25-OH-D, P1NP and β-CTX combined detection kit and preparation method thereof

Abstract

The present invention discloses a 25-OH-D, P1NP and β-CTX joint detection kit, including a reagent card and a dissociating agent; the reagent card includes a PVC rubber plate, a nitrocellulose membrane arranged on the PVC rubber plate, a conjugation pad, a sample pad, and a blotting paper sequentially arranged on the nitrocellulose membrane; the nitrocellulose membrane is coated with 25-OH-D antibody, P1NP antibody, β-CTX antibody and quality control antibody; the conjugation pad is marked with 25-OH-D antibody, P1NP antibody, and β-CTX antibody tracer by a coupling method; the dissociating agent is used to dissociate 25-OH-D in the form of a binding protein in serum or plasma, and does not affect the detection results of P1NP and β-CTX. A preparation method of the kit is also disclosed. The present invention can perform joint quantitative detection of 25-OH-D, P1NP, and β-CTX in blood.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro detection, and in particular to a method for preparing a 25-OH-D, P1NP and beta-CTX combined detection kit. Background Art

[0002] Vitamin D (VD) is a fat-soluble vitamin with multiple functions that is essential to humans. It is also an important steroid hormone that regulates bone metabolism. 25-hydroxyvitamin (25-OH-D) is the main storage form of VD in the human body. It is relatively stable in the circulation and is the best indicator for evaluating VD levels. The main physiological effects of VD and its metabolites are to promote the absorption of calcium and phosphorus in the intestine, inhibit the release of parathyroid hormone (PTH), maintain normal blood calcium and phosphorus levels, and thus ensure bone health and normal neuromuscular function. Its determination is to understand the storage level of VD in the human body. VD deficiency is related to many diseases, such as rickets, osteomalacia, osteoporosis, etc. Extraskeletal effects include effects on muscles, cardiovascular, metabolism, immunity, tumorigenesis, pregnancy and fetal development.

[0003] 90% of the organic components of bone are type I collagen, which is the main component for maintaining bone biomechanical function and bone integrity. Type I collagen is derived from type I procollagen. Type I procollagen is hydrolyzed by endopeptidases in the extracellular fluid, removing the carboxyl and amino-terminal additional peptides to form type I collagen monomers. The amino-terminal additional peptides removed are called type 1 procollagen N-terminal propeptide (type 1 procollagen amino terminal peptide, P1NP), also known as type I collagen amino-terminal extended peptide. When osteoblast activity increases, type I collagen synthesis increases, and the concentration of P1NP in the blood circulation increases, so it can reflect the level of bone formation. P1NP has good specificity, is less affected by physiological rhythms, and is not affected by diet and renal function. In the diagnosis and treatment of bone metabolic diseases, it is recommended to select at least one bone formation marker and one bone resorption marker. At present, IOF / IFCC recommends P1NP as the preferred bone formation marker and β-CTX as the preferred bone resorption marker. CTX, type I collagen carboxy-terminal peptide (CTX) is the most widely used collagen degradation marker. The level of CTX reflects the bone resorption activity of osteoclasts and is an effective marker for metabolic bone disease. It contains three different forms, namely CTX-MMP, α-CTX and β-CTX. β-CTX is an isomeric form of α-CTX. β-CTX has a compact structure, does not undergo further metabolism by the kidneys, and has good stability. Elevated β-CTX can be seen in menopausal women, high-conversion osteoporosis, bone tumors, prostate cancer bone metastasis, etc.; decreased β-CTX can be seen in postmenopausal osteoporosis patients treated with estrogen, etc.

[0004] Osteoporosis is a common bone metabolic disease. Many scholars have reported that changes in serum levels of 25-OH-D, P1NP, and β-CTX have an independent predictive role in assessing bone healing and potential fracture risk in patients. With the advent of an aging society, the incidence of fractures caused by osteoporosis in the elderly has increased. If effective and rapid assessments can be performed before the risk occurs and preventive protection and treatment can be carried out, it will reduce a significant burden on families and society. However, there is currently no popular rapid combined detection reagent used in clinical practice.

[0005] Bone metabolism markers are proteins or matrix degradation products produced and released by osteoblasts and osteoclasts at different stages of bone remodeling. Commonly used biochemical indicators of bone metabolism include calcium and phosphorus; bone turnover markers include bone formation markers and bone resorption markers. Therefore, it is urgent to provide a new type of bone metabolism biochemical marker 25-OH-D and bone turnover markers P1NP, β-CTX rapid combined detection kit to solve the above problems. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a 25-OH-D, P1NP and CTX combined detection kit and a preparation method thereof, which can perform combined quantitative detection of 25-OH-D, P1NP and β-CTX in blood and can be used in conjunction with single-channel immunoanalyzers, multi-channel and fully automatic immunoanalyzers.

[0007] In order to solve the above technical problems, a technical solution adopted by the present invention is: providing a 25-OH-D, P1NP and β-CTX combined detection kit, including a reagent card and a dissociation agent;

[0008] The reagent card comprises a PVC rubber plate, a nitrocellulose membrane arranged on the PVC rubber plate, a binding pad, a sample pad, and absorbent paper arranged on the nitrocellulose membrane in sequence; the nitrocellulose membrane is coated with 25-OH-D antibody, P1NP antibody, β-CTX antibody, and quality control antibody; the binding pad is labeled with a tracer with 25-OH-D antibody, P1NP antibody, and β-CTX antibody by a coupling method;

[0009] The dissociation agent is used to dissociate 25-OH-D present in the form of binding protein in serum or plasma, and does not affect the detection results of P1NP and β-CTX.

[0010] In a preferred embodiment of the present invention, the T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody at a concentration of 0.2-1 mg / ml; the T2 line is coated with P1NP antibody at a concentration of 0.2-1.25 mg / ml; the T3 line is coated with β-CTX antibody at a concentration of 0.1-0.6 mg / ml; the C line is coated with quality control antibody at a concentration of 0.1-0.5 mg / ml; and the coating amount of C, T1, T2, and T3 lines is 1 ul / cm.

[0011] In a preferred embodiment of the present invention, the coating solution of the nitrocellulose membrane, the reconstitution solution of the conjugate pad, and the sample pad treatment solution of the sample pad all include sodium lauryl glucose carboxylate and lectin.

[0012] In a preferred embodiment of the present invention, the dissociation agent includes 50-300mM, pH 6.0-8.0 phosphate buffer, 0.005-0.05% N,N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01mg-0.15mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1mg / ml sodium dodecyl sulfate, and 0.01-0.05mg / ml sodium chloride.

[0013] In order to solve the above technical problems, another technical solution adopted by the present invention is: to provide a method for preparing a 25-OH-D, P1NP and CTX combined detection kit as described in any one of the above items, comprising the following steps:

[0014] (1) Nitrocellulose membrane antibody coating:

[0015] (1.1) Prepare coating solution;

[0016] (1.2) Preparation of coating antibodies: Place 25-OH-D antibody, P1NP antibody, β-CTX antibody and quality control antibody in ultrafiltration tubes respectively, centrifuge and replace with prepared phosphate buffer, collect the antibodies in the ultrafiltration tubes into new centrifuge tubes, add an equal volume of 2× coating solution to dilute and mix, and prepare 25-OH-D coating antibody, P1NP coating antibody, β-CTX coating antibody and quality control coating antibody;

[0017] (1.3) Antibody coating: The T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody, the T2 line is coated with P1NP antibody, the T3 line is coated with β-CTX antibody, and the C line is coated with quality control antibody, and then dried to obtain a coated ruled PVC rubber plate;

[0018] (2) Preparation of bonding pad:

[0019] (2.1) Quantum dot activation and antibody labeling: add the quantum dot solution to 10 mM MES solution, add EDC and NHS, and react at room temperature; add 25-OH-D labeled antibody, P1NP labeled antibody, and β-CTX labeled antibody respectively, mix thoroughly, and react at room temperature using a vortex mixer; add 10% BSA solution and mix thoroughly, react at room temperature using a vortex mixer, centrifuge, discard the supernatant, and retain the precipitate to respectively label the 25-OH-D antibody, P1NP antibody, and β-CTX antibody; or

[0020] Preparation of colloidal gold and antibody labeling: Take the prepared colloidal gold solution, add K2CO3 solution to adjust the pH to 7.0-7.8, add 25-OH-D labeled antibody, P1NP labeled antibody, β-CTX labeled antibody respectively, stir the reaction thoroughly, then add BSA and stir the reaction thoroughly. Place the fully reacted colloidal gold labeled antibody in a centrifuge tube, centrifuge, discard the supernatant, and keep the precipitate to label the 25-OH-D antibody, P1NP antibody, and β-CTX antibody respectively;

[0021] (2.2) Prepare the reconstitution solution;

[0022] (2.3) Redissolving the labeled antibodies and spraying the membrane: Use the re-dissolving solution to re-dissolve the prepared 25-OH-D labeled antibody, P1NP labeled antibody, and β-CTX labeled antibody, mix the three labeled antibodies in a ratio of 1:1:1, spray them on the glass fiber membrane, and obtain the quantum dot binding pad after drying;

[0023] (3) Sample pad preparation:

[0024] preparing a sample pad treatment solution; soaking the glass fiber membrane in the sample pad treatment solution, and drying to obtain a sample pad;

[0025] (4) Paste the conjugate pad, sample pad, and absorbent paper onto the antibody-coated nitrocellulose membrane PVC plate in sequence, cut them into strips and put them into the cartridge, put them into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature.

[0026] In a preferred embodiment of the present invention, the coating solution is prepared by adding 0.05%-0.1% sodium lauryl glucose carboxylate to 0.01M-0.05M, pH 7.0-8.0 phosphate buffer and fully dissolving it; then adding 1%-5% trehalose and 0.5%-2% lectin.

[0027] In a preferred embodiment of the present invention, the reconstitution solution includes 0.01M phosphate buffer containing 0.01-0.08% sodium lauryl glucose carboxylate, 1% BSA, 0.05% Tween-20, 0.5% trehalose, 0.5%-2% lectin, and 0.5% PVP.

[0028] In a preferred embodiment of the present invention, the sample pad treatment solution includes 0.01M phosphate buffer containing 0.01-0.15% sodium lauryl glucose carboxylate, 0.5%-2% plant lectin, 0.5% BSA, 0.05% Triton X-100, and 0.5% PEG.

[0029] In a preferred embodiment of the present invention, the method further comprises preparing a dissociation agent, wherein the dissociation agent comprises 50-300 mM, pH 6.0-8.0 phosphate as a buffer, 0.005-0.05% N,N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01 mg-0.15 mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1 mg / ml sodium dodecyl sulfate, and 0.01-0.05 mg / ml sodium chloride.

[0030] In a preferred embodiment of the present invention, the method further comprises preparing a composite standard and a composite quality control preservation solution, wherein the composite standard and the composite quality control preservation solution comprises a 0.01M phosphate buffer containing 0.01-0.2% sodium lauryl glucose carboxylate at pH 7.0-8.0, 0.1-0.5% BSA, 0.5-1.0% trehalose, 0.05-0.1% PEG 20000, 0.01-0.05% sorbitol, 0.05-0.2% aminotriacetic acid, and 0.45-0.9% NaCl.

[0031] The beneficial effects of the present invention are:

[0032] (1) The present invention adopts a marker immunoassay system in which the same nitrocellulose membrane is used as a carrier to coat four antibodies, namely, 25-OH-D, P1NP, β-CTX antibodies and a quality control antibody, and a marker is used as a tracer to mark another antibody of 25-OH-D, P1NP and β-CTX. The system performs a combined quantitative detection of 25-OH-D, P1NP and β-CTX in the blood and can be used in conjunction with a single-channel immunoassay analyzer, a multi-channel and a fully automatic immunoassay analyzer, thereby solving the current problem of cumbersome single-item detection operation and large sample volume requirements;

[0033] (2) At the same time, the use of the 25-OH-D dissociator in the present invention does not affect the detection results of P1NP and β-CTX, and the development of the composite standard product and the composite control product enables the three-item joint detection to be realized;

[0034] (3) The present invention conducts a joint study on the bone metabolism biochemical marker 25-OH-D and the bone turnover markers P1NP and β-CTX that are clinically required to be detected, which is more conducive to the clinical and grassroots bone metabolism examination. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a flow chart of the method for preparing the kit of the present invention;

[0036] Figure 2 It is a schematic diagram of the 25-OH-D detection result of the reagent sample of the present invention after being treated with a dissociating agent;

[0037] Figure 3 It is a schematic diagram of the P1NP detection results of the reagent sample of the present invention after adding the dissociation agent to the sample;

[0038] Figure 4 It is a schematic diagram of the β-CTX detection result of the reagent sample of the present invention after being treated with a dissociating agent and then loaded;

[0039] Figure 5 is a schematic diagram of the detection results of 25-OH-D by the reagent sample without adding a dissociating agent;

[0040] Figure 6 is a schematic diagram of the detection results of the reagent sample for P1NP without adding a dissociating agent;

[0041] Figure 7 It is a schematic diagram of the detection results of β-CTX by the reagent sample without adding a dissociating agent. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0043] The embodiments of the present invention include:

[0044] A 25-OH-D, P1NP and β-CTX combined detection kit comprises a reagent card and a dissociation agent.

[0045] The reagent card comprises a PVC board, a nitrocellulose membrane arranged on the PVC board, a conjugation pad, a sample pad, and a blotting paper arranged on the nitrocellulose membrane in sequence. Specifically, the conjugation pad, the sample pad, and the blotting paper are sequentially attached to the nitrocellulose membrane coated with the antibody in an overlapping manner.

[0046] The nitrocellulose membrane is coated with four antibodies, namely, 25-OH-D antibody, P1NP antibody, β-CTX antibody and quality control antibody. In this example, the quality control antibody is rabbit anti-mouse IgG antibody. Specifically, the T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody at a concentration of 0.2~1mg / ml; the T2 line is coated with P1NP antibody at a concentration of 0.2~1.25mg / ml; the T3 line is coated with β-CTX antibody at a concentration of 0.1~0.6mg / ml; the C line is coated with quality control antibody at a concentration of 0.1~0.5mg / ml; the coating amount of C, T1, T2 and T3 lines is 1ul / cm.

[0047] The conjugate pad is labeled with 25-OH-D antibody, P1NP antibody, and β-CTX antibody by a coupling method. The tracer may be quantum dots, colloidal gold, time-resolved fluorescent substances, phycoerythrin, etc. In this example, the tracer is provided as quantum dots and colloidal gold.

[0048] The dissociation agent is used to dissociate 25-OH-D in the form of bound proteins in serum or plasma, and does not affect the detection results of P1NP and β-CTX. 25-OH-D detection requires the addition of a dissociation agent to the sample, and the three items are tested together. It is necessary to ensure that the dissociation agent can achieve the dissociation effect without affecting the detection effects of other P1NP and β-CTX. Therefore, the dissociation agent includes 50-300mM, pH 6.0-8.0 phosphate as a buffer, 0.005-0.05% N, N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01mg-0.15mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1mg / ml sodium dodecyl sulfate, and 0.01-0.05mg / ml sodium chloride.

[0049] See also Figure 1 The present invention also provides a method for preparing the kit, comprising the following steps:

[0050] (1) Nitrocellulose membrane antibody coating:

[0051] (1.1) Prepare coating solution: Add 0.05%-0.1% sodium lauryl glucose carboxylate to 0.01M-0.05M, pH 7.0-8.0 phosphate buffer and fully dissolve it; then add 1%-5% trehalose and 0.5%-2% lectin. Stir with a magnetic stirrer for 15 minutes until it is fully dissolved, and add to a volumetric flask to make up to volume.

[0052] (1.2) Preparation of coating antibody:

[0053] (1.2.1) Take 0.2 mg of 25-OH-D antibody and place it in an ultrafiltration tube. Centrifuge it with the prepared 0.01M-0.05M, pH 7.0-8.0 phosphate buffer. The centrifugation parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 1mg / ml;

[0054] (1.2.2) Take 0.25 mg of P1NP antibody and place it in an ultrafiltration tube. Centrifuge it with the prepared 0.01M-0.05M, pH 7.0-8.0 phosphate buffer. The centrifugation parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 1.25mg / ml;

[0055] (1.2.3) Take 0.12 mg of β-CTX antibody and place it in an ultrafiltration tube. Centrifuge it with the prepared 0.01M-0.05M, pH 7.0-8.0 phosphate buffer. The centrifugation parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 0.6mg / ml;

[0056] (1.2.4) Take 0.1 mg of rabbit anti-mouse IgG antibody and place it in an ultrafiltration tube. Centrifuge it with the prepared 0.01M-0.05M, pH 7.0-8.0 phosphate buffer. The centrifugation parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 0.5mg / ml.

[0057] (1.3) Coating antibody: The T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody at a concentration of 0.2-1 mg / ml; the T2 line is coated with P1NP antibody at a concentration of 0.2-1.25 mg / ml; the T3 line is coated with β-CTX antibody at a concentration of 0.1-0.6 mg / ml; the C line is coated with rabbit anti-mouse IgG antibody at a concentration of 0.1-0.5 mg / ml; the coating amount of the C, T1, T2, and T3 lines is 1 ul / cm, and the lines are placed face down in parallel so that the coated antibodies are concentrated on the surface of the nitrocellulose membrane, thereby improving the detection sensitivity, repeatability and linear range, and drying at 37°C for 2 hours to obtain a coated streaked rubber plate.

[0058] (2) Preparation of bonding pad:

[0059] (2.1) Using quantum dot activation and antibody labeling:

[0060] (2.1.1) Take 30-60ul of quantum dot solution and add it to 10mM MES solution (pH5.5), the final volume is 200ul. Add 20mg / ml EDC 200-400ul, 30mg / ml NHS 200-400ul, and react at room temperature for 30min. Add 15ug 25-OH-D labeled antibody and mix thoroughly, and react at room temperature for 2h using a vortex mixer. Add 20ul of 10% BSA solution and mix thoroughly, and react at room temperature for 1h using a vortex mixer. Centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0061] (2.1.2) Take 30-70ul of quantum dot solution and add it to 10mM MES solution (pH5.5), with a final volume of 200ul. Add 200-500ul of 20mg / ml EDC and 200-500ul of 30mg / ml NHS, and react at room temperature for 30min. Add 15ug of P1NP labeled antibody and mix thoroughly, and react at room temperature for 2h using a vortex mixer. Add 20ul of 10% BSA solution and mix thoroughly, and react at room temperature for 1h using a vortex mixer. Centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0062] (2.1.3) Take 20-50ul of quantum dot solution and add it to 10mM MES solution (pH5.5), with a final volume of 200ul. Add 100-400ul of 20mg / ml EDC and 100-400ul of 30mg / ml NHS, and react at room temperature for 30min. Add 15ug of β-CTX labeled antibody and mix thoroughly, and react at room temperature for 2h using a vortex mixer. Add 20ul of 10% BSA solution and mix thoroughly, and react at room temperature for 1h using a vortex mixer. Centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0063] Or (2.1') ​​using colloidal gold preparation and antibody labeling:

[0064] (2.1.1') Preparation of colloidal gold: Accurately measure 1L of purified water in a round-bottom flask and heat to boiling, add 15ml of 1% trisodium citrate solution, boil for 1min, then add 10ml of 1% chloroauric acid solution, continue heating for 10min after the color stabilizes, and adjust the volume to 1L after cooling;

[0065] (2.1.2') Antibody labeling:

[0066] Take 1ml of the prepared colloidal gold solution, add K2CO3 solution to adjust the pH to 7.0-7.5, add 10-15ug 25-OH-D antibody, stir and react for 15-30min, then add 10ul 10% BSA and stir and react for 15-30min. Place the fully reacted colloidal gold labeled antibody in a centrifuge tube, centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0067] Take 1ml of the prepared colloidal gold solution, add K2CO3 solution to adjust the pH to 7.0-7.8, add 10-15ug P1NP antibody, stir and react for 15-30min, then add 10ul 10% BSA and stir and react for 15-30min. Place the fully reacted colloidal gold labeled antibody in a centrifuge tube, centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0068] Take 1ml of the prepared colloidal gold solution, add K2CO3 solution to adjust the pH to 7.0-7.5, add 10-15ug β-CTX antibody, stir and react for 15-30min, then add 10ul 10% BSA and stir and react for 15-30min. Place the fully reacted colloidal gold labeled antibody in a centrifuge tube, centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate;

[0069] (2.2) Prepare the reconstitution solution: 0.01 M phosphate buffer (pH 7.0-8.0) containing 0.01-0.08% sodium lauryl glucose carboxylate, 1% BSA, 0.05% Tween-20, 0.5% trehalose, 0.5%-2% lectin, and 0.5% PVP;

[0070] (2.3) Redissolving and spraying labeled antibodies: Use the re-dissolving solution to re-dissolve the prepared 25-OH-D labeled antibody to a concentration of 0.5 mg / ml, the P1NP labeled antibody to a concentration of 0.6 mg / ml, and the β-CTX labeled antibody to a concentration of 0.5 mg / ml. The three labeled antibodies are fully mixed in a ratio of 1:1:1, sprayed on the glass fiber membrane at a parameter of 5-15 ul / cm, and placed face down in parallel so that the labeled antibodies are concentrated on the surface of the glass fiber membrane, which is more conducive to the release of the labeled antibodies during detection, improving the detection sensitivity, repeatability and linear range, and dried at 37°C to obtain the quantum dot binding pad.

[0071] (3) Sample pad preparation:

[0072] Prepare the sample pad treatment solution: 0.01 M phosphate buffer (pH 7.0-8.0) containing 0.01-0.15% sodium lauryl glucose carboxylate, 0.5%-2% lectin, 0.5% BSA, 0.05% Triton X-100, and 0.5% PEG;

[0073] Soak the glass fiber membrane in the sample pad treatment solution at a ratio of 1 glass fiber membrane: 30 ml sample pad treatment solution, place them face up and parallel, soak for 20 minutes. Place them face down and parallel, dry at 56℃ for 6-8 hours to obtain the sample pad.

[0074] (4) Paste the conjugate pad, sample pad, and absorbent paper onto the antibody-coated nitrocellulose membrane PVC plate in sequence, cut them into strips and put them into the cartridge, put them into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature.

[0075] (5) Preparation of dissociation agent:

[0076] 50-300mM, pH6.0-8.0 phosphate buffer, add 0.005-0.05% N,N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01mg-0.15mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1mg / ml sodium dodecyl sulfate, 0.01-0.05mg / ml sodium chloride.

[0077] (6) Preparation of composite standard and composite quality control product storage solutions

[0078] 0.01M phosphate buffer (pH 7.0-8.0) containing 0.01-0.2% sodium lauryl glucose carboxylate, 0.1-0.5% BSA, 0.5-1.0% trehalose, 0.05-0.1% PEG20000, 0.01-0.05% sorbitol, 0.05-0.2% aminotriacetic acid, 0.45-0.9% NaCl.

[0079] The reagent card in the 25-OH-D, P1NP and β-CTX joint detection kit of the present invention is coated with 25-OH-D, P1NP, β-CTX antibody and quality control antibody on the same nitrocellulose membrane carrier, and the test results need not interfere with each other. The measures taken include: 25-OH-D detection requires the addition of a dissociator to the sample, and the three items are jointly tested to ensure that the dissociator can achieve the dissociation effect of 25-OH-D without affecting the detection effects of the other two antibodies P1NP and β-CTX; at the same time, a certain concentration of sodium lauryl glucose carboxylate and lectin are added to the coating solution, the complex solution, and the sample pad treatment solution, and the front side is placed in parallel when the drying is prepared, so that when the three items are jointly tested, the interference is eliminated after adding the sample to the sample pad, the release effect of the binding pad is stable and complete, the coated antibody is stable, and the sensitivity, repeatability, repeatability, and linear range of the kit are significantly improved.

[0080] The detection method of the kit comprises the following steps:

[0081] Step 1: Place the reagent card at room temperature for 30-60 minutes (the sealed aluminum foil bag cannot be opened);

[0082] Step 2: Open the aluminum foil bag, take out the reagent card and place it parallel to the desktop, take 10ul-30ul of the sample to be tested and 5-10ul of the dissociation agent into the centrifuge tube, mix well and take 10-30ul into the sample well of the reagent card;

[0083] Step 3: After 5-10 minutes, place the reagent card in the instrument card slot for testing;

[0084] Step 4: Using the calibration curve of known concentration, substitute the obtained fluorescence value into the standard curve equation to calculate the concentration of 25-OH-D, P1NP, and β-CTX in the measured sample.

[0085] This kit solves the problem that the current single-item detection operation is cumbersome and the sample volume requirement is large. One sampling can realize the joint detection of 25-OH-D, P1NP, and β-CTX, and the sensitivity, repeatability, and accuracy are equal to or better than the single test results. At the same time, the use of the 25-OH-D dissociator in the present invention does not affect the detection results of P1NP and β-CTX, and the development of composite standard products and composite control products enables the three joint detections to be realized.

[0086] The performance of the kit was evaluated by the following experiments:

[0087] 1. Linearity and blank limits

[0088] Set up 20 replicate wells for the zero-concentration standard (S0) solution, and set up single wells for the other 5 concentration standards S1-S5. Draw a standard curve based on the theoretical concentration and luminescence value of the standard to obtain the dose-response curve of the kit. Calculate the mean (Mean) and standard deviation (SD) of the 20 S0 luminescence values, and calculate the concentration value of Mean+2SD based on the fitting equation, which is the blank limit of the kit.

[0089] Composite standard concentration:

[0090] S0: 0ng / ml;

[0091] S1: 25-OH-D (10ng / ml); P1NP (10ng / ml); β-CTX (0.1ng / ml)

[0092] S2: 25-OH-D (20ng / ml); P1NP (50ng / ml); β-CTX (0.5ng / ml)

[0093] S3: 25-OH-D (40ng / ml); P1NP (100ng / ml); β-CTX (2.0ng / ml)

[0094] S4: 25-OH-D (80ng / ml); P1NP (200ng / ml); β-CTX (5.0ng / ml)

[0095] S5: 25-OH-D (120ng / ml); P1NP (500ng / ml); β-CTX (20.0ng / ml)

[0096] The R2 of the dose-response curve was: 0.9997 (25-OH-D); 0.9999 (P1NP); 0.9998 (β-CTX).

[0097] The blank limits of the test kit (Table 1) are: 0.0097 ng / ml (25-OH-D); 0.0250 ng / ml (P1NP); 0.0371 ng / ml (β-CTX).

[0098] Table 1 Blank limits of test kit

[0099]

[0100] 2. Repeatability

[0101] Composite quality control products:

[0102] C1: 25-OH-D (15.0ng / ml); P1NP (15.0ng / ml); β-CTX (0.3ng / ml)

[0103] C2: 25-OH-D (60.0ng / ml); P1NP (60.0ng / ml); β-CTX (1.5ng / ml)

[0104] High and low concentration samples were taken, and each sample was tested 10 times using three batches of test kits.

[0105] The results are shown in Table 2-1, Table 2-2, and Table 2-3:

[0106] Intra-batch variation of the kit: 25-OH-D<5.1%; P1NP<6.1%; β-CTX<7.7%

[0107] The batch difference of the kit was as follows: 25-OH-D<6.0%; P1NP<5.7%; β-CTX<7.4%.

[0108] Table 2-1 Kit repeatability test results (25-OH-D)

[0109]

[0110] Table 2-2 Kit repeatability test results (P1NP)

[0111]

[0112] Table 2-3 Kit repeatability test results (β-CTX)

[0113]

[0114] 3. Specificity

[0115] To the low (ng / ml) concentration samples, type I procollagen C-terminal peptide (PICP), type I collagen N-terminal peptide cross-linking (NTX), and estrogen (E) were added to obtain the samples to be tested. Each sample was tested with batch 1 reagents, and the mean of the measurement results should be within the range of the target concentration mean (M) ± 2 standard deviations (SD).

[0116] The test results (Table 3) showed that the mean values ​​of 25-OH-D were between 14.54 ng / ml and 16.19 ng / ml, P1NP were between 14.60 ng / ml and 15.95 ng / ml, and β-CTX were between 0.26 ng / ml and 0.33 ng / ml, all within the target concentration range.

[0117] Table 3 Results of low concentration sample specificity experiments

[0118]

[0119] 4. Clinical sample correlation test

[0120] A total of 57 clinical samples were collected and tested and compared with the reagents developed by the present invention and the comparative reagents. The comparative reagent is a single detection reagent that uses the chemiluminescence principle for detection, and the comparative reagent detection method is strictly carried out in accordance with the instructions; the samples of the reagents of the present invention were treated with a dissociator and then loaded (20ul of each sample was added with 5ul of a dissociator, mixed and loaded). The results showed:

[0121] 25-OH-D correlation R2=0.9039; P1NP correlation R2=0.937; β-CTX correlation R2=0.9649, the correlation is good, respectively. Figure 2 , 3 , as shown in Figure 4.

[0122] In addition, 20ul of each of the 57 clinical samples was added to 5ul (50-300mM, pH6.0-8.0) phosphate buffer, mixed and loaded. The dissociation effect of the dissociation agent on 25-OH-D and its effect on the detection results of P1NP and β-CTX were evaluated. The results showed that Figure 5 , 6 As shown in Figures 7 and 8, when no dissociation agent was added, the 25-OH-D result was low and the correlation was poor (R2=0.5227), while the P1NP and β-CTX test results were not affected.

[0123] 5. Stability assessment of composite quality control products

[0124] Take 4 sets of composite quality control products, store 1 set at 2-8℃ and 3 sets at 37℃. After 5 days at 37℃, take the composite quality control products at 2-8℃ and 37℃ for 5 days, test them with batch 1 reagents, and record the results. Take the other two sets of composite quality control products at 37℃ for 9 days and 11 days, respectively, and record the results. The stability data (Table 4) show that the composite quality control products have good stability.

[0125] Table 4

[0126]

[0127] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0128] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A 25-OH-D, P1NP and β-CTX combined detection kit, characterized in that: Includes reagent cards and dissociation reagents; The reagent card comprises a PVC rubber plate, a nitrocellulose membrane arranged on the PVC rubber plate, a binding pad, a sample pad, and absorbent paper arranged on the nitrocellulose membrane in sequence; the nitrocellulose membrane is coated with 25-OH-D antibody, P1NP antibody, β-CTX antibody, and quality control antibody; the binding pad is labeled with a tracer with 25-OH-D antibody, P1NP antibody, and β-CTX antibody by a coupling method; The coating solution of the nitrocellulose membrane, the reconstitution solution of the conjugate pad, and the sample pad treatment solution of the sample pad all include sodium lauryl glucose carboxylate and plant lectin; The dissociation agent is used to dissociate 25-OH-D in the form of binding protein in serum or plasma without affecting the detection results of P1NP and β-CTX; the dissociation agent includes 50-300mM, pH 6.0-8.0 phosphate as a buffer, 0.005-0.05% N,N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01mg-0.15mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1mg / ml sodium dodecyl sulfate, and 0.01-0.05mg / ml sodium chloride.

2. The 25-OH-D, P1NP and β-CTX combined detection kit according to claim 1, characterized in that: The T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody at a concentration of 0.2-1 mg / ml; the T2 line is coated with P1NP antibody at a concentration of 0.2-1.25 mg / ml; the T3 line is coated with β-CTX antibody at a concentration of 0.1-0.6 mg / ml; the C line is coated with quality control antibody at a concentration of 0.1-0.5 mg / ml; and the coating amount of C, T1, T2 and T3 lines is 1 ul / cm.

3. A method for preparing a 25-OH-D, P1NP and β-CTX combined detection kit as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Nitrocellulose membrane antibody coating: (1.1) Preparation of coating solution: The coating solution is prepared by adding 0.05%-0.1% sodium lauryl glucose carboxylate to 0.01M-0.05M, pH 7.0-8.0 phosphate buffer and fully dissolving it; then adding 1%-5% trehalose and 0.5%-2% lectin; (1.2) Preparation of coating antibodies: Place 25-OH-D antibody, P1NP antibody, β-CTX antibody and quality control antibody in ultrafiltration tubes respectively, centrifuge and replace with prepared phosphate buffer, collect the antibodies in the ultrafiltration tubes into new centrifuge tubes, add an equal volume of 2× coating solution to dilute and mix, and prepare 25-OH-D coating antibody, P1NP coating antibody, β-CTX coating antibody and quality control coating antibody; (1.3) Antibody coating: The T1 line on the nitrocellulose membrane is coated with 25-OH-D antibody, the T2 line is coated with P1NP antibody, the T3 line is coated with β-CTX antibody, and the C line is coated with quality control antibody, and then dried to obtain a coated ruled PVC rubber plate; (2) Preparation of bonding pad: (2.1) Quantum dot activation and antibody labeling: add the quantum dot solution to 10 mM MES solution, add EDC and NHS, and react at room temperature; add 25-OH-D labeled antibody, P1NP labeled antibody, and β-CTX labeled antibody respectively, mix thoroughly, and react at room temperature using a vortex mixer; add 10% BSA solution and mix thoroughly, react at room temperature using a vortex mixer, centrifuge, discard the supernatant, and retain the precipitate to label the 25-OH-D antibody, P1NP antibody, and β-CTX antibody respectively; or Preparation of colloidal gold and antibody labeling: Take the prepared colloidal gold solution, add K2CO3 solution to adjust the pH to 7.0-7.8, add 25-OH-D labeled antibody, P1NP labeled antibody, β-CTX labeled antibody respectively, stir well for reaction, then add BSA and stir well for reaction; The colloidal gold-labeled antibody that has reacted completely is placed in a centrifuge tube, centrifuged, the supernatant is discarded, and the precipitate is retained, thereby labeling the 25-OH-D antibody, the P1NP antibody, and the β-CTX antibody respectively; (2.2) preparing a reconstitution solution: the reconstitution solution includes 0.01 M phosphate buffer containing 0.01-0.08% sodium lauryl glucose carboxylate, 1% BSA, 0.05% Tween-20, 0.5% trehalose, 0.5%-2% lectin, and 0.5% PVP; (2.3) Redissolving the labeled antibodies and spraying the membrane: Use the re-dissolving solution to re-dissolve the prepared 25-OH-D labeled antibody, P1NP labeled antibody, and β-CTX labeled antibody, mix the three labeled antibodies in a ratio of 1:1:1, spray them on the glass fiber membrane, and obtain the quantum dot binding pad after drying; (3) Sample pad preparation: Prepare a sample pad treatment solution: the sample pad treatment solution includes 0.01M phosphate buffer containing 0.01-0.15% sodium lauryl glucose carboxylate, 0.5%-2% plant lectin, 0.5% BSA, 0.05% Triton X-100, and 0.5% PEG; The glass fiber membrane is immersed in a sample pad treatment solution and dried to obtain a sample pad; (4) Paste the conjugate pad, sample pad, and absorbent paper onto the antibody-coated nitrocellulose membrane PVC plate in sequence, cut into strips and load into the cartridge, place into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature; (5) Preparation of dissociation agent: The dissociation agent includes 50-300 mM, pH 6.0-8.0 phosphate buffer, 0.005-0.05% N,N-dimethylformamide, 0.01-0.05% polysorbate-20, 0.01 mg-0.15 mg / ml 8-anilinonaphthalene-1-sulfonic acid ammonium salt, 0.01-0.1 mg / ml sodium dodecyl sulfate, and 0.01-0.05 mg / ml sodium chloride.

4. The method for preparing the 25-OH-D, P1NP and β-CTX combined detection kit according to claim 3, characterized in that: The method also includes preparing a composite standard and a composite quality control preservation solution, wherein the composite standard and the composite quality control preservation solution include a 0.01M phosphate buffer containing 0.01-0.2% sodium lauryl glucose carboxylate at pH 7.0-8.0, and 0.1-0.5% BSA, 0.5-1.0% trehalose, 0.05-0.1% PEG 20000, 0.01-0.05% sorbitol, 0.05-0.2% aminotriacetic acid, and 0.45-0.9% NaCl are added.

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