant region having 100% identity with the amino acid sequence shown in SEQ ID NO: 19, and / or comprises a light-chain constant region having 100% identity with the amino acid sequence shown in SEQ ID NO:

20.

4. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3.

5. A nucleic acid construct comprising the polynucleotide according to claim 4, and optionally at least one expression regulatory element operably linked to the polynucleotide.

6. A recombinant vector comprising the polynucleotide according to claim 4, or the nucleic acid construct according to claim 5.

7. A double-antibody sandwich immunoassay kit for detecting 25-hydroxyvitamin D, characterized in that, The kit comprises: (a) a monoclonal antibody specifically binding to 25-hydroxyvitamin D for coating, and (b) a monoclonal antibody specifically binding to 25-hydroxyvitamin D for labeling; The monoclonal antibody for coating comprises 3 light-chain complementarity-determining regions and 3 heavy-chain complementarity-determining regions, wherein the 3 heavy-chain complementarity-determining regions of the monoclonal antibody for coating comprise HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3, and the 3 light-chain complementarity-determining regions of the monoclonal antibody for coating comprise LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; The monoclonal antibody for labeling comprises 3 light-chain complementarity-determining regions and 3 heavy-chain complementarity-determining regions. Among them, the 3 heavy-chain complementarity-determining regions of the monoclonal antibody for labeling comprise HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11, and the 3 light-chain complementarity-determining regions of the monoclonal antibody for labeling comprise LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO:

15.

8. The double-antibody sandwich immunoassay kit according to claim 7, wherein the monoclonal antibody for coating (a) comprises a heavy-chain variable region with the amino acid sequence shown in SEQ ID NO: 7 and a light-chain variable region with the amino acid sequence shown in SEQ ID NO: 8; the monoclonal antibody for labeling (b) comprises a heavy-chain variable region with the amino acid sequence shown in SEQ ID NO: 12 and a light-chain variable region with the amino acid sequence shown in SEQ ID NO: 16; Optionally, the monoclonal antibody for coating (a) comprises a heavy-chain constant region with the amino acid sequence shown in SEQ ID NO: 17 and a light-chain constant region with the amino acid sequence shown in SEQ ID NO: 18; and the monoclonal antibody for labeling (b) comprises a heavy-chain constant region with the amino acid sequence shown in SEQ ID NO: 19 and a light-chain constant region with the amino acid sequence shown in SEQ ID NO:

20.

9. The double-antibody sandwich immunoassay kit according to any one of claims 7-8, characterized in that, The double-antibody sandwich immunoassay kit includes a double-antibody sandwich immunochromatographic test strip, and the double-antibody sandwich immunochromatographic test strip contains the monoclonal antibody for coating (a) that specifically binds to 25-hydroxyvitamin D and the monoclonal antibody for labeling (b) that specifically binds to 25-hydroxyvitamin D.

10. Use of the double-antibody sandwich immunoassay kit according to any one of claims 7-9 in the preparation of a detection kit for abnormal vitamin D concentration.

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