A sandwich method-based combined detection reagent for vitamin a and 25-hydroxyvitamin d and application thereof

Abstract

The present application belongs to the field of analytical chemistry and detection technology, and particularly relates to a vitamin A and 25-hydroxyvitamin D combined detection reagent based on a sandwich method and application thereof. Specifically, the present application effectively overcomes the defects of the known detection methods, such as not supporting whole blood, long analysis time, and poor sensitivity, by optimizing the combination of the detection reagents for vitamin A and 25-OH-VD, and simultaneously has the advantages of high detection sensitivity, low detection limit, wide linear range, simple operation, high stability, and normal temperature transportation, and has important value for the diagnosis and drug efficacy evaluation of vitamin A and 25-OH-VD.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry and detection technology, and particularly relates to a vitamin A and 25-hydroxyvitamin D combined detection reagent based on a sandwich method and application thereof. BACKGROUND

[0002] Vitamin A is a fat-soluble vitamin, usually representing retinol and its active substances, which cannot be synthesized by the human body itself and can only be taken exogenously. Vitamin A deficiency can cause dry eye disease, night blindness, impaired immune system, and increase the risk of infection and death in the population. In addition, compared with vitamin A deficiency, the incidence of marginal vitamin A deficiency is higher and is more easily ignored. In addition to affecting the growth and development of children, marginal vitamin A deficiency can also cause anemia, respiratory and digestive tract infections, and if there is no effective intervention, it is easy to progress to vitamin A deficiency. At the same time, the higher the content of vitamin A in the body is not necessarily better, as a fat-soluble vitamin, if a large amount is taken in a short period of time, it is easy to cause accumulation in the body and cause liver poisoning, therefore, early diagnosis of vitamin A, more scientific and effective evaluation of the body's vitamin A nutritional status and improvement are crucial to our health management.

[0003] Vitamin D is an essential fat-soluble vitamin for the human body. Vitamin D exists in the body mainly in the form of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D synthesized by direct sunlight on the skin is transported to the liver by binding to vitamin D binding protein (VDBP), while vitamin D obtained from food is first absorbed into the blood in the form of chylomicrons together with fat through the small intestinal wall, and then transported to the liver by binding to lipoproteins or vitamin D binding protein. Vitamin D absorbed by the two ways must undergo two hydroxylation reactions in the body to exert biological effects. First, it is transported to the liver by blood, where hydroxylation of C25 is initiated in the liver microsomes under the action of vitamin D-25-hydroxylase system and the participation of NADPH, O2, Mg 2+ , generating 25-hydroxyvitamin D (25-OH-VD) with weak physiological activity. Subsequently, 25-OH-VD in circulation is transported to the kidney by blood through binding to a-globulin, and is converted to the most physiologically active 1,25-dihydroxyvitamin D (1,25(OH)2D) (also known as calcitriol) in the kidney mitochondria catalyzed by 25-hydroxyvitamin D-1a hydroxylase, which then exerts hormone-like biological effects mediated by specific vitamin D receptors (VDR) in cells, and participates in various biological processes. 25-OH-VD and 1,25(OH)2D are the main metabolites for evaluating the vitamin D status in the body, and the main circulating metabolite is 25-OH-VD, while the active metabolite is 1,25(OH)2D.

[0004] In recent years, studies have shown that there is a correlation between vitamin A and vitamin D deficiency in children. However, there is still a lack of relevant reports on the combined detection of vitamin A and 25-hydroxyvitamin D in the prior art. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a vitamin A and 25-hydroxyvitamin D combined detection reagent based on the sandwich method and its application. Specifically, the detection reagent for vitamin A and 25-OH-VD is optimized and combined, thereby effectively overcoming the defects of the known detection methods, such as not supporting whole blood, long analysis time, and poor sensitivity. At the same time, the detection reagent has the advantages of high detection sensitivity, low detection limit, wide linear range, simple operation, high stability, and can realize normal temperature transportation. It has important value for the diagnosis and drug efficacy evaluation of vitamin A and 25-OH-VD. Based on the above research results, the present application is completed.

[0006] Specifically, the present application relates to the following technical solutions:

[0007] In a first aspect of the present application, a vitamin A and 25-hydroxyvitamin D combined detection reagent based on the sandwich method is provided, which at least includes a dissociation diluent for diluting a sample to be tested and a quantum dot fluorescence test strip for vitamin A and 25-OH-VD based on the sandwich method.

[0008] The dissociation diluent includes a buffer system, a polysaccharide, a reducing / denaturing agent, a perfluoroalkyl acid compound, a non-ionic surfactant, and an alcohol substance.

[0009] The sample to be tested is a blood sample, which can be whole blood, serum or plasma sample, without specific limitation.

[0010] In the present application, the quantum dot fluorescence test strip for vitamin A and 25-OH-VD based on the sandwich method includes a bottom plate, and a chromatographic structure is covered on the bottom plate. The chromatographic structure is provided with a sample pad, a binding pad, a coating film and a water absorption pad in sequence according to the sample flow direction, and the adjacent parts of the above components are connected by overlapping.

[0011] In a second aspect of the present application, a detection kit is provided, which contains the above detection reagent. Further, the detection kit has a shell protection outside the test strip.

[0012] In a third aspect of the present application, a method for detecting the content of vitamin A and 25-hydroxyvitamin D in blood based on the sandwich method is provided. The method obtains the concentrations of vitamin A and 25-hydroxyvitamin D in the blood sample of the subject based on the detection reagent of the first aspect or the detection kit of the second aspect.

[0013] In a fourth aspect of the present application, the above-mentioned detection reagent, detection kit or detection method is applied in any one or more of the following:

[0014] (a) detection of vitamin A and 25-hydroxyvitamin D content;

[0015] (b) preparation of products for evaluation of therapeutic effect of vitamin A and vitamin D drugs.

[0016] The products can be kits, detection devices or detection equipment, etc., which are not specifically limited herein.

[0017] The above one or more technical solutions have the following beneficial technical effects:

[0018] 1. The vitamin A and 25-hydroxyvitamin D detection reagent provided by the above technical solution uses a double-antibody sandwich method for detection of small molecules, thereby improving detection sensitivity and broadening the detection range.

[0019] 2. After the sample pad is pretreated with an RBC buffer, a freeze-drying method is used to maximize the activity of the RBC and to block the empty sites on the hemofiltration membrane, thereby avoiding adsorption of the target substance (25-OH-VD) in the blood sample, achieving effective filtration of red blood cells, and realizing detection of whole blood samples, and the detection results of whole blood samples are in good consistency with those of serum and plasma samples.

[0020] 3. Small molecules are unstable, and the reagent kit uses a freeze-drying method for membrane cutting and pad spraying involved in the antibody immobilization process, thereby maximizing the stability of antigens and antibodies.

[0021] 4. The dissociation diluent in the above technical solution can be applied to human whole sample types: whole blood, peripheral blood, serum, and plasma samples; during detection, the bound vitamin A and 25-hydroxyvitamin D in the human sample are dissociated into a free state that can be detected, thereby achieving stable and accurate detection.

[0022] Meanwhile, the detection test paper provided by the above technical solution has good stability and can be transported and stored at room temperature, thus having good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, and are incorporated in and constitute a part of this application. The embodiments of the present application illustrated herein and the descriptions thereof are presented for the purpose of explaining and teaching the present application, and are not intended to limit the present application unduly.

[0024] Figure 1 FIG. 1 is a structural schematic diagram of a test paper strip according to the present application.

[0025] FIG. 1 is a structural schematic diagram of a test paper strip according to the present application.Figure 2 Figure 1 is a schematic diagram of an external shell structure of a test strip according to the present application.

[0026] In the figure, 1 is a sample pad, 2 is a conjugate pad, 3 is an NC membrane, 4 is an absorbent pad, 5 is a bottom plate (PVC plate), 6 is a T1 detection line, 7 is a T2 detection line, 8 is a quality control line, 9 is a sample addition hole, and 10 is an observation window (viewing window). DETAILED DESCRIPTION

[0027] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] It should be noted that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should be further understood that the terms "comprise" and / or "include" as used herein indicate the presence of the features, steps, operations, devices, components and / or combinations thereof. If no specific conditions are specified in the following detailed description, the experimental methods are generally carried out according to the conventional methods and conditions of the art, and such techniques and conditions are fully explained in the literature.

[0029] As mentioned above, there is still a lack of relevant reports on the combined detection of vitamin A and 25-hydroxyvitamin D in the prior art.

[0030] In view of this, in one specific embodiment of the present application, a sandwich method-based combined detection reagent for vitamin A and 25-hydroxyvitamin D is provided, which at least comprises a dissociation diluent for diluting a sample to be tested (and dissociating the combined vitamin A and 25-hydroxyvitamin D in the sample into a free state that can be detected) and a sandwich method-based quantum dot fluorescence test strip for vitamin A and 25-OH-VD.

[0031] In another specific embodiment of the present application, the dissociation diluent comprises a buffer system, a polysaccharide, a reducing / denaturing agent, a perfluoroalkyl acid compound, a non-ionic surfactant and an alcohol substance.

[0032] The buffer system can use any one or more of a Tris-HCl buffer system, a phosphate buffer system or a barbitone-hydrochloric acid buffer system or other buffer systems; further preferably, the buffer system is a Tris-HCl buffer system with a pH of 8.0.

[0033] The high polysaccharide is selected from any one or more of β-glucan, microbial polysaccharide, water-soluble chitosan, and mannan; further preferably, the high polysaccharide is mannan.

[0034] The reducing / denaturing agent is selected from one or a combination of several of sodium borohydride, guanidine hydrochloride, guanidine isothiocyanate, urea, dithiothreitol, and mercaptoethanol; further, the reducing / denaturing agent is a mixture of sodium borohydride and guanidine isothiocyanate.

[0035] The perfluoroalkyl acid compound is selected from one or a combination of several of perfluorooctanoic acid, perfluorooctane sulfonic acid, perfluoroalkyl carboxylic acid, and perfluorooctanoyl chloride; further, the perfluoroalkyl acid compound is perfluorooctanoyl chloride.

[0036] The non-ionic surfactant is selected from one or a combination of several of S9, S17, S19, S20, and S21; further preferably, the non-ionic surfactant is S17.

[0037] The alcohol substance is selected from one or a combination of several of methanol, ethanol, n-propanol, isopropanol, ethylene glycol, and propylene glycol; further preferably, the alcohol substance is propylene glycol.

[0038] In another specific embodiment of the present application, the dissociation diluent has the following formulation: 0.05-0.15M Tris-HCl buffer system (pH 8.0) containing 1.0-1.5% mannan, 0.4-1.0% sodium borohydride, 0.3-1.2% guanidine hydrochloride, 0.2-1.5% perfluorooctanoyl chloride, 0.5-0.8% S17, and 0.5-1.2% propylene glycol.

[0039] The addition of mannan in the dissociation diluent of the present application can effectively adjust the viscosity of the diluent, thereby better controlling the chromatographic speed after sample loading, achieving the effect of uniform release. In the chromatographic process, sodium borohydride provides an external reducing environment, and guanidine hydrochloride and perfluorooctanoyl chloride can effectively destroy the binding of 25-OH-VD and the binding protein; mannan can also better protect the antibody from being damaged by the denaturing agent, thereby achieving sufficient reaction.

[0040] It should be noted that the sample to be tested can be a blood sample, which can be whole blood, serum, or plasma sample, which is not limited here.

[0041] In another specific embodiment of the present application, the quantum dot fluorescent detection test strip for vitamin A and 25-OH-VD based on the sandwich method includes a base plate, and a chromatographic structure is covered on the base plate. The chromatographic structure is provided with a sample pad, a binding pad, a coated membrane, and a water absorption pad in sequence in the sample flow direction, and the adjacent parts of the components are connected by overlapping.

[0042] The mixture of quantum dot labeled VA antibody, quantum dot labeled 25-OH-VD antibody and quantum dot labeled chicken IgY antibody is coated on the binding pad, and the mixture is dissolved and diluted by a complex solution and then sprayed on the binding pad, and the formula of the complex solution is as follows: 0.05-0.2M sodium barbital-hydrochloric acid buffer, 1.0-2.0% sodium casein, 1.0-2.0% BSA, 2-8% fish skin gelatin, 2-8% trehalose, 0.10-0.30% Proclin300.

[0043] It should be noted that, in order to maximize the high activity and stability of the quantum dot labeled antibody, the above binding pad is sprayed and then dried at low temperature, for example, freeze-dried.

[0044] The detection line (T line) and the quality control line (C line) are arranged in the chromatographic direction on the coated membrane in sequence; wherein the T line at least comprises T1 line and T2 line, the T1 line is coated with anti-VA complex antibody; the T2 line is coated with anti-25-OH-VD complex antibody, and the C line is coated with goat anti-chicken IgY antibody.

[0045] In another specific embodiment of the application, the sample pad, the binding pad and the coated membrane in the test strip are connected in sequence, and the water absorption pad is connected to the detection pad at the joint part.

[0046] In another specific embodiment of the application, the sample pad can be a glass cellulose membrane; the binding pad can be a polyester cellulose membrane; the coated membrane can be a nitrocellulose membrane; and the water absorption pad can be a material with good water absorption performance, such as glass fiber material, cotton pulp material or mixed material of glass fiber and cotton pulp, preferably cotton pulp material, or commercially available water absorption material, such as water absorption paper or water absorption board. Herein, no specific limitation is made.

[0047] In another specific embodiment of the application, the test strip uses a hydrophobic material with a certain hardness as the bottom plate; further, the bottom plate is a PVC plate.

[0048] In another specific embodiment of the present application, the sample pad contains anti-red blood cell antibodies (RBC) for filtering red blood cells in the whole blood sample; the pretreatment of the sample pad is prepared by spraying an RBC pretreatment solution, and the formula of the pretreatment solution is as follows: 1.5-3.0% NH4Cl, 1-5% trehalose, 0.5-1.0% sodium casein, 100-200 μg / mL anti-red blood cell antibodies (RBC), 0.5-2 mg / mL anti-mouse antibodies, and 1-3 mg / mL anti-ovine antibodies in 0.05-0.20 M HEPES buffer (pH 7.5); the sample pad is obtained by drying after spraying the pretreatment solution with the above formula; in order to preserve the activity of RBC, the above drying is preferably performed by low-temperature drying, such as freeze-drying, and the present application finds that, by using the freeze-drying method after the pretreatment of the sample pad, the activity of RBC can be maximally preserved, and the free sites on the sample pad can be blocked to avoid the adsorption of target substances in the blood sample, thereby achieving the purpose of improving sensitivity.

[0049] After the completion of the display board, the test card is placed into the detection instrument, and the fluorescence value T of the detection line region and the fluorescence value C of the quality control line region are further converted by software to obtain T / C (i.e., T1 / C, T2 / C as the output result), and the content of vitamin A and 25-OH-VD in the sample to be tested can be calculated through the standard curve.

[0050] In another specific embodiment of the present application, a detection kit is provided, which contains the above detection reagent, and further, the detection kit has a shell protection outside the test strip.

[0051] In another specific embodiment of the present application, a method for detecting the content of vitamin A and 25-hydroxyvitamin D in blood based on a sandwich method is provided, and the detection reagent or the detection kit is used to obtain the concentration of vitamin A and 25-hydroxyvitamin D in the blood sample of a subject.

[0052] In another specific embodiment of the present application, the specific steps of the detection method are as follows: the blood sample to be tested is added dropwise into a dissociation diluent, mixed, then added dropwise into the sample addition hole of the test strip, and placed, and after the display board is completed, the fluorescence content of the detection line and the quality control line in the detection pad is detected.

[0053] In another specific embodiment of the present application, the fluorescence content detection method includes but is not limited to fluorescence immunoassay instrument, flow cytometry, fluorescence microscope, confocal microscope, total internal reflection or two-photon microscope, etc.

[0054] In another specific embodiment of the present application, the above detection reagent, detection kit or detection method is applied in any one or more of the following:

[0055] (a) Vitamin A and 25-hydroxyvitamin D content detection;

[0056] (b) Preparation of products for evaluating the efficacy of vitamin A and vitamin D drug therapy.

[0057] The product can be a kit, a detection device or a detection apparatus, etc., which is not specifically limited here.

[0058] The present application is further explained by the following examples, which do not constitute a limitation of the present application. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0059] Example 1

[0060] This embodiment provides a quantum dot fluorescence detection method for vitamin A and 25-hydroxyvitamin D based on the sandwich method, which includes a test strip structure as shown in Figure 1 The test strip includes a bottom plate 5 and a chromatographic structure, and the bottom plate 5 carries the chromatographic structure.

[0061] The chromatographic structure has a sample pad 1, a binding pad 2, an NC membrane 3 and a water absorption pad 4 from the sample end to the sample chromatographic direction, the sample pad 1, the binding pad 2 and the NC membrane 3 are sequentially overlapped, and the part of the water absorption pad 4 contacting the NC membrane 3 is overlapped on the NC membrane 3.

[0062] In this embodiment, the quantum dot fluorescence detection method for 25-hydroxyvitamin D using the above test strip is used, and the specific operation steps are as follows:

[0063] 1) Preparation of coated membrane:

[0064] (A) Coating: Fix the non-spotting surface of the NC membrane to the designated position of the membrane sputtering instrument, take the anti-VA antibody with a concentration of 1.3 mg / mL and the anti-25-OH-VD antibody with a concentration of 1.8 mg / mL and the goat anti-chicken IgY antibody with a concentration of 0.9 mg / mL, apply the three-dimensional sputtering instrument to draw lines, and coat the anti-VA complex antibody (Biorbyt Company, SVT-18704), the anti-25-OH-VD complex antibody (Diasource Company, 5319716) and the goat anti-chicken IgY antibody on the test line T1, T2 and the control line C of the NC membrane respectively, and freeze-dry for 48 h.

[0065] 2) Preparation of binding pad

[0066] (A) Quantum dot labeled VA antibody

[0067] a: Take 80 μL of cadmium selenide quantum dots, add to 4 mL of MES buffer with an ionic strength of 50 mM (pH 5.5), add 70 μL of 20 mg / mL EDC and 20 mg / mL NHS respectively, and then add 250 μL of 5 mg / mL VA antibody (Biorbyt, SKU-B8962), and react at 30°C for 2.5 h in the dark.

[0068] b: To the quantum dot labeling solution prepared in step a, add BSA with a final concentration of 6 mg / mL and ethanolamine with a final concentration of 1 mg / mL, and react at 4°C for 4 h in the dark with stirring.

[0069] c: Transfer the labeled mixture in step b to a centrifuge tube, centrifuge at 14000 g / min for 20 min, discard the supernatant, add 4 mL of 50 mM PBS buffer (pH 7.4) as a washing solution to the centrifuge tube, mix well by blowing, centrifuge again at 14000 g / min for 20 min, and discard the supernatant, to obtain the labeled antibody.

[0070] d: Resuspend the labeled antibody obtained in step c using the resuspension solution, ultrasonic and mix well, to obtain the purified quantum dot labeled VA antibody.

[0071] (B) Quantum dot labeled 25-OH-VD antibody

[0072] a: Take 120 μL of cadmium selenide quantum dots, add to 5 mL of MES buffer with an ionic strength of 50 mM (pH 5.8), add 70 μL of 20 mg / mL EDC and 20 mg / mL NHS respectively, and then add 285 μL of 5 mg / mL 25-OH-VD antibody (Diasource, 5319706), and react at 30°C for 2.5 h in the dark.

[0073] b: To the quantum dot labeling solution prepared in step a, add BSA with a final concentration of 6 mg / mL and ethanolamine with a final concentration of 1 mg / mL, and react at 4°C for 4 h in the dark with stirring.

[0074] c: Transfer the labeled mixture in step b to a centrifuge tube, centrifuge at 14000 g / min for 20 min, discard the supernatant, add 4 mL of 50 mM PBS buffer (pH 7.4) as a washing solution to the centrifuge tube, mix well by blowing, centrifuge again at 14000 g / min for 20 min, and discard the supernatant, to obtain the labeled antibody.

[0075] d: Resuspend the labeled antibody obtained in step c using the resuspension solution, ultrasonic and mix well, to obtain the purified quantum dot labeled 25-OH-VD antibody.

[0076] (C) Quantum dot labeled chicken IgY

[0077] a: Take 750 μL of cadmium selenide quantum dots and add to 5 mL of MES buffer with an ionic strength of 50 mM (pH 5.5), add 20 mg / mL EDC, 20 mg / mL NHS, each 75 μL, add 5 mg / mL chicken IgY antibody 500 μL, respectively, and react at 30°C for 2.5 h in the dark.

[0078] b: To the quantum dot labeling solution prepared in step a, add casein with a final concentration of 6 mg / mL and ethanolamine with a final concentration of 1 mg / mL, and react at 4°C for 4 h in the dark with stirring.

[0079] c: Take the labeled mixture in step b and transfer to a centrifuge tube, centrifuge at 14000 g / min for 20 min, discard the supernatant; use 4 mL of 50 mM PBS buffer (pH 7.4) as the washing solution, add to the centrifuge tube, blow and mix evenly, centrifuge again at 14000 g / min for 20 min, discard the supernatant, and the labeled antibody is obtained.

[0080] d: Resuspend the labeled antibody obtained in step c with the resuspension solution, ultrasonic and mix well, and the purified quantum dot labeled chicken IgY antibody is obtained.

[0081] The resuspension solution formula is: 0.1 M barbital sodium-hydrochloric acid buffer (pH 8.0), 1.3% sodium casein, 1.7% BSA, 5% fish gelatin, 5% trehalose, and 0.15% Proclin 300.

[0082] (D) Spray pad

[0083] Mix the resuspended quantum dot labeled VA antibody, quantum dot labeled 25-OH-VD antibody, and quantum dot labeled chicken IgY in a ratio of 1:1:1, and apply a three-dimensional scribe gold spraying instrument to spray 3 μL / cm on a glass fiber pad, and freeze-dry for 48 h to obtain the combination pad.

[0084] 3) Sample pad pretreatment

[0085] a: Prepare a pretreatment solution: 0.1 M HEPES buffer (pH 7.5) containing 2.5% NH4Cl, 4% trehalose, 0.8% sodium casein, and 160 μg / mL anti-red blood cell antibody (RBC), 1 mg / mL anti-mouse antibody, and 2 mg / mL anti-ovine antibody.

[0086] b: Use the pretreatment solution prepared in step a to soak the blood filtration membrane at room temperature for 1 h.

[0087] c: The pretreated filter blood film in step b is dried by a freeze dryer, and the pretreated sample pad is obtained after 48h of freeze drying.

[0088] 4) Assembly:

[0089] The adhesive paper at the corresponding position on the bottom plate is torn off, and the water absorption paper is pasted on the NC film at the corresponding position, covering the NC film by 1mm. The binding pad is pasted under the NC film, covering the binding pad by 1mm. The sample pad is pasted under the binding pad, covering the binding pad by 2mm, and the assembled large plate is obtained.

[0090] 5) Cutting and casing: After setting the width of the test paper strip on the microcomputer automatic cutting machine, cutting is performed, and the prepared test paper strip is obtained. The test paper strip is placed in the card shell in the forward direction, and the shell is packaged, so that the detection card with shell protection is obtained (as shown in Figure 2 ). The detection card is placed in an aluminum foil bag and sealed, and the single-portion vitamin A and 25-hydroxyvitamin D detection card is obtained.

[0091] 6) Preparation of dissociation diluent. According to the optimal formula, 0.1M Tris-HCl buffer system (pH 8.0) contains 1% mannitol, 0.8% sodium borohydride, 0.8% guanidine hydrochloride, 0.5% perfluorooctanoyl chloride, 0.5% S17 and 0.5% propylene glycol.

[0092] 7) When used for detecting vitamin A and 25-hydroxyvitamin D in a sample, the detection method comprises the following steps:

[0093] a. 5μL of the sample to be tested (serum / plasma / whole blood) is taken and added to 100μL of dissociation diluent for mixing.

[0094] b. 50μL of the diluted sample is added to the sample well of the detection card, and the plate is incubated for 10min. Then, the detection card is inserted into the fluorescence immunoassay analyzer, and the fluorescence signal intensity of the detection card is detected. The fluorescence values of the detection line and the quality control line are represented by T1, T2 and C, respectively. The final detection result is provided in the form of a ratio (i.e., T1 / C and T2 / C as output results). The detection result ratio is substituted into the standard curve of vitamin A and 25-hydroxyvitamin D, respectively, and the concentration of vitamin A and 25-hydroxyvitamin D in the sample to be tested is obtained.

[0095] Comparative Example 1

[0096] The preparation method is the same as that of Example 1, except that no mannitol and denaturant are added when preparing the dissociation diluent of the vitamin A and 25-hydroxyvitamin D detection kit.

[0097] Comparative Example 2

[0098] The preparation method is the same as that of Example 1, except that no RBC is added in the sample pad treatment solution of the vitamin A and 25-hydroxy vitamin D detection kit.

[0099] Comparative Example 3

[0100] The preparation method is the same as that of Example 1, except that no anti-mouse antibody and goat-derived antibody are added in the sample pad treatment solution of the vitamin A and 25-hydroxy vitamin D detection kit.

[0101] Comparative Example 4

[0102] The preparation method is the same as that of Example 1, except that the coating film, binding pad, and sample of the vitamin A and 25-hydroxy vitamin D detection kit are dried by a conventional 37°C drying method instead of a freeze-drying method.

[0103] Effect verification

[0104] 1. Detection of clinical samples in Example 1

[0105] The coincidence rates of the results of the detection of vitamin A and 25-OH-VD in Example 1 with the results of the clinical detection of vitamin A and 25-OH-VD are both 100%, and the correlations are 0.993 and 0.992, respectively.

[0106] Table 1. Comparison of the detection results of Example 1 with the clinical results

[0107]

[0108] 2. Detection of clinical samples in Comparative Example 1

[0109] The coincidence rates of the results of the detection of vitamin A and 25-OH-VD in Comparative Example 1 with the results of the clinical detection of vitamin A and 25-OH-VD are 57% and 47%, respectively, and the correlations are 0.302 and 0.409, respectively.

[0110] Table 2. Comparison of the detection results of Comparative Example 1 with the clinical results

[0111]

[0112] 3. Detection of clinical samples in Comparative Example 2

[0113] The coincidence rates of the results of the detection of vitamin A and 25-OH-VD in Comparative Example 2 with the results of the clinical detection of vitamin A and 25-OH-VD are 77% and 67%, respectively, and the correlations are 0.528 and 0.582, respectively; and severe hemolysis occurs in the detection of whole blood samples in Comparative Example 2.

[0114] Table 3. Comparison of the detection results of Comparative Example 2 with the clinical results

[0115]

[0116] 4. Detection of clinical samples in Comparative Example 3

[0117] The coincidence rates of the results of the detection of vitamin A and 25-OH-VD in Comparative Example 3 with the results of the clinical detection of vitamin A and 25-OH-VD were 83% and 87%, respectively, and the correlations were 0.568 and 0.805, respectively.

[0118] Table 4. Comparison of the detection results in Comparative Example 3 with the clinical results

[0119]

[0120] 5. Detection of clinical samples in Comparative Example 4

[0121] The coincidence rates of the results of the detection of vitamin A and 25-OH-VD in Comparative Example 4 with the results of the clinical detection of vitamin A and 25-OH-VD were 87% and 90%, respectively, and the correlations were 0.945 and 0.982, respectively.

[0122] Table 5. Comparison of the detection results in Comparative Example 4 with the clinical results

[0123]

[0124] 6. Technical indexes of the test strip in the present application:

[0125] Vitamin A item:

[0126] Accuracy: the relative deviation is not more than ±5%;

[0127] Repeatability: the CV is not more than ±5%;

[0128] Detection limit: not higher than 0.01 mg / L;

[0129] Linear range: the correlation r is not less than 0.990 within the range of [0.02, 5] mg / L.

[0130] 25-hydroxyvitamin D item:

[0131] Accuracy: the relative deviation is not more than ±5%;

[0132] Repeatability: the CV is not more than ±5%;

[0133] Detection limit: not higher than 1 ng / mL;

[0134] Linear range: the correlation r is not less than 0.990 within the range of [2, 200] ng / mL.

[0135] 7. Stability verification of the test strip

[0136] The sample with known vitamin A and 25-OH-VD concentrations of 0.38 mg / L and 31.95 ng / mL respectively is used to test three different batches of test strips, and the results are shown in Tables 6 and 7, which show that the test strip has good room temperature storage stability, is simple to operate, and can realize room temperature transportation.

[0137] Table 6 Test strip stability test results for vitamin A

[0138]

[0139] Table 7 Test strip stability test results for 25-OH-VD

[0140]

[0141] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A combined detection reagent for vitamin A and 25-hydroxyvitamin D based on a sandwich method, characterized in that, The detection reagent includes at least a dissociation diluent for diluting the sample to be tested and a quantum dot fluorescent test strip for vitamin A and 25-OH-VD based on a sandwich method; The formulation of the dissociation diluent is as follows: a 0.05-0.15M Tris-HCl buffer system containing 1.0-1.5% mannan, 0.4-0.8% sodium borohydride, 0.3-0.8% guanidine hydrochloride, 0.2-1.5% perfluorooctanoyl chloride, 0.5-0.8% S17 and 0.5-1.2% propylene glycol; The quantum dot fluorescent test strip for vitamin A and 25-OH-VD based on the sandwich method includes a base plate covered with a chromatographic structure. The chromatographic structure is arranged in sequence according to the sample flow direction, including a sample pad, a conjugation pad, a coating membrane, and an absorbent pad. The above components are overlapped and connected at adjacent locations. The sample pad was pretreated by spraying RBC pretreatment solution, which was formulated as follows: 0.05-0.20M HEPES buffer containing 1.5-3.0% NH4Cl, 1-5% trehalose, 0.5-1.0% sodium caseinate, 100-200 μg / mL anti-erythrocyte antibody, 0.5-2 mg / mL anti-mouse antibody, and 1-3 mg / mL anti-sheep antibody; The coated membrane is sequentially arranged with a detection line (T line) and a control line (C line) along the chromatography direction; wherein, the T line includes at least a T1 line and a T2 line, the T1 line is coated with an anti-VA complex antibody, the T2 line is coated with an anti-25-OH-VD complex antibody, and the C line is coated with a goat anti-chicken IgY antibody.

2. The detection reagent as described in claim 1, characterized in that, The sample to be tested is a blood sample, which may be whole blood, serum, or plasma.

3. The detection reagent as described in claim 1, characterized in that, The conjugate pad is coated with a mixture of quantum dot-labeled VA antibody, quantum dot-labeled 25-OH-VD antibody, and quantum dot-labeled chicken IgY antibody. The mixture is dissolved and diluted with a reconstitution solution before being sprayed onto the conjugate pad. The reconstitution solution has the following formulation: 0.05-0.2M sodium barbital-hydrochloric acid buffer, 1.0-2.0% sodium caseinate, 1.0-2.0% BSA, 2-8% fish skin gelatin, 2-8% trehalose, and 0.10-0.30% Proclin 300.

4. The detection reagent as described in claim 1, characterized in that, The sample pad, conjugate pad, and coating membrane are overlapped in sequence, and the portion of the absorbent pad that is in contact with the test pad overlaps on the test pad. The sample pad contains anti-erythrocyte antibodies.

5. A test kit, characterized in that, The test kit comprises the test reagent according to any one of claims 1-4, wherein the test strip is protected by a shell.

6. The use of the test reagent according to any one of claims 1-4 or the test kit according to claim 5 in the preparation of products for evaluating the efficacy of vitamin A and vitamin D drugs.

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