Monoclonal antibody specifically binding to 25-hydroxyvitamin D and application thereof

Abstract

The invention provides a monoclonal antibody with high specificity and high affinity for 25-hydroxy vitamin D (25-OH-VD), and an application of the monoclonal antibody in immunodetection. The monoclonal antibody is successfully prepared through an innovative immune procedure and a cell fusion technology, the limitation of a traditional detection method is broken through, and the detection sensitivity and accuracy are remarkably improved. A monoclonal antibody combination is obtained based on screening, an immunodetection kit of a double-antibody sandwich method is developed, and experimental results prove that compared with a traditional detection method, the kit has extremely high coincidence rate and shows good clinical correlation and consistency, a new technical means is provided for quantitative analysis of 25-OH-VD, and the kit has good application prospects. Important clinical application value and market potential are realized.

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 that can specifically bind to 25-hydroxyvitamin D. In addition, the present invention also provides a method for immunological detection using the monoclonal antibody, and a detection kit containing the monoclonal antibody, providing a new technical means for 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. Vitamin D plays an important role in maintaining human health. Vitamin D deficiency can lead to bone health, respiratory diseases, cardiovascular diseases, digestive diseases, endocrine metabolic diseases, neuropsychiatric diseases and urinary system diseases. 25-Hydroxyvitamin D is formed by the 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 discovered and treated in a timely manner. The detection of 25(OH)D levels helps to assess the individual's bone health status and to detect and prevent bone 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 to be 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 double antibody sandwich method for immunoassay. Therefore, the development of highly specific and high-affinity monoclonal antibodies 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 is expensive and complicated to operate, and there may be deviations in the test results between different laboratories, which requires the 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 test results.

[0005] Competitive protein binding assay (CPBA) may lead to inaccurate test results due to its lack of specificity. Therefore, the development of monoclonal antibodies with high specificity and affinity is crucial to improve the accuracy and reliability of the test. This will 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] In order to solve the above problems, the present invention provides a highly specific and high affinity monoclonal antibody that can specifically bind to 25(OH)D and can be used to develop a double antibody sandwich immunoassay kit. The development of this monoclonal antibody breaks through the limitations of traditional immunoassay technology and improves the sensitivity and accuracy of detection.

[0007] The first aspect of the present invention is to provide an anti-25-hydroxyvitamin D monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or its antigen-binding fragment has three light chain complementary determining regions and three heavy chain complementary determining regions, wherein the three light chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment 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 or its antigen-binding fragment comprise HCDR1 shown in SEQ ID NO:4, HCDR2 shown in SEQ ID NO:5, and HCDR3 shown in SEQ ID NO:6; or the three light chain complementary determining regions of the monoclonal antibody or its antigen-binding fragment 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 or its antigen-binding fragment comprise HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, and HCDR3 shown in SEQ ID NO:15.

[0008] Further, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:8;

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

[0010] Further, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 17, and / or comprises a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 18; or

[0011] The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 19, and / or comprises a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 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 antigen-binding fragment thereof 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 control element operably linked to the polynucleotide.

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

[0015] The kit comprises:

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

[0017] (b) a monoclonal antibody that specifically binds to 25-hydroxyvitamin D for labeling;

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

[0019] The three light chain complementary determining regions of the monoclonal antibody used for coating 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 three heavy chain complementary determining regions of the monoclonal antibody used for coating include 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, wherein 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) used for coating comprises a light chain variable region of the amino acid sequence 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) used for labeling comprises a light chain variable region of the amino acid sequence 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 (a) monoclonal antibody for coating and the (b) monoclonal antibody 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 comprises a double antibody sandwich immunochromatographic test strip, in which the double antibody sandwich immunochromatographic test strip comprises a monoclonal antibody (a) that specifically binds to 25-hydroxyvitamin D for coating, and a monoclonal antibody (b) that specifically binds to 25-hydroxyvitamin D for labeling.

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

[0027] Furthermore, the vitamin D abnormalities include osteoporosis and osteomalacia.

[0028] Furthermore, the vitamin D abnormality is vitamin D deficiency.

[0029] More preferably, the vitamin D deficiency includes diseases associated with diabetes, different types of cancer, cardiovascular and cerebrovascular diseases, and autoimmune diseases.

[0030] The seventh aspect of the present invention provides a method for preparing an immunoassay test strip, firstly bonding a coating pad to a PVC plate, then overlapping an absorbent paper at one end of the quality control line c line near the coating pad, and overlapping a marker pad at one end of the T line near the coating pad, wherein the yellow portion of the marker pad contains fluorescent microspheres-ab and fluorescent microspheres-goat anti-rabbit secondary antibody (see Figure 4 ), the coating pad is coated with T line and C line in sequence; wherein the labeled antibody numbered MAb2135 of the present invention is used to prepare the fluorescent microsphere-antibody complex and is placed on the marker 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 paper prepared by the method of the seventh aspect.

[0032] Advantages of the present invention:

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

[0034] 2. Breaking through the limitations of traditional technology: This invention overcomes the limitations of traditional immunoassay technology, 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 detection methods such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), the immunoassay method of the present invention is simple to operate, easy to automate, and has low cost, making it 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 high compliance rate compared with the traditional Roche competitive method, showing good clinical relevance. This means that the method can provide doctors and patients with more accurate test results, which helps guide clinical decision-making and disease management.

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

[0038] Definition of terms:

[0039] Antibodies: Antibodies are proteins produced by the immune system. Their 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 are highly specific and can bind to specific antigen epitopes, protecting the body from infection through a variety of mechanisms (such as neutralization, opsonization, and 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: Monoclonal antibodies (mAb) are antibodies produced by a single clone of B cell lines. These antibodies are homogeneous in molecular structure and only target specific antigenic 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 multiply indefinitely in vitro and produce large quantities of identical antibodies. Monoclonal antibodies are very useful in medical research, diagnosis and treatment because of their high specificity and uniformity.

[0041] Immunoassays: Immunoassays are methods that use antigen-antibody reactions to detect specific substances (usually proteins or pathogens). This testing can be used to diagnose disease, monitor treatment response, or assess immune status. There are many different methods for immunoassays, including enzyme-linked immunosorbent assay (ELISA), immunofluorescence, immunoblotting (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 immunoassays make them an indispensable tool in medical research and clinical diagnosis.

[0042] Affinity, binding affinity: In immunology, the binding affinity or binding affinity of an antibody usually refers to how tightly it binds to a specific antigen epitope. High affinity means that the antibody is able to bind to the antigen with high specificity and strength, while low affinity means that the binding is weak and may be easily affected by competing molecules. Affinity can be measured by a variety of experimental methods, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and enzyme-linked immunosorbent assay (ELISA). These methods can provide kinetic parameters about the binding of antibodies to antigens, such as the binding constant (Kd) or the dissociation constant (Koff), which can be used to assess the magnitude of affinity. The binding constant Kd is an inverse indicator of affinity, that is, the smaller the Kd value, the stronger the affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a gel image of 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 This is the HPLC spectrum of the MAb1024 monoclonal antibody in Example 3.

[0045] Figure 3 This is the HPLC spectrum of the MAb2135 monoclonal antibody in Example 3.

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

[0047] Figure 5 This is a real shot of the colloidal gold immunochromatographic test strip prepared in the embodiment of the present invention. Specific embodiments

[0048] The present invention is further described below in conjunction with specific embodiments. The described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. It should be understood that the following embodiments are provided to provide a complete disclosure and description of how to use the methods and compositions of the present invention to general professionals in the technical field to which the present invention belongs, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0049] Example 1 Preparation of monoclonal antibodies specifically binding to 25-OH-VD

[0050] Experimental methods:

[0051] (1) Immunization program design:

[0052] - The coupled protein 25-OHVD-BSA (purchased from Nanjing Kaiertai Biotechnology Co., Ltd., catalog number AG0124) was used as the antigen, which was fully emulsified with an equal amount of Freund's complete adjuvant and then injected subcutaneously to immunize goats.

[0053] -Use the immune complex of 25-OH-VD antigen and its labeled antibody (mixed with the labeled antibody obtained by screening in this project) as the antigen, fully emulsify it with an equal amount of Freund's complete adjuvant, and then inject it subcutaneously to immunize rabbits (species: New Zealand white rabbit, female, 6-8 weeks old).

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

[0055] -5 to 8 days after the last booster immunization, venous blood was collected and the antibody titer in the serum was assessed by enzyme-linked immunosorbent assay (ELISA).

[0056] - Set 128 as the standard line, and start preparing peripheral blood mononuclear cell (PBMC) suspension when the serum titer is detected to be over 128K.

[0057] (2) Preparation of PBMC suspension:

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

[0059] - Slowly pour the mixture onto the Ficoll layering solution along the tube wall and centrifuge at 400g for 30 minutes.

[0060] -After centrifugation, remove most of the upper liquid, retain about 1 mL of the sediment, 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, centrifuge at 300g for 10 minutes, and repeat the washing twice.

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

[0063] - The supernatant was discarded, and finally RPMI1640 medium containing 10% fetal bovine serum (FBS) was added to resuspend the cells and count them.

[0064] (3) B cell screening:

[0065] - Using PBMC suspension, add FITC-labeled antigen (25-OHVD-BSA, or 25-OH-VD antigen and labeled antibody obtained in this project), and use flow sorting technology to screen B cells that specifically respond 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, which were all IgG types after testing.

[0068] Table 1. Sources of monoclonal antibodies against 25-OH-VD obtained by screening

[0069] Clone Name Screening methods MAb1024 (coated) Flow cytometry screening and sequencing MAb2135 (labeled) Flow cytometry screening and sequencing

[0070] Example 2 Molecular Construction and Production of Complete Antibodies

[0071] The monoclonal antibodies constructed for coating and labeling in Table 1 were sequenced, wherein the amino acid sequences of the variable regions of the two lead antibodies are shown in Table 2 (the CDR regions are underlined, and the analysis system is the IMGT system), and the heavy chain and light chain constant region sequences are shown in Table 3.

[0072] Table 2

[0073]

[0074]

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

[0076]

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

[0078]

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

[0080] Experimental methods:

[0081] (1) Cell culture:

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

[0083] - Prior to transfection, ensure that the viability of the cells is greater than 80-90%, which can be assessed by trypan blue exclusion assay or similar cell viability assay.

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

[0085] (2) Cell transfection:

[0086] -After the cell density and viability reach the required level, perform cell transfection. Before transfection, calculate the amount 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 determined in Example 2, these gene fragments were chemically synthesized.

[0088] -The synthetic light chain and heavy chain genes were cloned into the pcDNA3.4 plasmid vector to construct a recombinant expression vector.

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

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

[0091] -Gently mix the culture system to ensure that the transfection mixture is fully in 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. This step can be performed by centrifugation to remove cell debris.

[0094] - Purification of monoclonal antibodies 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. During the dialysis process, PBS should be replaced regularly to ensure complete replacement.

[0097] - Use a Nanodrop or similar spectrophotometer to measure the antibody concentration to ensure that the correct amount of antibody is used in subsequent experiments.

[0098] (4) Antibody purity test:

[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 samples for reducing SDS-PAGE: Add β-mercaptoethanol or DTT to the loading buffer and perform SDS-PAGE under reducing conditions.

[0102] -Prepare non-reducing SDS-PAGE samples: No reducing agent is added to maintain the native disulfide bond status of the antibody.

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

[0104] -After electrophoresis, the PAGE gel was taken out and stained with Coomassie Brilliant Blue for 15 minutes.

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

[0106] (4.2) HPLC detection:

[0107] -Analyze the antibody samples using a high performance liquid chromatography (HPLC) system equipped with appropriate columns and detectors.

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

[0109] -Analyze HPLC profiles to assess the purity of the antibody and possible impurities.

[0110] Experimental results:

[0111] The electrophoresis results are as follows Figure 1As shown in the figure: under reducing conditions, the monoclonal antibodies numbered MAb1024 and MAb2135 both have two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands are consistent with the size of their respective light and heavy chains; under non-reducing conditions, the monoclonal antibodies both have a single band, and the corresponding molecular weights of the electrophoretic bands are consistent with the size of their respective complete antibodies; these results indicate that the monoclonal antibodies are accurately expressed. In addition, all electrophoretic bands have clear edges and no mixed bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps have a high purity.

[0112] HPLC profile: HPLC results showed that the purity of the monoclonal antibodies was greater than 99%.

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

[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 labeling antibody to prepare a colloidal gold immunochromatographic test strip for detecting 25-OH-VD using the double antibody sandwich method. The schematic diagram of the structure of the test strip is shown in FIG. Figure 3 As shown, MAb2135 labeled antibody is used to prepare fluorescent microsphere-antibody complex and placed on the marker pad, and MAb1024 coated antibody is used for T-line coating.

[0115] The test strips are prepared as follows:

[0116] (1) Preparation of fluorescent microsphere markers:

[0117] - Weigh 0.02 g of latex fluorescent microspheres and centrifuge at 10,000 rpm for 30 minutes to remove excess liquid.

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

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

[0120] - At the same time, 2 mg of carbodiimide was added and stirred at room temperature for 1 hour to promote the binding of the antibody to the microspheres.

[0121] -After the reaction, centrifuge again at 10,000 rpm for 15 min and discard the supernatant.

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

[0123] (2) Preparation of marker pad:

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

[0125] - According to the operating method of the three-dimensional plane dot film gold spraying instrument, the mixed working solution of the marker is sprayed on the glass cellulose membrane, with a spray volume of 5 μl per cm.

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

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

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

[0129] - Both the coating antibody and goat anti-rabbit working solution were coated at a volume of 1 μl per cm.

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

[0131] (4) Test strip assembly:

[0132] - Cover the end of the marker pad close to the test line T and the end of the absorbent paper close to the quality control line C.

[0133] -Cut the bottom plate with the strips pasted into test strips with a width of 4±0.2 mm.

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

[0135] (5) Test strip detection performance test:

[0136] -For the fluorescent immunochromatographic test strip prepared in this example, a clinical serum sample was diluted with a sample diluent (whose composition is: 10 mM PBS + 0.5% Triton-100 + 0.5% Tween-20 + 0.5% sodium salicylate + 0.1% dithiothreitol + 0.1% proclin 300, Ph 7.4) and reacted at room temperature for 10 minutes.

[0137] - Add 75 μl of diluted sample to the test strip and test using a fluorescent immunoassay analyzer after 15 minutes.

[0138] -Read the test values ​​and process the data to obtain the 25-OH-VD concentration in each dilution sample.

[0139] -For the Roche test kit, follow the instructions to dilute the serum sample and test it 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 the antibody. The detection performance of the test strip in clinical serum samples was verified and compared with the detection performance 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 fluorescent immunochromatographic test strip prepared based on the paired antibodies of the present invention has a high coincidence rate (r=0.9579) compared with the traditional Roche competitive method, and therefore has better clinical relevance.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against 25-hydroxyvitamin D, characterized in that: The monoclonal antibody or antigen-binding fragment thereof has three light chain complementary determining regions and three heavy chain complementary determining regions, wherein The three light chain complementary determining regions of the monoclonal antibody or antigen-binding fragment thereof 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 three heavy chain complementary determining regions of the monoclonal antibody or antigen-binding fragment thereof 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 three 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 three 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.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; The monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:12, and a heavy chain variable region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 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, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 17, and / or comprises a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 18; or The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 19, and / or comprises a light chain constant region that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to 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 to 3.

5. A nucleic acid construct comprising the polynucleotide of claim 4 and, optionally, at least one expression control 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 that specifically binds to 25-hydroxyvitamin D for coating, and (b) a monoclonal antibody that specifically binds to 25-hydroxyvitamin D for labeling; The monoclonal antibody used for coating comprises 3 light chain complementary determining regions and 3 heavy chain complementary determining regions, wherein The three light chain complementary determining regions of the monoclonal antibody used for coating 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 three heavy chain complementary determining regions of the monoclonal antibody used for coating include HCDR1 shown in SEQ ID NO: 4, HCDR2 shown in SEQ ID NO: 5, and HCDR3 shown in SEQ ID NO: 6; The monoclonal antibody for labeling comprises three light chain complementary determining regions and three heavy chain complementary determining regions, wherein 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.

8. The double antibody sandwich immunoassay kit according to claim 7, characterized in that: The monoclonal antibody (a) used for coating comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 8; The monoclonal antibody (b) used for labeling comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO: 12, and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 16; Optionally, the (a) monoclonal antibody for coating and the (b) monoclonal antibody 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.

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

10. Use of the double antibody sandwich immunoassay kit according to any one of claims 7 to 9 in the preparation of a detection kit for diseases caused by abnormal vitamin D; preferably, the diseases caused by abnormal vitamin D include osteoporosis and osteomalacia.

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