Portable hydroxyl vitamin D detection kit

Abstract

The utility model relates to the technical field of vitamin D detection, and discloses a portable hydroxyl vitamin D detection kit. The technical problems that in the prior art, detection sensitivity is not high, a container does not have an oscillation function, and operation is complex and tedious are solved. The sample releasing device comprises an oscillation releasing structure, the oscillation releasing structure comprises an oscillator, and a matched container is placed on the oscillator to contain a sample and a releasing agent; the container comprises a coated plate, a plurality of micropores are formed in the coated plate, solid-phase antibodies are arranged in the micropores, each solid-phase antibody sequentially comprises a microporous plate substrate, a coating layer and a sealant covering layer from bottom to top, the coating layer comprises 25-hydroxyvitamin D antibodies, and the sealant covering layer comprises bovine serum albumin. According to the utility model, the detection sensitivity is improved, non-specific binding is reduced, the use is flexible, and the operation is simple and convenient.

Description

TECHNICAL FIELD

[0001] The utility model relates to the detection technical field of vitamin D especially relates to a portable hydroxyl vitamin D detection kit. BACKGROUND

[0002] Vitamin D is a sterol derivative, and is a necessary nutrient for human metabolism. The main ways for human body to obtain VD are: food intake and synthesis through sunlight irradiation of the skin. The vitamin D obtained by the human body is transported to the liver in the body through blood circulation, and is converted into 25-OHD under the action of liver enzymes. 25-OHD is transported to the kidney, and is converted into active 1, 25-dihydroxyvitamin D through second hydroxylation. 1, 25-dihydroxyvitamin D exerts its powerful biological effect by binding to target cell vitamin D receptors. The half-lives of vitamin D, 25-OHD and 1, 25-dihydroxyvitamin D in the human body are 24h, 3 weeks and 4h respectively. Among them, 25-OHD has a longer half-life and smaller fluctuation, so the serum 25-OHD content is currently internationally recognized as an indicator for measuring the nutritional status of the body. The classic effect of vitamin D is to regulate calcium and phosphorus levels and affect bone health. However, recent studies have found that in addition to the traditional bone effect, vitamin D is also related to autoimmune diseases, diabetes, cardiovascular diseases, cancer and other diseases. At present, vitamin D deficiency is very common. According to surveys, about 1 billion people in the world are in a state of VD deficiency. The optimal level of 25-OHD in serum has not been clearly defined, but many documents report that the serum 25-OHD level below 20ng / mL is generally defined as vitamin D deficiency, 21-29ng / mL is insufficient, and greater than or equal to 30ng / mL is sufficient. The existing 25-OHD detection methods include radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), chemiluminescence (CLIA) and liquid chromatography-tandem mass spectrometry (LC-MS). Among them, chemiluminescence has become a development trend due to its high sensitivity, wide linear range, no pollution and easy operation.

[0003] The Chinese patent document 202020891520.7 in the prior art discloses a detection kit for detecting 25-hydroxyvitamin D index, which comprises a top box, a middle box and a bottom box. The middle box has a moving structure, an upper and lower through slot is arranged on the inner bottom surface of the middle box, a vertical plug-in plate is arranged in the slot, the upper end of the plug-in plate is provided with a limiting plate, the lower end of the plug-in plate is provided with a stabilizing plate, a plug hole is arranged on the limiting plate, a sleeve is arranged in the plug hole, a plurality of springs arranged in a ring are connected to the outer surface of the sleeve, and the ends of the springs away from the sleeve are connected to the inside of the plug hole.

[0004] However, the above-mentioned scheme has at least the following technical problems in the implementation process: the detection sensitivity is not high, the container does not have a shaking function, and the operation is complex and tedious. Therefore, it is urgent to propose a portable hydroxyl vitamin D detection kit. SUMMARY

[0005] In view of the above technical problems, the present disclosure provides a portable hydroxy vitamin D detection kit, which solves the technical problems of low detection sensitivity, no shaking function of the container and complex operation in the prior art.

[0006] According to one aspect of the present disclosure, a portable hydroxy vitamin D detection kit is provided, which comprises a shaking release structure, the shaking release structure comprising a vibrator, a matched container being placed on the vibrator to contain a sample and a release agent; the container comprising a coated plate, the coated plate being provided with a plurality of micropores, a solid-phase antibody being arranged in the micropores, the solid-phase antibody comprising, from bottom to top, a micropore plate base, a coating layer and a blocking agent covering layer, the coating layer comprising 25-hydroxy vitamin D antibodies, and the blocking agent covering layer comprising bovine serum albumin.

[0007] In some embodiments of the present disclosure, the coating layer comprises 25-hydroxy vitamin D antibodies dissolved in a 0.1 mol / L phosphate buffer.

[0008] In some embodiments of the present disclosure, the blocking agent covering layer comprises a 2% bovine serum albumin and 5% sucrose phosphate buffer.

[0009] In some embodiments of the present disclosure, the vibrator comprises a motor, a power output shaft of the motor being connected to an eccentric wheel, an outer surface of the eccentric wheel being point-contacted to a transmission rod, the other end of the transmission rod being connected to a shaking platform through a support structure.

[0010] In some embodiments of the present disclosure, the power output shaft of the motor is provided with a driving sprocket, the driving sprocket being connected to a driven sprocket through a transmission chain, and the driven sprocket being connected to the eccentric wheel through a rotating shaft.

[0011] In some embodiments of the present disclosure, the transmission rod is U-shaped and surrounds the support structure.

[0012] In some embodiments of the present disclosure, a wear-resistant sleeve is arranged at the connection between the transmission rod and the support structure.

[0013] In some embodiments of the present disclosure, the support structure comprises a support plate, a pin shaft being arranged in the middle of the support plate to support the shaking platform.

[0014] The present utility model has the advantages of:

[0015] The portable design enables the detection of hydroxy vitamin D to be performed anytime and anywhere, and is suitable for on-site rapid detection; the shock release structure accelerates the detection process and shortens the detection time by rapidly mixing the sample and the release agent through the vibrator; the solid-phase antibody, the coating layer of the 25-hydroxy vitamin D antibody, provides high sensitivity and specificity of the detection, and ensures the accuracy of the detection result; the bovine serum albumin (BSA) in the blocking agent covering layer effectively reduces the non-specific binding, reduces the background signal, and improves the accuracy of the detection; the 2% BSA and 5% sucrose in the blocking agent covering layer provide a stable environment for the antibody, and help to maintain the stability of the kit and prolong the shelf life; the design of the shock release structure simplifies the operation steps, and enables non-professionals to easily perform the detection; the wear-resistant sleeve arranged at the connection between the transmission rod and the support structure, and the pin shaft design penetrating through the middle of the support plate improve the durability and service life of the equipment. BRIEF DESCRIPTION OF DRAWINGS

[0016] Fig. 1 It is a structure schematic view of the portable hydroxy vitamin D detection kit;

[0017] Fig. 2 It is another view of the structure schematic view of the portable hydroxy vitamin D detection kit;

[0018] Fig. 3 It is a left view of the portable hydroxy vitamin D detection kit;

[0019] Names of components in the figure: 1, coated plate; 2, microwell; 3, motor; 4, eccentric wheel; 5, transmission rod; 6, driving sprocket; 7, transmission chain; 8, driven sprocket; 9, rotating shaft; 10, wear-resistant sleeve; 11, support plate; 12, pin shaft; 13, shock platform. DETAILED DESCRIPTION

[0020] The preferred embodiments of the utility model are described below in combination with the drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the utility model, and are not used to limit the utility model. Example 1

[0021] This example discloses a portable hydroxy vitamin D detection kit, referring to Figs. 1 to 3 ; including a shock release structure, the shock release structure includes a vibrator, and a matching container is placed on the vibrator to contain a sample and a release agent; the container includes a coated plate 1, a plurality of microwells 2 are arranged on the coated plate, solid-phase antibodies are arranged in the microwells 2, the solid-phase antibodies sequentially include a microwell plate base, a coating layer, and a blocking agent covering layer from bottom to top, the coating layer includes 25-hydroxy vitamin D antibodies, and the blocking agent covering layer includes bovine serum albumin.

[0022] The coating layer 1 includes 25-hydroxy vitamin D antibody dissolved in 0.1 mol / L phosphate buffer solution.

[0023] The closed covering layer includes 2% bovine serum albumin and 5% sucrose in phosphate buffer solution.

[0024] The oscillator includes a motor 3, the power output shaft of the motor 3 is connected to an eccentric wheel 4, the outer surface of the eccentric wheel 4 is point-contact connected to a transmission rod 5, the other end of the transmission rod 5 is connected to the oscillation platform 13 through the support structure.

[0025] The power output shaft of the motor 3 is installed with a driving sprocket 6, the driving sprocket 6 is connected to a driven sprocket 8 through a transmission chain 7, and the driven sprocket 8 is connected to the eccentric wheel 4 through a rotating shaft 9.

[0026] The transmission rod 5 is U-shaped and surrounds the outside of the support structure.

[0027] A wear-resistant sleeve 10 is arranged at the connection between the transmission rod 5 and the support structure.

[0028] The support structure includes a support plate 11, a pin shaft 12 is arranged in the middle of the support plate 11 to support the oscillation platform.

[0029] During the working process of the oscillator, the motor 3 starts to rotate after being connected to the power supply and generates power. The power output shaft of the motor is connected to the eccentric wheel 4 through the transmission chain 7, the driving sprocket 6, and the driven sprocket 8, and the eccentric wheel rotates with the rotation of the motor. Due to the uneven mass distribution of the eccentric wheel, its rotation will generate a periodic centrifugal force. The centrifugal force of the eccentric wheel is transmitted to the transmission rod 5 through point contact. The U-shaped design of the transmission rod allows it to swing around the pin shaft 12 outside the support plate 11. The other end of the transmission rod is connected to the oscillation platform, and the swinging of the transmission rod makes the oscillation platform vibrate. The sample container placed on the oscillation platform vibrates with the vibration of the platform. Such vibration helps to promote the mixing of the sample and the releasing agent. At the connection between the transmission rod and the support structure, the wear-resistant sleeve 10 reduces the wear of the transmission rod when it swings, protects the equipment and prolongs the service life. The working parameters such as the vibration frequency and amplitude of the oscillator can be adjusted by controlling the rotating speed of the motor or adjusting the eccentric amount of the eccentric wheel.

[0030] Preparation method of coated plate

[0031] Dissolve 25-hydroxy vitamin D antibody in 0.1 mol / L PB buffer solution (pH 7.0), dilute to the optimal concentration, and stir with a magnetic stirrer for 30 min to ensure that the coating solution is fully mixed. Add 100 μL of coating solution to each well of the coated plate, and let it stand at 2°C~8°C overnight. Pour out the coating solution, wash the plate once with the washing solution, add 200 μL of blocking solution (containing 2% BSA and 5% sucrose in PB buffer solution) to each well, and let it stand at 2°C~8°C overnight. Pour out the blocking solution, blow dry, and seal.

[0032] Sample pretreatment method

[0033] Since almost all circulating 25-hydroxy vitamin D in serum exists in combination with vitamin D binding protein (DBP) and albumin. Before determining the concentration of 25-hydroxy vitamin D in serum, it is necessary to release it from DBP and albumin first. The treatment method is: 30 μL of serum is added with 100 μL of releasing agent, shaken (500 bp) for 120 min, which can make the 25-hydroxy vitamin D in serum completely released.

[0034] Principle of kit

[0035] The kit adopts a competitive chemiluminescence method to quantitatively detect the concentration of 25-hydroxy vitamin D in serum in vitro. After adding calibrators, serum samples and releasing agents in a microwell plate pre-coated with 25-OHD monoclonal antibodies, the released 25-OHD in the samples is combined with the 25-OHD antibodies on the coated plate. After washing to remove the uncombined substances, an enzyme conjugate (a mixture of biotinylated 25-OHD and SA-HRP) is added to be combined with the 25-OHD antibodies, so as to form a competitive immune complex. Subsequently, after washing to remove the uncombined substances, a chemiluminescence substrate is added, and a chemiluminescence immunoassay instrument is used to read the relative light intensity (RLU).

[0036] Operation steps of kit

[0037] Firstly, 30 μL of calibrators, serum samples and 100 μL of releasing agents are added in a microwell plate pre-coated with 25-OHD antibodies, and shaken (500 bp) for 120 min on a plate shaker. The enzyme conjugate working solution (a mixture of biotinylated 25-OHD and SA-HRP) is prepared within 10 min after the sample addition is completed, and is placed in the dark. Then, the plate is washed 5 times to remove the uncombined substances, 200 μL of the enzyme conjugate working solution is added to each well, and is reacted for 30 min on the plate shaker (500 bp). After washing the plate 5 times, 100 μL of the substrate (50 μL of substrate A and 50 μL of substrate B) is added, and a chemiluminescence immunoassay instrument is used to read the relative light intensity (RLU).

[0038] Although some preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0039] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A portable kit for the detection of hydroxyvitamin D, characterized by: The application relates to a shock release structure, which comprises a vibrator, a matched container is placed on the vibrator to contain samples and a release agent; the container comprises a coated plate, a plurality of micropores are arranged on the coated plate, solid-phase antibodies are arranged in the micropores, the solid-phase antibodies sequentially comprise a micropore plate base, a coating layer and a blocking agent covering layer from bottom to top, the coating layer comprises 25-hydroxy vitamin D antibodies, and the blocking agent covering layer comprises bovine serum albumin.

2. The portable kit for the detection of hydroxyl vitamin D according to claim 1, characterized in that: The vibrator comprises a motor, a power output shaft of the motor is connected with an eccentric wheel, the eccentric wheel is connected with a transmission rod through point contact on the outer surface of the eccentric wheel, and the transmission rod is connected with a shock platform through a support structure at the other end.

3. The portable kit for the detection of hydroxyl vitamin D according to claim 2, wherein: The power output shaft of the motor is provided with a driving sprocket, the driving sprocket is connected with a driven sprocket through a transmission chain, and the driven sprocket is connected with the eccentric wheel through a rotating shaft.

4. The portable kit for hydroxyvitamin D of claim 2, wherein: The transmission rod is U-shaped and is arranged around the support structure.

5. The portable kit for hydroxyvitamin D of claim 2, wherein: A wear-resistant sleeve is arranged at the connection position of the transmission rod and the support structure.

6. The portable kit for the detection of hydroxyl vitamin D according to claim 5, wherein: The support structure comprises a support plate, a pin shaft is arranged in the middle of the support plate to support the shock platform.

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