Urine-based kit for semi-quantitatively detecting content of vitamin D and detection method

Abstract

The invention discloses a urine-based kit for semi-quantitatively detecting the content of vitamin D and a detection method, which can be used for detecting a urine sample. The kit for semi-quantitatively detecting the content of vitamin D based on urine is obtained by combining a nitrocellulose membrane, an antibody labeling technology, an antibody-antigen specific binding reaction and an immunomembrane chromatography technology. The kit comprises a vitamin D detection reagent strip comprising sample filtering paper, a colloidal gold combination pad, a nitrocellulose membrane and absorbent paper which are arranged in sequence, has the characteristics of simplicity and convenience in operation, rapidness in detection, high sensitivity and strong specificity, and can be used for semi-quantitative detection and judgment of the concentration of vitamin D in urine; therefore, whether the content of the vitamin D in the urine is normal or not is accurately distinguished, efficient and sensitive vitamin D detection is achieved, and a new and reliable solution is provided for vitamin D detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a kit and a detection method for semi-quantitatively detecting vitamin D content based on urine. Background Art

[0002] Vitamin D plays an important role in human health, and its deficiency or excess may lead to a variety of diseases. At present, the methods for detecting vitamin D mainly include liquid chromatography-tandem mass spectrometry, electrochemiluminescence and immunoturbidimetry. However, these methods are all for serum and plasma samples, which usually require complex sample pretreatment and expensive instruments and equipment, and also require professional operators, so it takes longer testing time, higher costs and more expensive fees, especially the immunoturbidimetry, which also needs to be operated in a professional laboratory and requires more complex equipment to read the results through complex operations. Therefore, the above existing detection methods limit the application of vitamin D detection in primary medical institutions and families.

[0003] After searching, the existing Chinese invention application document with publication number CN202310753543.X discloses a 1,25-dihydroxyvitamin D detection material and preparation method, a 1,25-dihydroxyvitamin D detection method and a detection kit, which can measure the content of trace 1,25-dihydroxyvitamin D. However, the detection material needs to be used in combination with liquid-mass spectrometry detection technology, and professional operators need to use expensive instruments and equipment to ensure the detection results, which has certain limitations.

[0004] In summary, in the process of vitamin D detection, how to design a kit and a detection optimization method to improve the convenience and efficiency of detection and reduce costs has become a technical problem that needs to be solved urgently by those skilled in the art. The vitamin D detection method of the present invention adopts colloidal gold technology, and the specimen is urine, which is convenient for sampling and testing. It can not only accurately distinguish whether the human body lacks vitamin D or has an excess, but also can achieve self-detection anytime and anywhere. Summary of the invention

[0005] In order to solve the problems existing in the prior art mentioned above, the purpose of the present invention is to provide a kit and a detection method for semi-quantitative detection of vitamin D content based on urine. The present invention improves the sensitivity, specificity and accuracy of detection by combining nitrocellulose membrane, antibody labeling technology, antibody-antigen specific binding reaction and immunomembrane chromatography technology, and realizes rapid, simple and semi-quantitative detection of vitamin D concentration in urine, providing a new and reliable solution for the detection of vitamin D.

[0006] In order to achieve the above-mentioned purpose and other related purposes, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided a kit for semi - quantitatively detecting the content of vitamin D based on urine, including a vitamin D test strip. The vitamin D test strip includes a sample filter paper, a colloidal gold conjugate pad, a nitrocellulose membrane, and a blotting paper arranged in sequence on a rubber plate, wherein:

[0008] The colloidal gold conjugate pad is coated with a colloidal gold - labeled vitamin D antibody. The colloidal gold - labeled vitamin D antibody is used to specifically bind to vitamin D in the urine sample when the urine sample flows through, and form a colloidal gold - labeled antigen - antibody complex;

[0009] On the nitrocellulose membrane, a detection T - line and a quality control C - line are arranged in sequence along the chromatography direction;

[0010] The detection T - line is coated with a vitamin D - BSA conjugate. The vitamin D - BSA conjugate is used to capture the colloidal gold - labeled vitamin D antibody;

[0011] The quality control C - line is coated with a goat anti - mouse antibody. The goat anti - mouse antibody is used to bind the colloidal gold - labeled antigen - antibody complex and the free colloidal gold - labeled vitamin D antibody from the colloidal gold conjugate pad.

[0012] As a preferred technical solution, the dosage of the colloidal gold - labeled vitamin D antibody is 5mg - 20mg of vitamin D antibody per liter of colloidal gold solution; the coating concentration of the vitamin D - BSA conjugate at the detection T - line is 0.5 - 2mg / ml; the coating concentration of the goat anti - mouse antibody at the quality control C - line is 0.5 - 1.5mg / ml.

[0013] As a preferred technical solution, the colloidal gold - labeled vitamin D antibody adopts a 1,25 - dihydroxyvitamin D antibody - colloidal gold conjugate; the vitamin D - BSA conjugate adopts a conjugate formed by coupling 1,25 - dihydroxyvitamin D with the carrier protein BSA.

[0014] In the second aspect of the present invention, there is provided the application of the above - mentioned kit in the semi - quantitative detection of the vitamin D concentration in human urine.

[0015] In the third aspect of the present invention, there is provided a detection method for semi - quantitatively detecting the content of vitamin D based on urine. The above - mentioned kit is used to detect the urine sample to be tested, including the following steps:

[0016] Add the urine sample to be tested to the filter paper on the vitamin D test strip. After immunochromatographic reaction, observe the color development at the test T line and the quality control C line respectively. Taking the color development at the quality control C line as a reference, obtain the semi-quantitative test result of the vitamin D content in the urine sample to be tested according to the comparison of the color depth between the color development at the test T line and the color development at the quality control C line.

[0017] As a preferred technical solution, the judgment criteria for the semi-quantitative test result of the vitamin D content in the urine sample to be tested are as follows:

[0018] If brown-red bands appear on both the test T line and the quality control C line, and the band color at the test T line is darker than the band color at the quality control C line, it is determined that the vitamin D concentration in the urine sample to be tested is lower than the normal range;

[0019] If brown-red bands appear on both the test T line and the quality control C line, and there is a color change at the test T line and the band color is not darker than the band color at the quality control C line, it is determined that the vitamin D concentration in the urine sample to be tested is within the normal range;

[0020] If the test T line does not show color and only the quality control C line shows a brown-red band, it is determined that the vitamin D concentration in the urine sample to be tested is higher than the normal range;

[0021] If the quality control C line does not show color, regardless of whether the test T line shows color or not, the result is determined to be invalid.

[0022] As a preferred technical solution, the darker the band color at the test T line, the lower the judged vitamin D concentration.

[0023] As described above, the present invention combines a nitrocellulose membrane, an antibody labeling technique, an antibody-antigen specific binding reaction, and an immunochromatographic technique to obtain a kit for semi-quantitatively detecting the vitamin D content based on urine, which has the advantages of simple operation, rapid detection, high sensitivity, strong specificity, etc. It can semi-quantitatively detect and judge the concentration of vitamin D in urine, so as to accurately distinguish whether the content of vitamin D in urine is normal, realizing efficient and sensitive detection of vitamin D, and providing a new and reliable solution for the detection of vitamin D. Specifically, it has the following beneficial effects:

[0024] (1) Through the nitrocellulose membrane with a porous structure, the present invention can guide the fluid to move in a specific direction and adsorb and immobilize macromolecular substances such as proteins, so that the samples and reagents in the detection process can be evenly distributed, ensuring the accuracy of the detection results. At the same time, the nitrocellulose membrane can be used as a solid phase for antigen-antibody specific binding reactions. Moreover, by labeling colloidal gold, a tracer substance, on the antibody and utilizing the specific binding of the antibody to the antigen, the present invention improves the specificity, sensitivity, and accuracy of the detection. At the same time, after the label binds to the target antigen, the detection is carried out through color development or fluorescence signals, making the detection results more intuitive and reliable, and the specific binding reaction can also more effectively distinguish the target antigen from other substances, reducing false positive results.

[0025] (2) The sample solution detected by the present invention is the urine to be tested. Compared with the serum and plasma samples in the prior art, urine samples are more convenient and risk-free to collect, the detection is faster and more accurate. At the same time, compared with the professional laboratory environment required in existing methods such as mass spectrometry and immunoturbidimetry, the kit of the present invention does not require a special operating environment, and the operation is simpler. It can be self-tested anytime and anywhere, and the reagent has a validity period of up to two years. Therefore, this method is more suitable for promotion in primary medical institutions and families, and can be applied to some special groups. By detecting vitamin D in urine, it can quickly, accurately, and risk-free detect whether the subject lacks vitamin D or has excessive vitamin D, thus solving the problem of whether vitamin supplementation is needed, and can also guide special populations when to supplement vitamins, with wide application value.

[0026] (3) Through the immunochromatography technique, the present invention utilizes capillary action to promote the movement of urine samples on the nitrocellulose membrane, thereby achieving the detection of target substances through the chromatography principle. Specifically, the present invention utilizes the immunocompetition inhibition method, enabling vitamin D in the urine sample to be tested to competitively bind to the vitamin D antibody labeled with colloidal gold with vitamin D-BSA immobilized on the nitrocellulose membrane. This competitive binding reaction enables the color development intensities of the test line and the quality control line to reflect the concentration of vitamin D in the sample, and semi-quantitative detection of the vitamin D content in urine samples can be achieved, thereby accurately distinguishing whether the human body lacks vitamin D or has excessive vitamin D. Brief Description of the Drawings

[0027] Figure 1 is a schematic structural diagram of the vitamin D detection reagent strip in the present invention.

[0028] Figure 2 is a schematic diagram for determining the detection results when the kit in the present invention detects the vitamin D concentration in urine samples.

[0029] Figure 3 is the actual detection result diagram of the kit in the present invention.

[0030] Among them, the specific descriptions of the attached drawing reference numerals are as follows: 1. Vitamin D detection reagent strip; 2. Glue board; 3. Filter paper; 4. Colloidal gold conjugate pad; 5. Nitrocellulose membrane; 6. Absorbent paper; 7. Detection T line; 8. Quality control C line. Specific implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0032] All chemical reagents in the following embodiments are purchased from Sigma Corporation.

[0033] Example 1

[0034] This example provides a kit for semi-quantitatively detecting the content of vitamin D based on urine, and the applicable sample of the kit is a urine sample.

[0035] As Figure 1 shown, the kit includes a vitamin D detection reagent strip 1, and the vitamin D detection reagent strip 1 includes a filter paper 3, a colloidal gold conjugate pad 4, a nitrocellulose membrane 5, and an absorbent paper 6 sequentially arranged on a glue board 2, wherein:

[0036] The colloidal gold conjugate pad 4 is coated with a colloidal gold-labeled vitamin D antibody, and the dosage of the colloidal gold-labeled vitamin D antibody is 5 mg - 20 mg of vitamin D antibody per liter of colloidal gold solution, and the colloidal gold-labeled vitamin D antibody adopts a 1,25-dihydroxyvitamin D antibody colloidal gold conjugate to specifically bind to vitamin D in the urine sample when the urine sample flows through, and form a colloidal gold-labeled antigen-antibody complex.

[0037] On the nitrocellulose membrane 5, a detection T line 7 and a quality control C line 8 are sequentially arranged along the chromatography direction. The detection T line 7 is coated with a vitamin D-BSA conjugate, and the coating concentration is 0.5-2 mg / ml. The vitamin D-BSA conjugate is a carrier conjugate formed by conjugating 1,25-dihydroxyvitamin D with the carrier protein BSA, and is used to capture the colloidal gold-labeled vitamin D antibody. The quality control C line 8 is coated with a goat anti-mouse antibody, and the coating concentration is 0.5-1.5 mg / ml, and is used to bind the colloidal gold-labeled antigen-antibody complex and the free colloidal gold-labeled vitamin D antibody from the colloidal gold conjugate pad 4.

[0038] In this embodiment, based on the normal range of vitamin D content in human urine being 30 to 80 ng / ml, through exploration and repeated experiments, the above-mentioned preparation concentrations of each reagent in the vitamin D test strip 1 are obtained, so that when the vitamin D content in the urine sample is 30 ng / ml, the lowest detection limit within the normal range, the color development depth of the detection T line 7 is equivalent to that of the quality control C line 8, and when the vitamin D content in the urine sample is greater than 80 ng / ml, the highest detection limit above the normal range, the detection T line 7 does not develop color.

[0039] This embodiment also provides a preparation method of the kit, including the following steps:

[0040] Label 1,25-dihydroxyvitamin D antibody with colloidal gold and then co-spray it on the colloidal gold conjugate pad 4; coat the carrier conjugate formed by conjugating 1,25-dihydroxyvitamin D with the carrier protein BSA at the detection T line 7 on the nitrocellulose membrane 5; at the same time, coat the goat anti-mouse antibody at the quality control C line 8 on the nitrocellulose membrane 5 to obtain the vitamin D test strip 1.

[0041] Specifically as follows:

[0042] 1. Prepare antibodies

[0043] (1) Dilute the antibody to 1 mg / ml with the labeling buffer.

[0044] 2. Method for labeling antibody with colloidal gold

[0045] (1) Measure 100 ml of colloidal gold solution.

[0046] (2) Add 10 ml of labeling buffer and mix well.

[0047] (3) Add the diluted antibody and mix quickly.

[0048] (4) Add 1 ml of 10% BSA solution and mix well.

[0049] (5) Centrifuge at 12,000 rpm for 20 minutes, and discard the supernatant.

[0050] (6) Collect the precipitate for later use to obtain the labeled colloidal gold storage solution.

[0051] 3. Preparation of the colloidal gold conjugate pad 4

[0052] (1) Appropriately dilute the labeled colloidal gold storage solution with the basic buffer.

[0053] (2) Spread the diluted buffer solution prepared above on the glass fiber pad.

[0054] (3) Vacuum-dry the glass fiber pad.

[0055] (4) Cut the dried glass fiber pad to the specified size.

[0056] 4. Coating of the immunochromatographic membrane

[0057] (1) Dilute vitamin D-BSA with the buffer to an appropriate concentration for later use.

[0058] (2) Dilute the goat anti-mouse antibody to an appropriate concentration for later use.

[0059] (3) Use a membrane spraying machine to spray the above solutions on the nitrocellulose membrane to form the quality control C line 8 and the test T line 7, and dry for later use.

[0060] 5. Assembly of the vitamin D test strip 1

[0061] (1) Paste the prepared immunochromatographic membrane, colloidal gold conjugate pad 4, sample filter paper 3, and absorbent paper 6 together in an orderly manner through the PVC plate 2.

[0062] (2) Use a strip cutting machine to cut into strips of a specific width to form the vitamin D test strip 1, and place it in a plastic shell.

[0063] (3) Add a desiccant to the plastic shell containing the vitamin D test strip 1 and seal it in an aluminum foil bag for storage.

[0064] Example 2

[0065] This example provides a detection method when the kit described in Example 1 is used for semi-quantitative detection of the vitamin D concentration in human urine, including the following steps:

[0066] Add the urine sample to be tested to the filter paper 3 on the vitamin D test strip 1. After immunochromatographic reaction, observe the color development at the test T line 7 and the quality control C line 8 respectively, and take the color development at the quality control C line 8 as a reference. According to the comparison of the color depth between the color development at the test T line 7 and the color development at the quality control C line 8, obtain the semi-quantitative test result of the vitamin D content in the urine sample to be tested.

[0067] As Figure 2 shown, the judgment criteria for the semi-quantitative test result of the vitamin D content in the urine sample to be tested are as follows:

[0068] If brown-red color development bands appear on both the test T line 7 and the quality control C line 8, and the band color at the test T line 7 is darker than the band color at the quality control C line 8, it is determined that the vitamin D concentration in the urine sample to be tested is lower than the normal range, and the test result of the kit is negative;

[0069] If brown-red color development bands appear on both the test T line 7 and the quality control C line 8, and there is a color change at the test T line 7 and the band color is not darker than the band color at the quality control C line 8, it is determined that the vitamin D concentration in the urine sample to be tested is within the normal range, and the test result of the kit is negative-positive;

[0070] If the test T line 7 does not show color and only the quality control C line 8 shows a brown-red color development band, it is determined that the vitamin D concentration in the urine sample to be tested is higher than the normal range, and the test result of the kit is positive;

[0071] If the quality control C line 8 does not show color, regardless of whether the test T line 7 shows color or not, the result is determined to be invalid.

[0072] Moreover, on the same vitamin D test strip 1, relative to the band color at the quality control C line 8, the darker the band color at the test T line 7, the lower the judged vitamin D concentration. That is, by comparing the band color at the test T line 7 with the band color at the quality control C line 8, the vitamin D concentration can be further judged.

[0073] As Figure 3 shown, the judgment of the test results of each kit is as follows: The left two kits show that the T line color concentration is darker than the C line color concentration, so the test result of the kit is negative, indicating that the vitamin D content in the sample is lacking. The middle two kits show that the T line color concentration is the same as the C line color concentration, so the test result of the kit is negative-positive, indicating that the vitamin D content in the sample is normal. The right two kits show that the T line color concentration is lighter than the C line color concentration, so the test result of the kit is positive, indicating that the vitamin D content in the sample is excessive.

[0074] The working principle of the detection method is as follows:

[0075] This application combines antibody labeling technology, antigen-antibody specific binding reaction, and immunochromatography technology to achieve semi-quantitative detection of the concentration of vitamin D in urine.

[0076] The antibody labeling technology refers to labeling antibodies with nanoparticles such as colloidal gold, latex particles, and fluorescent particles as tracer markers. After these tracer markers bind to the target antigen, detection is carried out through color development or fluorescence signals. In this embodiment, colloidal gold is used as the label, and the 1,25-dihydroxyvitamin D antibody is labeled on the colloidal gold for binding to 1,25-dihydroxyvitamin D.

[0077] The test strip 1 in this application uses a nitrocellulose membrane 5. Utilizing the porous structure of the nitrocellulose membrane 5, which can guide the fluid to move in a specific direction and has the property of adsorbing and fixing macromolecular substances such as proteins, after adsorbing the conjugate formed by coupling 1,25-dihydroxyvitamin D with the carrier protein BSA onto the nitrocellulose membrane 5, the nitrocellulose membrane 5 is used as a solid phase for the antigen-antibody specific binding reaction.

[0078] Meanwhile, this application also combines immunochromatography technology, using capillary action to promote the movement of the sample on the nitrocellulose membrane 5, thereby achieving the detection of the target substance through the chromatography principle. Specifically, after the 1,25-dihydroxyvitamin D in the urine sample collected in this application binds to the 1,25-dihydroxyvitamin D antibody labeled with colloidal gold, during the chromatography process, when passing through the test T line 7, it specifically binds to the conjugate formed by coupling 1,25-dihydroxyvitamin D with the carrier protein BSA fixed on the nitrocellulose membrane 5, forming a visible color reaction at the test T line 7, showing a brownish-red color band. The quality control C line 8 is used to verify the reliability of the kit in this application and the correctness of the operation during the detection process. In this embodiment, on the quality control C line 8, a goat anti-mouse antibody known for binding the colloidal gold-labeled antigen-antibody complex and the free colloidal gold-labeled vitamin D antibody from the colloidal gold conjugate pad 4 is added. And when the urine sample is correctly operated, the sample addition amount is sufficient, and all components of the kit work properly on the vitamin D test strip 1, the goat anti-mouse antibody binds to the colloidal gold-labeled 1,25-dihydroxyvitamin D antibody or complex of the standard reference, and a corresponding color signal will appear on the quality control C line 8 to indicate whether the reaction system of the kit is working properly, specifically manifested as a brownish-red color band appearing at the quality control C line 8. At this time, it indicates that the kit is working properly. If the quality control C line 8 does not show color, regardless of whether the test T line 7 shows color or not, it indicates that there may be improper operation during the detection process or the vitamin D test strip 1 in the current kit has failed, and re-detection is required.

[0079] It should be further noted here that a conventional colloidal gold detection kit can only judge the presence or absence of the object to be detected in a sample by observing whether the test T line 7 and the quality control C line 8 show color. In other words, the judgment criteria for the test results of a conventional colloidal gold detection kit are as follows: when only the quality control C line 8 shows color, the test result is judged to be negative; when both the test T line 7 and the quality control C line 8 show color, the test result is judged to be positive; when the quality control C line 8 does not show color, the test result is judged to be invalid. For the kit of the present application, by comparing the color depths of the test T line 7 and the quality control C line 8, the concentration of vitamin D in the sample can be semi-quantitatively judged more intuitively: when the color of the test T line 7 is darker than that of the quality control C line 8, it indicates that the concentration of vitamin D in the urine sample is lower than the lowest detection limit; when the test T line 7 shows color and is not darker than the quality control C line 8, it indicates that the concentration of vitamin D in the sample falls within the normal range of the detection limit. In this embodiment, the normal range of the detection limit is 30-80 ng / ml, that is, the normal range of the vitamin D content in human urine; when the test T line 7 shows color and is not darker than the quality control C line 8, it indicates that the concentration of vitamin D in the sample is higher than the highest detection limit; when the quality control C line 8 does not show color, the test result is judged to be invalid; at the same time, in the present application, by comparing the color depths of the test T line 7 and the quality control C line 8, the concentration of vitamin D can be further judged: the darker the color of the test T line 7, the lower the concentration of vitamin D; the lighter the color of the test T line 7, the higher the concentration of vitamin D. This semi-quantitative detection method can provide a preliminary diagnosis result, which is convenient for screening vitamin D deficiency or excess. To sum up, the color development of the quality control C line 8 in the present application can not only verify the correctness of the detection process and ensure the reliability of the test results, but also the color of the quality control C line 8 can be used as a reference benchmark for comparing the color depths of the T line color development, so as to ensure that even if there are slight differences in the coating concentration in different production batches, resulting in different color depths of the quality control C line 8 and the test T line 7, the color reference benchmarks of the same kit are consistent.

[0080] Example 3

[0081] This embodiment provides sensitivity and specificity tests for the kit described in Example 1.

[0082] 1. Sensitivity experiment:

[0083] Vitamin D was configured into different concentrations with physiological saline, which were 80 ng / ml, 30 ng / ml, and 0 ng / ml respectively, and then tested according to the detection method described in Example 2, and each concentration was repeated twice. The results are as Figure 3 shown: Each group of two kits is a group. From right to left, the concentrations of each group are 80 ng / ml, 30 ng / ml, and 0 ng / ml respectively. Among them, 0 ng / ml is physiological saline, indicating that the sensitivity of this kit is good.

[0084] 2. Specificity experiment: Vitamin B2, vitamin C, and vitamin E were used for testing according to the detection method described in Example 2. The results showed negative results, that is, the color of the band at the T line 7 was darker than the color of the band at the control C line 8, indicating good specificity of this kit.

[0085] Example 4

[0086] This example provides the quality inspection of the kit described in Example 1.

[0087] 1. Minimum detection limit test and maximum detection limit test

[0088] Method: Normal saline was used for spiking experiments, and the spiking levels were 0 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, and 80 ng / ml respectively.

[0089] Results showed that: 0 ng / ml and 20 ng / ml were negative, the color of the T line detected was darker than the color of the C line; 30 ng / ml was negative-positive, the color of the T line detected was basically the same as the color of the C line; 40 ng / ml and 80 ng / ml were positive, the color of the T line detected was lighter than the color of the C line, and at 80 ng / ml, the color of the T line detected was close to colorless. This result indicates that: the minimum detection limit of this vitamin D detection reagent strip 1 is 30 ng / ml, and the maximum detection limit is 80 ng / ml.

[0090] 2. Negative sample compliance rate

[0091] Twenty negative urine samples with known vitamin D concentrations ranging from 5 ng / ml to 25 ng / ml were obtained by mass spectrometry and tested with this vitamin D detection reagent strip 1. The results showed a compliance rate of 100%.

[0092] 3. Positive sample compliance rate

[0093] Thirty positive urine samples with known vitamin D concentrations ranging from 35 ng / ml to 80 ng / ml were obtained by mass spectrometry and tested with this test strip. All the results were positive, and the compliance rate was 100%.

[0094] 4. Parallelism detection

[0095] Normal saline was taken, spiked with a concentration of 40 ng / ml, and tested 20 times repeatedly. The results were all positive, indicating good repeatability.

[0096] The above detection results indicate that the detection results of the kit of the present invention are accurate and reliable.

[0097] Example 5

[0098] This example provides an investigation of the stability of the kit described in Example 1.

[0099] After the vitamin D test strip 1 prepared in Example 1 of the present invention was placed at 4°C and 37°C for 26 days respectively, a comparative test was then carried out with a 40 ng standard product. The test results showed no obvious difference. According to the conventional conversion method in the art (one day at 37°C is equivalent to one month at 4°C), therefore, the above results indicate that the test strip can be stably stored at 4 - 30°C for 2 years.

[0100] The present invention has been disclosed in the above preferred embodiments, but it is not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation are within the protection scope of the present invention.

Claims

1. A kit for semi-quantitative detection of vitamin D content based on urine, characterized in that: The invention comprises a vitamin D detection reagent strip (1), wherein the vitamin D detection reagent strip (1) comprises a filter paper (3), a colloidal gold binding pad (4), a nitrocellulose membrane (5) and a water-absorbing paper (6) which are sequentially arranged on a rubber plate (2), wherein: The colloidal gold binding pad (4) is coated with colloidal gold-labeled vitamin D antibodies, and the colloidal gold-labeled vitamin D antibodies are used to specifically bind to vitamin D in the urine sample when the urine sample flows through, and form a colloidal gold-labeled antigen-antibody complex; The nitrocellulose membrane (5) is provided with a detection T line (7) and a quality control C line (8) in sequence along the chromatography direction; The detection T line (7) is coated with a vitamin D-BSA conjugate, and the vitamin D-BSA conjugate is used to capture the colloidal gold-labeled vitamin D antibody; The quality control C line (8) is coated with goat anti-mouse antibodies, which are used to bind to the colloidal gold-labeled antigen-antibody complex and the free colloidal gold-labeled vitamin D antibody from the colloidal gold binding pad (4).

2. A kit for semi-quantitative detection of vitamin D content based on urine according to claim 1, characterized in that: The amount of the colloidal gold-labeled vitamin D antibody is 5 mg to 20 mg of vitamin D antibody per liter of colloidal gold solution; The coating concentration of the vitamin D-BSA conjugate at the detection T line (7) is 0.5-2 mg / ml; The coating concentration of the goat anti-mouse antibody at the quality control line C (8) is 0.5-1.5 mg / ml.

3. A kit for semi-quantitative detection of vitamin D content based on urine according to claim 2, characterized in that: The colloidal gold-labeled vitamin D antibody adopts a 1,25-dihydroxyvitamin D antibody colloidal gold conjugate; The vitamin D-BSA conjugate is a carrier conjugate formed by coupling 1,25-dihydroxyvitamin D with a carrier protein BSA.

4. Use of the kit according to any one of claims 1 to 3 for semi-quantitative detection of vitamin D concentration in human urine.

5. A method for semi-quantitative detection of vitamin D content based on urine, characterized in that: Using the kit according to any one of claims 1 to 3 to detect the urine sample to be tested, comprising the following steps: The urine sample to be tested is added to the filter paper (3) on the vitamin D detection reagent strip (1). After the immunochromatographic reaction, the color development at the detection T line (7) and the quality control C line (8) are observed respectively, and the color development at the quality control C line (8) is used as a reference. According to the color depth comparison between the color development at the detection T line (7) and the color development at the quality control C line (8), a semi-quantitative detection result of the vitamin D content in the urine sample to be tested is obtained.

6. The method for semi-quantitative detection of vitamin D content based on urine according to claim 5, characterized in that: The criteria for determining the semi-quantitative test results of the vitamin D content in the urine sample to be tested are as follows: If brown-red bands appear on both the test T line (7) and the quality control C line (8), and the color of the band at the test T line (7) is darker than the color of the band at the quality control C line (8), it is determined that the vitamin D concentration in the urine sample to be tested is lower than the normal range; If brown-red bands appear on both the test T line (7) and the quality control C line (8), and there is a color change at the test T line (7) and the color of the band is not darker than the color of the band at the quality control C line (8), then it is determined that the vitamin D concentration in the urine sample to be tested is within the normal range; If the test T line (7) does not show color and only the quality control C line (8) shows a brown-red band, it is determined that the vitamin D concentration in the urine sample to be tested is higher than the normal range; If the quality control C line (8) does not show color, the result will be considered invalid regardless of whether the test T line (7) shows color.

7. The method for semi-quantitative detection of vitamin D content based on urine according to claim 6, characterized in that: The darker the color of the strip at the detection T line (7), the lower the vitamin D concentration is.

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