A 25-hydroxyvitamin D detection kit

Abstract

The invention belongs to the field of chemical immunoassay, and particularly relates to a 25-hydroxyvitamin D detection kit. The 25-hydroxyvitamin D detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 contains a CAPS-glycine-EDTA buffer, a composite sensitizer, a composite surfactant, and 1,3-dihydroxymethyl-5,5-dimethylhydantoin; the reagent R2 contains a CAPS-glycine-EDTA buffer, a composite latex stabilizer, a latex particle solution coated with a mouse anti-human 25OH vitamin D monoclonal antibody, a composite surfactant, and 1,3-dihydroxymethyl-5,5-dimethylhydantoin; and the novel composite sensitizer, the composite surfactant, and the composite latex stabilizer are used to optimize the content of each component and scientifically proportion the components, thereby significantly improving the stability and linear range of the kit, and significantly enhancing the sensitivity and accuracy of the kit.

Description

Technical Field

[0001] The invention belongs to the field of chemical immunoassay, and specifically relates to a 25-hydroxyvitamin D detection kit. Background Art

[0002] 25-Hydroxyvitamin D (25-OH VD) is the main form of vitamin D in the body. Vitamin D is a steroid derivative, a fat-soluble vitamin, and a cyclopentane polyhydrogen phenanthrene compound. Vitamin D not only affects calcium and phosphorus metabolism, but also has a wide range of physiological effects. It is an indispensable substance for maintaining human health, cell growth and development, and is closely related to many diseases. There are two forms of vitamin D in the human body, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D is converted into 25-hydroxyvitamin D (25-OHVD) through hydroxylation in the liver, and then converted into active 1,25-hydroxyvitamin D in the kidneys. The concentration of 25-hydroxyvitamin D is considered to be the most reliable indicator for determining the overall vitamin D status, and can therefore be used to determine whether a patient has vitamin D deficiency.

[0003] In clinical practice, the main methods for detecting 25-hydroxyvitamin D include liquid chromatography-tandem mass spectrometry (LC-MS / MS) and immunoassay.

[0004] Among them, the LC-MS / MS method has expensive instruments, strict installation conditions, and requires a larger site to meet the requirements; sample pre-treatment is complicated, and the detection throughput is relatively low, making it difficult to adapt to the needs of large quantities of clinical samples; manual operations are relatively complicated, and operators need to undergo relatively professional training, making it difficult to promote; although recognized as the gold standard for detecting 25-OH VD, the LC-MS / MS method is still susceptible to cross-influence of isomers such as 3-epi-25OH D3 and 3-epi-25OH D2, and is particularly prone to false positives in infants and young children; in addition, due to the non-standardization of chromatographic and mass spectrometry conditions, different laboratories have different processing methods and chromatographic conditions, which will result in different clinical evaluations of the same individual.

[0005] The test kit prepared by latex immunoturbidimetry in the immunoassay can be applied to a fully automatic biochemical analyzer to achieve automated operation. Serum samples can be tested without further pretreatment steps, and the result is available in a short time, with the concentration generally being able to be measured within 10 minutes.

[0006] However, the latex immunoturbidimetry method involves many components involved in the reaction, and they are biologically active substances, such as substrates, enzymes, antibodies, etc. Since it involves the interaction between antibodies and enzymes, the screening of key raw materials such as antibodies and enzymes is crucial to the establishment of the method. On the one hand, the proportion coordination between the components has a great influence on the linearity and sensitivity of the method. Therefore, the raw material screening and formula development of this method are very difficult. At the same time, due to the high requirements of related enzymes and antibodies, the raw materials themselves are not easy to obtain, and the difficulty of inspection at the production end is also correspondingly increased, which also increases the research and development and manufacturing costs of this method.

[0007] Compared with LC-MS / MS, immunoassays are prone to the following problems: ① Matrix effects and cross-reactions can easily lead to high test results; ② The test results are for total 25-OH VD, which can easily underestimate the level of 25-OH VD2, resulting in low total 25-OH VD test results; ③ It is impossible to distinguish between 25-OH VD2 and 25-OH VD3, resulting in poor specificity of immunoassays. Even LC-MSMS faces the problem of completely separating 25-OH VD from binding proteins.

[0008] In short, no immunoassay kit currently available can match the LC-MS / MS25-OHVD detection kit in terms of detection accuracy.

[0009] Therefore, it is an urgent problem to find an optimized latex-enhanced immunoturbidimetry to prepare a 25-hydroxyvitamin D detection kit, while giving full play to the advantages of latex-enhanced immunoturbidimetry and enhancing its detection accuracy.

[0010] References: [1]. Pan Xiaofang, Wu Jingbiao, Huang Yanhong, et al. Detection methods and research progress of 25-hydroxyvitamin D in my country. Journal of Molecular Diagnosis and Therapy, 2020, 12(12): 5. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a 25-hydroxyvitamin D detection kit, which adopts an optimized latex enhanced immunoturbidimetry, and by improving the preparation process of the antibody-coated latex particle solution, the analytical sensitivity and anti-interference ability of the reagent are effectively enhanced; in addition, by optimizing the buffer system, adding a composite surfactant, a composite sensitizer and a composite latex stabilizer, scientifically proportioning, optimizing the reaction system, and greatly improving the stability of the reagent. The 25-hydroxyvitamin D kit of the present invention is easy to use, can realize automated operation, and is convenient for hospitals at all levels to promote and use.

[0012] Specifically, the technical solution of the present invention is as follows:

[0013] A 25-hydroxyvitamin D detection kit, comprising reagent R1 and reagent R2;

[0014] The reagent R1 includes the following components:

[0015]

[0016] The reagent R2 includes the following components:

[0017]

[0018] The components of the mouse anti-human 25-hydroxyvitamin D monoclonal antibody coated latex particle solution include: a composite blocking agent, latex particles, and mouse anti-human 25-hydroxyvitamin D monoclonal antibody;

[0019] The components of the composite surfactant include monooleate polyethylene glycol 400 phosphate, isopropylene phosphoric acid copolymer, isomeric tridecyl alcohol ether CLD-1303 and acylated amino polyether silicone oil;

[0020] The components of the composite latex stabilizer include sodium monostearate glyceryl sulfate and brown algae polysaccharide;

[0021] The preservative is 1,3-dihydroxymethyl-5,5-dimethylhydantoin.

[0022] Preferably, the volume ratio of the reagent R1 to the reagent R2 is 4:1.

[0023] Preferably, in reagent R1, the buffer A is CAPS-glycine-EDTA buffer, and the pH value is 6.53 at 25°C; and in reagent R2, the buffer B is CAPS-glycine-EDTA buffer, and the pH value is 7.22 at 25°C.

[0024] Furthermore, the preparation method of the CAPS-glycine-EDTA buffer is as follows: weigh 5.1-12.2 g of 3-(cyclohexylamine)-1-propanesulfonic acid, completely dissolve it in 900 mL of deionized water, add 1.5 g of glycine, stir to dissolve, then add disodium ethylenediaminetetraacetic acid dihydrate, adjust the pH to 6.53 or 7.22 with NaOH or HCl, and finally dilute to 1 L with deionized water.

[0025] Furthermore, the preparation method of the composite sensitizer is: weigh polyethylene glycol 10000 and add it to water, stir and dissolve it at 30-60°C, cool it, add ethylene glycol and polyethylene glycol 400, stir and completely dissolve it; then add choline chloride, calcium chloride and potassium chloride, stir until completely dissolved, and obtain the composite sensitizer.

[0026] Furthermore, the preparation method of the composite surfactant is as follows: monooleate polyethylene glycol 400 phosphate is added to deionized water, stirred at 30-45° C., isopropylene phosphate copolymer, isomeric tridecyl alcohol ether CLD-1303 and acylated amino polyether silicone oil are added in sequence, stirred at 30-45° C. for 8-24 hours, adjusted to pH 7.22 with NaOH or HCl, and cooled to 20-25° C. to obtain a composite surfactant.

[0027] Furthermore, the preparation method of the composite latex stabilizer is: weigh sodium monostearate glycerol sulfate, add it to deionized water, stir to dissolve, add brown algae polysaccharide and maltodextrin, stir at 37°C until completely dissolved, cool to 20-25°C, add lithium chloride, stir at 20-25°C until completely dissolved, and obtain the composite latex stabilizer.

[0028] Furthermore, the preparation steps of the mouse anti-human 25-hydroxyvitamin D monoclonal antibody coated latex particle solution are as follows:

[0029] (1) Take 3 mL of 122 nm carboxyl latex particles and 1 mL of 320 nm carboxyl latex particles, add 21 mL of 5 mmol / L CAPS-glycine-EDTA buffer, pH = 7.22 at 25°C, and then stir and mix at 30°C for 45 min;

[0030] (2) Add 5-10 mg of mouse anti-human 25-hydroxyvitamin D monoclonal antibody and stir at 35°C for 60 min; cool to 20°C, add 3-5.5 mg of EDC, and shake at 200 r / min at 20°C for 5 h;

[0031] (3) Add 5 mL of a composite blocking agent, wherein the main components of the composite blocking agent are: 3.5 g / L BSA, 1 g / L ethylenediamine, 5 g / L PEG200, and 5 g / L NaCl; oscillate at 200 r / min at 30°C for 12 h; the resulting solution is supplemented to 50 mL with a CAPS-glycine-EDTA buffer having a pH of 7.22 and a concentration of 5 mmol / L at 25°C. The final solution obtained is a latex particle solution coated with mouse anti-human 25-hydroxyvitamin D monoclonal antibody.

[0032] The dosage of the components of the present invention is not limited to the specific values ​​described above, and the ratios between the corresponding components are also within the protection scope of the present invention.

[0033] The 25-hydroxyvitamin D detection kit of the present invention adopts a fully automatic biochemical analyzer with a dual reagent function, preferably a Hitachi 7180 fully automatic analyzer, the main detection wavelength is 600nm, and the detection method is an endpoint method. Those skilled in the art believe that feasible fully automatic biochemical analyzers are within the protection scope of the present invention.

[0034] The beneficial effects of the present invention are:

[0035] 1. The present invention adopts optimized latex enhanced immunoturbidimetry, a novel coupling buffer system (i.e., CAPS-glycine-EDTA buffer) and a composite blocking agent, which significantly improves the coupling efficiency. The whole process does not require centrifugation or ultrasound, which is simple and convenient. In addition, 122 nm and 320 nm carboxyl microspheres are used in combination. Due to the increase in the density of latex particles in the buffer system, the reaction sensitivity and linear range of the reagent are greatly enhanced.

[0036] 2. The use of composite sensitizers can promote latex dispersion and significantly improve the analytical sensitivity and linear range of the reagents;

[0037] 3. The present invention adopts a new type of composite surfactant, which can better maintain the stability of antibodies, keep the latex in a dispersed state, and prevent the system from becoming turbid compared to traditional surfactants. The accuracy, sensitivity and stability experiments of reagent detection have proved that this composite surfactant significantly enhances the analytical sensitivity, stability and anti-interference ability of the reagent;

[0038] 4. The composite latex stabilizer is used to optimize the content of each component and scientifically proportion it to promote and maintain the stability of the antibody, keep the latex reagent dispersed, prevent microspheres from agglomerating, and significantly enhance the stability of the reagent;

[0039] 5. The reagent is easy and fast to operate and is suitable for automated analysis. It is a more stable, sensitive, and wide linear range 25-hydroxyvitamin D reagent. The reagent has good accuracy and stability, strong anti-interference ability, and is easy to use, which can fully meet clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0041] Figure 1 It is a line graph comparing the reagent stability of Examples 1-3 and Comparative Example 1. DETAILED DESCRIPTION

[0042] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0043] In a preferred embodiment of the present invention, the buffer in reagent R1 is a CAPS-glycine-EDTA buffer at 25°C and a pH of 6.53; the buffer in reagent R2 is a CAPS-glycine-EDTA buffer at 25°C and a pH of 7.22. The preparation method is as follows: weigh 5.1 to 12.2 g of 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS), dissolve it completely with 900 mL of deionized water, then add 1.5 g of glycine, stir to dissolve, then add EDTA disodium salt dihydrate, adjust the pH to 6.53 or 7.22 with an appropriate amount of sodium hydroxide or HCl, and finally dilute to 1 L with deionized water.

[0044] In a preferred embodiment of the present invention, the preparation method of the composite sensitizer is as follows: weigh 20g of polyethylene glycol 10000 and add it to 400mL of water, stir and dissolve it at 45°C for 1h, cool it to 25°C, add 50mL of ethylene glycol and 50mL of polyethylene glycol 400, stir at 25°C for 30min to completely dissolve it; then add 5g of choline chloride, 5g of calcium chloride, and 15g of potassium chloride, stir at 25°C for 2h, and set aside after complete dissolution.

[0045] In a preferred embodiment of the present invention, the preparation method of the composite surfactant is as follows: 10 g of monooleate polyethylene glycol 400 phosphate is added to 200 mL of deionized water, stirred at 30° C. for 1 h, and then 5.5 g of isopropylene phosphate copolymer, 2 g of isomeric tridecyl alcohol ether CLD-1303 and 1 g of acylated amino polyether silicone oil are added in sequence, stirred at 35° C. for 12 h, the pH is adjusted to 7.22 with HCl or NaOH, and cooled to room temperature (25° C.) for standby use.

[0046] In a preferred embodiment of the present invention, the preparation method of the composite latex stabilizer is: weigh 5g of sodium monostearate glycerol sulfate, add it to 250mL of water, stir and dissolve it at 25°C for 30min, add 10g of brown algae polysaccharide and 5g of maltodextrin, stir at 37°C for 1h to completely dissolve it; after cooling to 25°C, add 5g of lithium chloride, stir at 25°C for 20min, and set aside after it is completely dissolved.

[0047] In a preferred embodiment of the present invention, the preparation method of the latex particle solution coated with mouse anti-human 25-hydroxyvitamin D monoclonal antibody is as follows: 3 mL of 122 nm carboxyl latex particles and 1 mL of 320 nm carboxyl latex particles are taken, 21 mL of 5 mmoL / L CAPS-glycine-EDTA buffer (pH 7.22, 25° C.) is added, and the mixture is stirred at 30° C. for 45 min; then 5 mg of mouse anti-human 25-hydroxyvitamin D monoclonal antibody is added, the temperature is adjusted to 35° C., and the mixture is stirred for 60 min. After the temperature is adjusted to 20° C., 3 mg of EDC is added, and the mixture is shaken at 20° C. for 5 h (speed 200 r / min); then 5 mL of composite blocking agent (main components: 3.5 g / L BSA, 1 g / L ethylenediamine, 5 g / L PEG200, 5 g / L NaCl) is added, the shaking rate is adjusted to 200 r / min, the temperature is 30° C., and the mixture is shaken for 12 h. The resulting solution was supplemented to 50 mL with 5 mmol / L CAPS-glycine-EDTA buffer (pH 7.22, 25° C.), and the final solution obtained was the latex particle solution coated with mouse anti-human 25-hydroxyvitamin D monoclonal antibody.

[0048] The dosage of the components of the present invention is not limited to the specific values ​​described above, and the ratios between the corresponding components are also within the protection scope of the present invention.

[0049] Example 1

[0050] 25-hydroxyvitamin D detection kit, comprising reagent R1 and reagent R2;

[0051] The reagent R1 includes the following components:

[0052]

[0053] The reagent R2 includes the following components:

[0054]

[0055] Method of using the kit of this embodiment:

[0056] The 25-hydroxyvitamin D detection kit of this embodiment is used with a Hitachi 7180 fully automatic analyzer with a dual reagent function, and the endpoint method is used for determination. Reagent R1 and reagent R2 are placed in the corresponding reagent positions in a ratio of 4:1, and distilled water, standard products and samples are placed in the corresponding positions of the sample tray. The operation is as shown in Table 1.

[0057] Table 1 Reagent detection method table

[0058]

[0059] Calculation: 25-hydroxyvitamin D content (ng / mL) = (ΔA assay ÷ ΔA standard) × C standard.

[0060] Example 2

[0061] A 25-hydroxyvitamin D detection kit comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following components:

[0062]

[0063] The reagent R2 includes the following components:

[0064]

[0065] The use and detection methods are the same as in Example 1.

[0066] Example 3

[0067] A 25-hydroxyvitamin D detection kit comprises a reagent R1 and a reagent R2; the reagent R1 comprises the following components:

[0068]

[0069] The reagent R2 includes the following components:

[0070]

[0071] The use and detection methods are the same as in Example 1.

[0072] Comparative Example 1

[0073] This comparative example adopts a 25-hydroxyvitamin D detection kit (latex enhanced immunoturbidimetric method) commonly used in the market.

[0074] The ingredients and contents of reagent 1 are as follows:

[0075]

[0076] Wherein, “%” means mass / volume.

[0077] The ingredients of reagent 2 are as follows:

[0078] Human 25-hydroxyvitamin D antigen, carbonyldiimidazole, MES solution, 223 nm polystyrene latex particles, diaminodipropylimide;

[0079] The use and detection methods are the same as in Example 1.

[0080] Comparative Example 2

[0081] 25-hydroxyvitamin D detection kit, comprising reagent R1 and reagent R2;

[0082] The reagent R1 includes the following components:

[0083]

[0084] The reagent R2 includes the following components:

[0085]

[0086] Except for using Tween 20 as a common surfactant, the other components of this comparative example are completely consistent with those of Example 1, and the use and detection methods are the same as those of Example 1.

[0087] Comparative Example 3

[0088] 25-hydroxyvitamin D detection kit, comprising reagent R1 and reagent R2;

[0089] The reagent R1 includes the following components:

[0090]

[0091] The reagent R2 includes the following components:

[0092]

[0093] Except for using stabilizers glycerol and sucrose, the other components of this comparative example are completely consistent with those of Example 1, and the use and detection methods are the same as those of Example 1.

[0094] Accuracy comparison test

[0095] Take a high-value quality control material (target value 45.38 ng / mL) and a low-value quality control material (target value 12.55 ng / mL) with traceability, respectively, use the reagents of Example 1-3 and Comparative Example 1-3, prepare a 25-hydroxyvitamin D detection kit for control detection, each test 5 times, calculate the average value, and compare it with the target value of the quality control material. The results are shown in Table 2.

[0096] Table 2.1 High value quality control material accuracy test data table

[0097] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Maximum 45.65 45.61 45.59 50.89 48.40 48.12 Minimum 45.21 45.15 45.11 47.48 46.31 45.11 average value 45.41 45.32 45.29 49.51 47.39 47.25

[0098] Table 2.2 Low value quality control material accuracy test data table

[0099] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Maximum 12.91 12.85 12.74 11.65 11.98 12.21 Minimum 12.32 12.45 12.41 10.58 10.69 11.21 average value 12.65 12.61 12.51 10.89 11.16 11.36

[0100] It can be seen from the test results in Table 2 that, compared with Comparative Examples 1-3, the test values ​​of the reagents prepared in Example 1, Example 2, and Example 3 are closer to the target values, and the difference between the average value and the target value is within 0.5, which has a higher accuracy. This shows that the present invention optimizes the preparation method of 25-hydroxyvitamin D antibody-coated latex particles, optimizes the reagent buffer system, and adds a composite surfactant, a composite sensitizer, and a composite latex stabilizer. Through scientific proportioning, the reaction system is optimized, and the accuracy of the reagent is greatly improved.

[0101] Comparative test of reagent sensitivity

[0102] The traceable calibrator was diluted from low to high concentrations to obtain 7 samples, and the reagents of Examples 1-3 and Comparative Examples 1-3 were used to prepare 25-hydroxyvitamin D detection reagents for control detection, and the detection results were compared with the theoretical concentrations. The results are shown in Table 3.

[0103] Table 3 Sensitivity comparison test data table (ng / mL)

[0104] Theoretical concentration Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 0.5 0.55 0.51 0.53 0.15 0.34 0.43 1 1.05 0.99 1.01 0.81 0.89 0.91 10 10.11 10.01 9.98 9.01 9.35 9.45 50 50.21 49.98 50.11 50.55 50.12 50.01 100 100.6 100.63 100.47 100.42 99.87 100.31 150 151.11 152.01 150.21 144.25 146.35 149.12 350 351.25 352.11 350.12 289.36 301.16 333.21

[0105] As can be seen from the test results in Table 3, when the sample concentration is as low as 0.5ng / mL, the detection value of Comparative Examples 1-3 is 0.15-0.43ng / mL, while the reagents prepared by the formulas of Examples 1, 2 and 3 can still detect the accurate value of the sample; and compared with Comparative Examples 1-3, the accuracy of the reagents prepared by the formulas of Examples 1, 2 and 3 when detecting low-value samples (0.5-10ng / mL) close to the linear lower limit is higher. In addition, when the sample concentration is at the linear upper limit of 350ng / mL, the detection value of the comparative example is between 289.36-333.21ng / mL, and the test value is obviously low, while the reagents prepared by the formulas of Examples 1, 2 and 3 can still detect the accurate value of the sample, which shows that the reagents prepared by the formulas of Examples 1, 2 and 3 have higher analytical sensitivity, accuracy and a wider linear range. Therefore, it can be explained that the present invention effectively improves the linearity and analytical sensitivity of the reagent by optimizing the preparation method of 25-hydroxyvitamin D antibody-coated latex particles; in addition, the reagent buffer system is optimized, and a composite surfactant, a composite sensitizer and a composite latex stabilizer are added, and the reaction system is optimized through scientific proportioning, which further greatly improves the accuracy and analytical sensitivity of the reagent. In summary, through the above measures, the reaction system is optimized, and the analytical sensitivity, accuracy and linear range of the reagent are greatly improved.

[0106] Comparative test of reagent stability

[0107] The reagents in Examples 1-3 and Comparative Examples 1-3 were evenly divided into 17 groups, each with a reagent volume of 20 mL for reagent R1 and 5 mL for reagent R2. They were placed in a refrigerator at 2-8°C. On the same day of each month, a group of reagents was taken out to test the 25-hydroxyvitamin D quality control product (target value was 45.38 ng / mL). The test results were as follows: Figure 1 As shown, the reagents of Examples 1-3 are more stable than the comparative example under storage conditions of 2-8°C.

[0108] It can be seen from the test results that when stored for 17 months, the detection values ​​of comparative examples 1-3 are 33.77-41.1, which differ from the target value by 4.28-11.61, and the detection value shows an obvious decreasing trend with the extension of storage time; while the detection values ​​of the reagents prepared in the formulas of Examples 1, 2 and 3 are 44.65-46.82, which differ from the target value by 0.73-1.44; This shows that the reagents prepared in the formulas of Examples 1, 2 and 3 have higher stability, and the validity period can reach 1.5 years, which is much higher than the common similar products in the market. Therefore, it can be explained that the present invention optimizes the preparation method of 25-hydroxyvitamin D antibody-coated latex particles, optimizes the buffer system, and adds composite surfactants, composite sensitizers and composite latex stabilizers, optimizes the reaction system through scientific proportioning, and greatly improves the stability of the reagent.

[0109] In summary, through verification, the reagent provided by the present invention has high sensitivity, wide linear range and high accuracy compared to the comparison ratio, and is a more stable and good 25-hydroxyvitamin D detection kit.

Claims

1. A 25-hydroxyvitamin D detection kit, comprising reagent R1 and reagent R2; The reagent R1 includes the following components: The reagent R2 includes the following components: The components of the latex particle solution coated with mouse anti-human 25-hydroxyvitamin D monoclonal antibody include: Composite blocking agent, latex particles, mouse anti-human 25-hydroxyvitamin D monoclonal antibody; The main components of the composite sealant are: BSA, ethylenediamine, PEG200 and NaCl; wherein, by mass ratio, BSA: ethylenediamine: PEG20: NaCl = 3.5:1:5:5; The components of the composite surfactant include monooleate polyethylene glycol 400 phosphate, isopropylene phosphoric acid copolymer, isomeric tridecyl alcohol ether CLD-1303 and acylated amino polyether silicone oil; The components of the composite latex stabilizer include sodium monostearate glyceryl sulfate and brown algae polysaccharide; The preservative is 1,3-dihydroxymethyl-5,5-dimethylhydantoin; The volume ratio of the reagent R1 to the reagent R2 is 4:1; In reagent R1, the buffer A is a CAPS-glycine-EDTA buffer, and its pH value is 6.53 at 25°C; in reagent R2, the buffer B is a CAPS-glycine-EDTA buffer, and its pH value is 7.22 at 25°C.

2. A 25-hydroxyvitamin D detection kit as claimed in claim 1, characterized in that: The preparation method of the CAPS-glycine-EDTA buffer is as follows: weigh 5.1-12.2 g of 3-(cyclohexylamine)-1-propanesulfonic acid, completely dissolve it with 900 mL of deionized water, add 1.5 g of glycine, stir and dissolve it, then add disodium ethylenediaminetetraacetic acid dihydrate, adjust the pH to 6.53 or 7.22 with NaOH or HCl, and finally dilute to 1 L with deionized water.

3. A 25-hydroxyvitamin D detection kit as claimed in claim 1, characterized in that: The preparation method of the composite sensitizer is as follows: weighing polyethylene glycol 10000 and adding it into water, stirring and dissolving it at 30-60° C., cooling it, adding ethylene glycol and polyethylene glycol 400, stirring and dissolving them completely; and then adding choline chloride, calcium chloride and potassium chloride, stirring and dissolving them completely to obtain the composite sensitizer.

4. A 25-hydroxyvitamin D detection kit as claimed in claim 1, characterized in that: The preparation method of the composite surfactant is as follows: monooleate polyethylene glycol 400 phosphate is added to deionized water, stirred at 30-45° C., isopropylene phosphoric acid copolymer, isomeric tridecyl alcohol ether CLD-1303 and acylated amino polyether silicone oil are added in sequence, stirred at 30-45° C. for 8-24 hours, adjusted to pH 7.22 with NaOH or HCl, and cooled to 20-25° C. to obtain the composite surfactant.

5. A 25-hydroxyvitamin D detection kit as claimed in claim 1, characterized in that: The preparation method of the composite latex stabilizer is as follows: weighing sodium monostearate glyceryl sulfate, adding it into deionized water, stirring to dissolve, adding brown algae polysaccharide and maltodextrin, stirring at 37° C. until completely dissolved, cooling to 20-25° C., adding lithium chloride, stirring at 20-25° C. until completely dissolved, and obtaining the composite latex stabilizer.

6. A 25-hydroxyvitamin D detection kit as claimed in claim 1, characterized in that: The preparation steps of the mouse anti-human 25-hydroxyvitamin D monoclonal antibody coated latex particle solution are as follows: (1) Take 122 nm carboxyl latex particles and 320 nm carboxyl latex particles, add 5 mmol / L CAPS-glycine-EDTA buffer at 25°C, pH = 7.22, and then stir to mix; (2) Add mouse anti-human 25-hydroxyvitamin D monoclonal antibody and stir to mix; cool to 20- 25°C, add EDC, shake at 20-25°C; (3) adding a composite sealing agent, wherein the main components of the composite sealing agent are: 3.5 g / L BSA, 1 g / L ethylenediamine, 5 g / L PEG200, and 5 g / L NaCl; oscillating at 30°C; and making up the resulting solution with a CAPS-glycine-EDTA buffer having a pH of 7.22 and a concentration of 5 mmol / L at 25°C. The final solution obtained is a latex particle solution coated with mouse anti-human 25-hydroxyvitamin D monoclonal antibody.

7. A 25-hydroxyvitamin D detection kit according to any one of claims 1 to 6, characterized in that: The 25-hydroxyvitamin D detection kit was detected by a fully automatic biochemical analyzer, the main detection wavelength was 600nm, and the detection method was the endpoint method.

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