Fluorescence immunoassay kit for detecting canine vitamin D3 and preparation method thereof

Abstract

The invention relates to the technical field of vitamin D detection, and discloses a fluorescence immunoassay kit for detecting canine vitamin D3 and a preparation method of the fluorescence immunoassay kit. The kit comprises a dissociation agent and a reagent card, the dissociation agent is non-toxic and adapts to a fluorescence immunochromatography method, and adverse effects on an NC membrane and microsphere aggregation can be avoided. A test strip in the reagent card is sequentially provided with a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, the nitrocellulose membrane is provided with a detection area T line (coated with a 25-hydroxyvitamin D3 murine monoclonal antibody) and a quality control area C line (coated with a rabbit IgG antibody), and the combination pad is sprayed with a corresponding antibody marked by fluorescent microspheres. The kit and the test strip are simple, convenient and rapid to operate, low in sample consumption, low in cost, high in sensitivity, good in repeatability, capable of accurately detecting the canine vitamin D3, and suitable for basic level and field detection.

Description

Technical Field

[0001] The present invention relates to the technical field of vitamin D detection, in particular to a fluorescent immunoassay kit for detecting canine vitamin D3 and a preparation method thereof. Background Art

[0002] In human health research, serum 25-hydroxyvitamin D3 levels are often considered an important indicator of vitamin D storage status and are closely correlated with clinical symptoms of vitamin D deficiency. Interestingly, studies have found that canine vitamin D metabolism is similar to that of humans, so serum 25-hydroxyvitamin D3 levels can also be used to assess canine vitamin D status. Vitamin D deficiency can significantly impact canine health, with puppies susceptible to rickets and adult dogs prone to osteomalacia.

[0003] Currently, common methods for determining 25-hydroxyvitamin D3 include chromatography and immunology. Although chromatography can accurately measure the content, it has many disadvantages, such as large sample requirements, complex and time-consuming pre-treatment, high instrument operation difficulty, strict operator requirements, and expensive instruments. Among immunological methods, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLI), and electrochemiluminescence immunoassay (ECLI) are commonly used for vitamin D3 detection. Although ELISA, CLI, and ECL are widely used in the detection field, they have certain limitations. They often require professionals to operate specific instruments, the detection steps are cumbersome and time-consuming, and the instruments are expensive, with high operating and maintenance costs. They also have relatively stringent requirements for the laboratory environment, limiting their application in complex environments such as grassroots or on-site.

[0004] During the actual testing process, since 25-hydroxyvitamin D3 in canine serum is primarily bound to 25-hydroxyvitamin D binding protein (VDBP), dissociation must be performed using a dissociating agent before testing. Commonly used vitamin D dissociating agents are often composed of organic solvents (such as DMSO), strong acids and bases, fluorides, ANS, guanidine hydrochloride, and salt ions. While these agents provide excellent dissociation, they are toxic, posing biosafety and environmental risks. While a non-toxic agent, 8-anilino-1-naphthalenesulfonic acid (ANS), has emerged that also exhibits dissociation effects, ANS is only suitable for liquid-phase assays, such as enzyme-linked immunosorbent assays, chemiluminescence, and electrochemiluminescence.

[0005] Another type of fluorescent immunochromatography in immunological testing is simple and fast to operate. It can be used by personnel with simple training and can quickly produce test results. It uses a small amount of sample and is suitable for situations where sample collection is difficult. It is relatively low-cost and does not require expensive instruments, making it economically feasible. It is also highly portable and does not require complex laboratory conditions, allowing on-site testing in a variety of complex environments. However, there is currently no fluorescent immunochromatography kit suitable for canine vitamin D3 detection, and there is a problem with the fluorescent immunochromatography method. Most dissociation agents (such as organic solvents, strong acids and bases, and fluoride-containing compounds) will have an adverse effect on the NC membrane, the carrier of the fluorescent immunochromatography. Excessive concentrations or long-term exposure will cause the cellulose component of the NC membrane to swell, changing the microstructure and affecting its adsorption and chromatographic performance for biological molecules. The hydrophobic ANS will cause chromatographic abnormalities, causing the fluorescent microspheres to aggregate on the membrane during chromatography and release unevenly, resulting in high background, poor sensitivity and repeatability, and other problems.

[0006] In response to the above-mentioned related technologies, the inventors believe that it is necessary to develop a canine test strip suitable for fluorescent immunochromatography, which can solve the problems of uniform microsphere release, good stability and high sensitivity, fill the market gap, and realize accurate detection of vitamin D3 levels in dogs. Summary of the Invention

[0007] In order to solve the technical defects of the prior art, the present application provides a fluorescent immunoassay kit for detecting canine vitamin D3 and a preparation method thereof.

[0008] In a first aspect, the present application provides a fluorescent immunoassay kit for detecting canine vitamin D3, which adopts the following technical solution: A fluorescent immunoassay kit for detecting canine vitamin D3, comprising a dissociator and a reagent card, wherein the dissociator comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, EDTA-Na2, polyvinylpyrrolidone, sodium lauryl sulfate, sodium deoxycholate, sodium sulfosuccinate dioctyl ester and dithiothreitol; the reagent card comprises a shell and a fluorescent immunochromatographic test strip for detecting 25-hydroxyvitamin D3 built into the shell; the test strip comprises a PVC bottom plate and a sample pad, a A conjugate pad, a nitrocellulose membrane and absorbent paper, wherein the nitrocellulose membrane is provided with a quality control area C line and a detection area T line spaced adjacent to each other, the detection area T line is coated with 25-hydroxyvitamin D3 mouse monoclonal antibody, and the quality control area C line is coated with rabbit IgG antibody; the conjugate pad is sprayed with an antibody solution labeled with fluorescent microspheres, and the antibody solution labeled with fluorescent microspheres specifically contains a mixture of 25-hydroxyvitamin D3 mouse monoclonal antibody labeled with fluorescent microspheres and quality control goat anti-rabbit antibody labeled with fluorescent microspheres.

[0009] By adopting the above technical solution, the dissociation agent has a unique formula, including ingredients such as 4-hydroxyethylpiperazineethanesulfonic acid, which not only has a good dissociation effect, but also improves the flow and diffusion properties of the sample on the NC membrane. Among them, sodium deoxycholate has both dissociation and blocking effects, reducing nonspecific adsorption, lowering background signals, and improving detection sensitivity. The test strip structure of the reagent card is rationally designed. The fluorescent microsphere-labeled antibody on the binding pad is combined with the T-line and C-line antibodies on the NC membrane. The fluorescent immunochromatography method is simple to operate, requires less sample, is low-cost, and can be tested on-site. It solves the adverse effects of existing dissociation agents on the NC membrane and the problem of microsphere aggregation, and realizes the accurate detection of canine vitamin D3.

[0010] Preferably, the fluorescent microspheres are XC050 fluorescent microspheres from Merck.

[0011] By adopting the above technical solution, Merck's XC050 fluorescent microspheres were selected, which have stable performance, appropriate fluorescence intensity and stable luminescence. They can effectively combine with antibodies to ensure the labeling effect, make the detection signal more stable and reliable, and improve the detection accuracy of the test kit.

[0012] Preferably, the dissociation agent includes 0.05M 4-hydroxyethylpiperazineethanesulfonic acid buffer (pH 7.4), 0.3M sodium chloride, 0.005M EDTA-Na2, 1wt% polyvinylpyrrolidone, 1wt% sodium lauryl sulfate, 0.25wt% sodium deoxycholate, 0.25wt% sodium dioctyl sulfosuccinate and 0.2wt% dithiothreitol.

[0013] By employing this technical solution, the specific formulation of the dissociation agent has been optimized, resulting in higher dissociation efficiency and more complete dissociation of 25-hydroxyvitamin D3 bound to VDBP in canine serum. The optimal proportions of the various components ensure effective dissociation without adversely affecting the NC membrane, further reducing background interference and enhancing detection sensitivity and reproducibility.

[0014] In a second aspect, the present invention applies to protect the use of the above-mentioned fluorescent immunoassay kit for detecting canine vitamin D3 in detecting canine vitamin D3.

[0015] In a third aspect, the present invention provides a method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3, using the following technical solution: A method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3 comprises the following specific steps: S1: Preparation of fluorescent microsphere-labeled antibodies Adding 25-hydroxyvitamin D3 mouse monoclonal antibody to the fluorescent microsphere solution, mixing and reacting to obtain fluorescent microsphere-labeled 25-hydroxyvitamin D3 mouse monoclonal antibody; Add the quality control goat anti-rabbit antibody to the fluorescent microsphere solution, and after mixed reaction, obtain the quality control goat anti-rabbit antibody labeled with fluorescent microspheres; S2: Preparation of conjugate pad Spraying the bonding pad pretreatment liquid on the glass fiber and forming a pretreated bonding pad after drying; Then spray the conjugate pad onto the pre-treated conjugate pad and form a conjugate pad after drying; The conjugate pad pretreatment solution comprises phosphate buffer, trehalose, bovine casein sodium salt, Tween-20, liquid biological preservative, hydroxypropyl methylcellulose and polyacrylic acid; The conjugate pad working solution includes trehalose, casein, mannitol, Tween, antibacterial preservative, phosphate buffer, 25-hydroxyvitamin D3 mouse monoclonal antibody labeled with fluorescent microspheres, and quality control goat anti-rabbit antibody labeled with fluorescent microspheres; S3: Preparation of nitrocellulose membrane (NC membrane) The 25-hydroxyvitamin D3 mouse monoclonal antibody was diluted with a coating solution containing trehalose and TRIS buffer and streaked on the NC membrane and dried to prepare the detection area T line; Rabbit IgG antibody was diluted with a coating solution containing trehalose and TRIS buffer and streaked on the NC membrane, and then dried to prepare the quality control area C line; S4: Preparation of sample pad The sample pad working solution is sprayed on the glass fiber and formed into a sample pad after drying; The sample pad working solution comprises trehalose, Tween 20, casein sodium salt, disodium hydrogen phosphate, anti-erythrocyte antibody and mouse IgG antibody; S5: Assembly of test strips A sample pad, a conjugate pad, an NC membrane, and absorbent paper were sequentially attached to a PVC bottom plate to obtain a fluorescent immunochromatographic test strip for detecting vitamin D3.

[0016] By employing the above technical solutions, the effectiveness of fluorescent labeling was ensured. Pretreatment of the conjugate pad and spraying of the working solution enhanced the adsorption and loading of the fluorescent marker, enabling more uniform release of the microspheres and preventing membrane clogging. The coating of T and C lines on the NC membrane and the treatment of the sample pad ensured antibody stability and detection specificity. Optimization of each process step improved the reproducibility and stability of the test strips, providing a reliable method for mass production of the test kit.

[0017] Preferably, the fluorescent microspheres in step S1 are cleaned and activated before use.

[0018] By adopting the above technical solution, the fluorescent microspheres are cleaned and activated before use to remove impurities on the surface of the microspheres. After activation, the coupling efficiency with the antibody can be improved, nonspecific binding can be reduced, and the antibodies labeled with the fluorescent microspheres can be made purer, thereby improving the specificity of the detection.

[0019] Preferably, in step S2, the spraying volume of the conjugate pad pretreatment solution and the conjugate pad working solution is 4 μL / cm.

[0020] By adopting the above technical solution, the spraying volume of the conjugate pad pretreatment liquid and working liquid is 4μL / cm. This amount can ensure that the conjugate pad fully adsorbs the relevant reagents, avoiding excessive use leading to reagent waste or chromatographic obstruction, and preventing insufficient use from affecting microsphere loading and release, thereby ensuring the stability of the detection.

[0021] Preferably, in step S3, the line concentrations of the detection area T line and the quality control area C line are both 1 μL / cm.

[0022] By adopting the above technical solution, the line concentration of the detection area T line and the quality control area C line is 1 μL / cm. At this concentration, the antibody coating amount is appropriate and can effectively bind to the target substance, making the signal intensity of the T line and C line moderate, facilitating accurate reading of the results and improving the accuracy of the test.

[0023] Preferably, the spraying volume of the sample pad working solution in step S4 is 4 μL / cm.

[0024] By adopting the above technical solution, the spray volume of the sample pad working liquid is 4μL / cm, which can ensure that the sample pad can fully play its role, effectively process the sample, reduce the interference of impurities, and ensure the smooth chromatography of the sample, thereby improving the detection efficiency.

[0025] In a fourth aspect, the present invention provides a fluorescent immunochromatographic test strip for detecting canine vitamin D3, which adopts the following technical solution: A fluorescent immunochromatographic test strip for detecting canine vitamin D3 is prepared by the method for preparing the fluorescent immunochromatographic test strip for detecting canine vitamin D3.

[0026] By adopting the above technical solution and using the above-mentioned preparation method, the fluorescent immunochromatographic test strip has a stable structure and well-connected components. The conjugate pad can evenly release the fluorescent microsphere-labeled antibody, and the T and C lines on the NC membrane can specifically bind to the target substance and quality control antibody, achieving efficient detection of canine 25-hydroxyvitamin D3. This test strip is compatible with the fluorescent immunochromatographic method and offers advantages such as rapid operation, high sensitivity, and excellent reproducibility. It is a core component of the test kit for accurate detection.

[0027] In summary, this application has the following beneficial effects: 1. The present invention focuses on the detection of canine vitamin D3, significantly improving accuracy and reliability. In the test kit, the unique formula of the dissociation agent effectively dissociates the sample, allowing 25-hydroxyvitamin D3 to be fully free. The various components of the reagent card work closely together, with the sample pad receiving, the conjugate pad carrying antibodies, the nitrocellulose membrane for detection and quality control, and the absorbent paper assisting liquid flow to ensure accurate detection. The test strip preparation process is rigorous. From fluorescent microspheres labeling antibodies to the processing of various components, all contribute to accurate detection. Microsphere cleaning and activation enhance antibody binding, precise spraying of the conjugate pad and sample pad ensures uniform composition, and the nitrocellulose membrane is accurately marked to control the amount of antibodies, so that the test strip can stably and accurately reflect the canine vitamin D3 content.

[0028] 2. The present invention has outstanding advantages in operation and application. In terms of operation, the test kit is simple in design, and the dissociation agent is matched with the reagent card. It does not require complex equipment and professional skills, and ordinary personnel can operate it after simple training. The detection is based on the double antibody sandwich method, and the results can be obtained in a short time with high efficiency. It is also suitable for various scenarios, such as pet hospitals, grassroots veterinary stations and farms. The detection of canine vitamin D3 fills the gap in the field, provides key support for canine nutritional assessment, and meets the diverse detection needs of different users. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the chromatography effect diagram of Example 1 and Comparative Example 8. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] According to one aspect of the present invention, the present invention provides a kit for detecting canine vitamin D3, wherein the vitamin D3 is 25-hydroxyvitamin D3.

[0032] The kit provided by the present invention includes a dissociation agent and a reagent card. The dissociation agent includes 0.05M 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer (pH 7.4), 0.3M sodium chloride, 0.005M EDTA-Na2, 1wt% polyvinylpyrrolidone, 1wt% sodium dodecyl sulfate (SDS), 0.25wt% sodium deoxycholate (SDC), 0.25wt% sodium dioctyl sulfosuccinate and 0.2wt% dithiothreitol.

[0033] This formula not only has an excellent dissociation effect on the sample, but the specially added sodium deoxycholate, dioctyl sodium sulfosuccinate and dithiothreitol can significantly improve the flow and diffusion properties of the sample on the nitrocellulose membrane (NC membrane) by reducing the surface tension. It is worth noting that sodium deoxycholate plays a dual role in this system. On the one hand, it has the ability to dissociate, helping to effectively dissociate related substances in the sample; on the other hand, in the reaction system of fluorescent immunochromatography, it acts as a blocking agent.

[0034] Specifically, sodium deoxycholate can be adsorbed onto the surface of chromatographic membranes and other solid phase carriers, forming a dense protective film. This protective film effectively prevents nonspecific proteins or impurities in the sample from adsorbing to the solid phase carrier, thereby greatly reducing background interference and lowering background signals, ultimately achieving a significant improvement in detection sensitivity.

[0035] The reagent card includes a shell and a fluorescent immunochromatographic test strip for detecting 25-hydroxyvitamin D3 built into the shell. The test strip is based on a PVC base plate, on which a sample pad, a conjugation pad, a nitrocellulose membrane and a blotting paper are tightly connected in sequence. Among them, the nitrocellulose membrane is provided with a quality control area C line and a detection area T line spaced adjacent to each other. The detection line is coated with a 25-hydroxyvitamin D3 mouse monoclonal antibody, and the quality control line is coated with a rabbit IgG antibody; the conjugation pad is sprayed with an antibody solution labeled with fluorescent microspheres, and the fluorescent microsphere-labeled antibody solution specifically contains a mixture of a 25-hydroxyvitamin D3 mouse monoclonal antibody labeled with fluorescent microspheres and a quality control goat anti-rabbit antibody labeled with fluorescent microspheres.

[0036] The specific steps of the preparation method of the fluorescent immunochromatographic test strip for detecting 25-hydroxyvitamin D3 are as follows: S1: Preparation of fluorescent microsphere-labeled antibodies (1) Fluorescent microsphere cleaning: Add 1 mL of fluorescent microspheres to a 2 mL centrifuge tube and centrifuge at 14,000 g for 10 minutes. Discard the supernatant. Add 1 mL of solution A consisting of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 6.0) to the centrifuge tube and resuspend the microspheres by ultrasonication for 2 seconds with a 5-second pause. Repeat this operation 5 times.

[0037] (2) Microsphere activation: Add 50-150 μL of activator A (20-80 mg / mL N-hydroxysuccinimide (NHS) in 0.05 M MES buffer, pH adjusted to 6.0-7.4) and 50-150 μL of activator B containing 20-80 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 0.05 M MES buffer to the centrifuge tube containing the washed microsphere particles. Vortex to mix thoroughly, and then place on a rotary incubator for 20 minutes.

[0038] (3) Post-activation cleaning: The activated microspheres were centrifuged again at 14,000 g for 10 minutes, the supernatant was removed, and the precipitate was retained. 1 mL of a cleaning solution consisting of 0.05 M MES buffer (pH adjusted to 6.0-7.4) was added to the precipitate and resuspended by ultrasonication for 2 seconds, followed by a 5-second pause, and repeated 5 times.

[0039] (4) 25-hydroxyvitamin D3 mouse monoclonal antibody conjugation: After activation, cleaning, and sonication, add 20-60 μg of 25-hydroxyvitamin D3 mouse monoclonal antibody to the solution. Vortex to mix thoroughly and place on a rotating incubator for 2 hours. After the reaction is complete, centrifuge at 14,000 g for 10 minutes, remove the supernatant, and retain the precipitate.

[0040] (5) Quality control goat anti-rabbit antibody coupling: Repeat the above washing, activation, and post-activation washing steps (i.e., steps (1)-(3)). Add 20-60 μg of quality control goat anti-rabbit antibody to the solution after the last sonication. Vortex mix thoroughly and place on a rotating incubator for 2 hours. After the reaction is completed, centrifuge at 14,000 g for 10 minutes, remove the supernatant, and retain the precipitate.

[0041] (6) Blocking treatment: 1 mL of blocking solution (0.05 M TRIS buffer, 0.1-0.5 M ethanolamine, 0.5-2 wt% casein, pH adjusted to 8.0) was added to each of the precipitates obtained in steps (4) and (5), and the mixture was resuspended by ultrasound using a 2-second cycle followed by a 5-second pause, repeated 5 times. After resuspension, the mixture was vortexed and placed in a rotary incubator for 1 hour.

[0042] (7) Storage: After the above blocking reaction is completed, centrifuge at 14,000 g for 10 minutes, remove the supernatant, and retain the precipitate. Add 1 mL of fluorescent microsphere particle storage solution (containing 0.2-2 wt% casein, 5-10 wt% trehalose, and 0.1 M BIS-TRIS buffer at pH 7.4-pH 8.0) to the precipitate, and resuspend by ultrasonication for 2 seconds, followed by a 5-second pause, to obtain fluorescent microsphere-labeled 25-hydroxyvitamin D3 mouse monoclonal antibody and fluorescent microsphere-labeled quality control goat anti-rabbit antibody.

[0043] S2: Preparation of conjugate pad Conjugate Pad Pretreatment: Cut the glass fiber into 30 x 1.6 cm squares. Add 0.2-0.5 wt% Evans blue dye to the conjugate pad pretreatment solution. Using a spray pad machine, apply the conjugate pad pretreatment solution three times in parallel onto the cut glass fiber at a spray rate of 4 μL / cm, with a 0-second interval between each spray. After spraying, dry the glass fiber in a drying oven at 50°C for 18-24 hours.

[0044] Add 0.2-0.5 wt% Evans Blue dye to the conjugate pad working solution. Using the same sprayer, apply the conjugate pad working solution three times in parallel onto the dried glass fiber at a spray rate of 4 μL / cm, with 0-second intervals between each application. After spraying, dry the glass fiber again in a drying oven at 50°C for 18-24 hours.

[0045] Purpose and Principle of Pretreatment: Pretreatment of the conjugate pad is intended to increase the active sites on the glass fiber surface, enabling it to better adsorb and load fluorescent markers, antibodies, and other biological reagents. This results in more uniform release of the microspheres, avoids membrane clogging, ensures the proper chromatography process, and improves test reproducibility. The addition of Evans Blue dye helps monitor the uniformity of the spray pad during production, promptly identifying any breakpoints and preventing leaks, which can affect the precision of the test strip.

[0046] Conjugate pad pretreatment solution formula: Conjugate pad pretreatment solution includes 0.01M phosphoric acid (PB) buffer (pH 7.4) The composition includes 10wt% trehalose, 0.5wt% bovine casein sodium salt, 0.5wt% Tween-20, 0.02wt% liquid biopreservative, 0.5wt% hydroxypropyl methylcellulose (HPMC), and 0.5wt% polyacrylic acid. HPMC interacts with the surface groups of the fluorescent marker and antibody conjugate, forming a protective barrier to prevent denaturation, aggregation, or dissociation during storage and detection, ensuring the reliability of test results.

[0047] Formula for the conjugate pad working solution: Use a fluorescent microsphere-labeled antibody coating solution containing 10wt% trehalose, 1wt% casein, 0.5wt% mannitol, 0.5wt% Tween, 0.4wt% PC-300 antibacterial preservative, and 0.01M PB buffer (pH 7.4), and dilute the fluorescent microsphere-labeled 25-hydroxyvitamin D3 mouse monoclonal antibody and the fluorescent microsphere-labeled quality control goat anti-rabbit antibody to 1 mg / mL and 0.5 mg / mL, respectively, to obtain the conjugate pad working solution.

[0048] S3: Preparation of nitrocellulose membrane (NC membrane) Preparation of base plate and NC membrane: Select a PVC base plate with a specification of 7.7×30 cm, and stick a nitrocellulose membrane (NC membrane) with a width of 2.5 cm in the middle of the PVC base plate.

[0049] Marking the control and test lines: On the NC membrane, draw the control line (C line) and the test line (T line) using the C-line coating working solution and T-line coating working solution, respectively. The two lines are placed parallel and adjacent, with a 4 mm interval. The test line (T line) is located near the conjugate pad, while the control line (C line) is located away from the conjugate pad and closer to the absorbent pad. The marking concentration for both lines is 1 μL / cm. After marking, dry the prepared NC membrane in a drying oven at 50°C for 24-72 hours.

[0050] Coating working solution formula: T-line coating working solution: Dilute 25-hydroxyvitamin D3 mouse monoclonal antibody to 0.5 mg / mL with a coating solution containing 5% trehalose and 0.01 M TRIS buffer (pH 8.0) to obtain the T-line working solution.

[0051] C-line coating working solution: Dilute rabbit IgG antibody to 0.5 mg / mL using a coating solution containing 5% trehalose and 0.01 M TRIS buffer (pH 8.0). This is the C-line coating working solution.

[0052] S4: Preparation of sample pad Add 0.5 wt% Evans Blue dye to the sample pad working solution. Use a spray pad machine to spray the sample pad working solution onto the glass fiber three times in parallel at a spray rate of 4 μL / cm, with 0-second intervals between each application. After spraying, dry the glass fiber in a drying oven at 50°C for 18-24 hours to obtain the sample pad.

[0053] Sample pad working solution formula: Use a coating solution containing 10wt% trehalose, 0.5wt% Tween 20, 0.5wt% casein sodium salt, and 0.01M disodium hydrogen phosphate (pH 7.4), and prepare anti-RBC (anti-red blood cell antibody) and mouse IgG antibody into a solution with a concentration of 0.5-1 mg / mL as the sample pad working solution.

[0054] S5: Assembly of test strips The prepared sample pad, conjugate pad, NC membrane, and absorbent paper are connected and assembled in the order of PVC base plate, sample pad, conjugate pad, NC membrane, and absorbent paper to obtain a fluorescent immunochromatographic test strip for detecting vitamin D3.

[0055] The raw materials used in the present invention are all commercially available products, including HEPES, sodium chloride, EDTA-Na2, polyvinyl pyrrolidone, sodium dodecyl sulfate SDS, sodium deoxycholate SDC, sodium sulfosuccinate dioctyl ester, disodium hydrogen phosphate, sodium dihydrogen phosphate, trehalose, bovine casein sodium salt, Tween-20, PC-300 and liquid biological preservative (model ProClin TM 950) was purchased from Aladdin Reagent Company in Shanghai; Hydroxypropyl methylcellulose (HPMC) and polyacrylic acid were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Fluorescent microspheres were purchased from Merck, Germany, the specific model is XC050; Nitrocellulose membrane was purchased from Sartorius, Germany, the specific model is CN95; 25-Hydroxyvitamin D-coated antibody was purchased from Jiangsu Dongkang Biological Company; The quality control line antibody rabbit IgG and quality control goat anti-rabbit antibody were purchased from Shanghai Shenggong Company; S9 (Tetronic 1307), S14 (TRTTONX-100), S19 (TWEEN 20), S20 (TWEEN 80), S21 (BRIJ 35), dithiothreitol, PEG 6000, NP40, CHAPS, and PVA polyvinyl alcohol were purchased from Shanghai Yiheng Biotechnology.

[0056] Performance Testing 1. Dissociation agent test In order to verify the effect of the dissociation agent of the present invention on the detection effect, the above dissociation agent and test strips were used as Example 1. Based on Example 1, comparative examples with different dissociations were designed. The test strips of Comparative Examples 1-9 were consistent with those of Example 1.

[0057] Comparative Example 1 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that S9 in the BIODOT series of surfactants is used to replace the original sodium dodecyl sulfate (SDS).

[0058] Comparative Example 2 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that S14 in the BIODOT series of surfactants is used to replace the original sodium dodecyl sulfate (SDS).

[0059] Comparative Example 3 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that S19 in the BIODOT series of surfactants is used to replace the original sodium dodecyl sulfate (SDS).

[0060] Comparative Example 4 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that S20 in the BIODOT series of surfactants is used to replace the original sodium dodecyl sulfate (SDS).

[0061] Comparative Example 5 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that S21 in the BIODOT series of surfactants is used to replace sodium dodecyl sulfate (SDS).

[0062] Comparative Example 6 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that polyethylene glycol 6000 (PEG6000) is used to replace the original dithiothreitol.

[0063] Comparative Example 7 The difference between the dissociator of this comparative example and the dissociator of Example 1 is that 3-[(3-cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) is used to replace sodium deoxycholate (SDC).

[0064] Comparative Example 8 The dissociation agent of this comparative example is formulated as follows: 0.05M HEPES (pH 7.4), 0.3M sodium chloride, 0.005M EDTA-Na2, 1wt% polyvinylpyrrolidone, 1wt% dimethylethanolamine, 0.25wt% dimethylformamide, 0.25wt% dimethyl sulfoxide (DMSO), 0.2wt% guanidine thiocyanate, and 0.25wt% perfluorooctanoic acid. This dissociation agent is a combination of an organic solvent and a strong acid.

[0065] Test method: (1) First, a high-value canine serum sample rich in vitamin D3 was selected and serially diluted using newborn calf serum as the dilution matrix to prepare sample solutions with concentrations of 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1.5 ng / ml, respectively. The specific dilution was performed at a ratio of 1:20.

[0066] Subsequently, for each concentration of sample solution, different formulations of dissociation agents were used for dilution and sample loading. During the experiment, each concentration point was tested three times to ensure the reliability of the experimental data, and the reaction time for each test was set at 15 minutes.

[0067] The samples were then tested using the Healvet HV-FIV3000Plus dry-type fluorescent immunoassay instrument. The instrument's excitation wavelength was set to 470 nm and its emission wavelength to 525 nm. The instrument converts the fluorescence intensity of the T and C lines in the reagent into electrical signals. These signals are collected using the accompanying software and further calculated to produce the T / C ratio. The results are detailed in Table 1.

[0068] Table 1 Test results From the data in Table 1, it can be seen that the linear range of the dissociation agent in Example 1 is 1.5-80 ng / ml, the sensitivity can reach 1.5 ng / ml, and the R of the linear equation is 2 >0.99, specifically R 2 =0.9977. This means that the dissociation agent of Example 1 can maintain a good linear relationship in a wide concentration range, greatly improving the sensitivity and accuracy of the detection, and providing a more reliable solution for related detection work.

[0069] When S9, S14, S19, S20 or S21 were added to the dissociation agent formula of Example 1 (i.e., Comparative Examples 1-5), the dissociation effect did not meet expectations. Specifically, the corresponding linear range only fell between 20-80 ng / ml, which indicates that the sample concentration range that the dissociation agent can accurately detect is relatively narrow. At the same time, the linear equation obtained by linear regression analysis has a determination coefficient R 2 All of them are less than 0.99, indicating that the linear relationship between concentration and detection signal is not ideal, which means that the dissociation agent has poor performance in terms of sensitivity.

[0070] The effects of the dissociation agents in Comparative Examples 6 and 7 were also unsatisfactory. The linear range they exhibited was also limited to 20-80 ng / ml, and the R 2 The linear range of the two dissociation agents is also less than 0.99. This shows that when the two dissociation agents are used to detect samples, not only the linear range is narrow, but also the correlation between the concentration and the detection signal is weak, which affects the sensitivity and accuracy of the detection. The dissociation effect of the dissociation agent in Comparative Example 8 is improved in some aspects compared with the dissociation agents in Comparative Examples 1-7. Its linear range is extended to 5-80 ng / ml, and the R of the linear equation is 2 =0.9824.

[0071] However, from Figure 1It can be clearly seen from the chromatography effect diagram that there are some problems with the dissociator of comparative example 8. During the chromatography process, the microspheres showed aggregation. This is because the formula of the organic solvent and the strong acid in the dissociator is superimposed, which has a destructive effect on the nitrocellulose membrane (NC membrane). Compared with the chromatogram of Example 1, the dissociator of this formula shows a situation where the baseline starting point is higher and tilted, and most of the microspheres gather at the front end of the NC membrane and fail to be fully released, resulting in aggregation. This phenomenon fully illustrates that the above dissociator formulas are not conducive to the smooth chromatography of the sample on the reagent strip, and will have an adverse effect on the test results. The dissociator of Example 1 of the present invention shows relatively excellent performance. Its baseline is smooth, the starting point is lower than 20,000, and the fluorescent microspheres can be completely released. This phenomenon shows that the dissociator formula of the present invention effectively solves the problem of microspheres easily aggregating and blocking the membrane during the chromatography process, especially between the conjugate pad and the NC membrane, thereby avoiding the background increase and limited sensitivity caused by microsphere aggregation.

[0072] 2. Repeatability test Comparative Example 9 The difference between this comparative example and Example 1 is that the conjugate pad pretreatment solution used in the preparation of the test strips in this comparative example is different. The conjugate pad pretreatment solution in this comparative example includes 0.01M PB buffer (pH 7.4), 10wt% trehalose, 0.5wt% bovine casein sodium salt, 1wt% polyvinylpyrrolidone (PVP40), 0.5wt% Tween-20 and 0.5wt% mannitol.

[0073] In order to further explore the repeatability of this detection system, high, medium and low concentration samples of 25-hydroxyvitamin D3 in dog serum were selected and determined by liquid chromatography to carry out relevant repeatability tests.

[0074] The specific detection method is as follows: select high, medium and low value samples of canine serum with vitamin D3 concentrations of 80ng / ml, 10ng / ml and 1.5ng / ml respectively, dilute them at a ratio of 1:20, and then use the dissociation agent of Example 1 for further dilution and loading. For each concentration point, 10 repeated tests were performed, and the reaction time for each test was set to 15 minutes. The Healvet dry fluorescent immunoassay instrument HV-FIV3000Plus was used for detection. The instrument was set to an excitation wavelength of 470nm and an emission wavelength of 525nm, which can convert the fluorescence intensity of the T line and C line in the reagent into electrical signals. These test signals were collected by supporting software, and the coefficient of variation (CV) of the T / C value was calculated.

[0075] Table 2 Repeatability test results Where AV is the mean, SD is the standard deviation, and CV is the coefficient of variation. The calculation formula is SD / AV.

[0076] The data in Table 2 clearly show that when the test strips were treated with the conjugate pad pretreatment solution of Comparative Example 1, the coefficient of variation (CV) was greater than 15%, indicating that the detection repeatability of this pretreatment solution was poor. However, when the test strips were treated with the conjugate pad pretreatment solution of the present invention, the coefficient of variation (CV) was less than 15%, indicating that the test strips of the present invention had good detection repeatability and performed excellently in terms of repeatability.

[0077] Compared to existing technologies, this invention utilizes the classic and effective double-antibody sandwich method for immunoassay detection. Specifically, a precisely defined volume of canine serum sample is dripped onto a sample pad. Due to the capillary action of the sample pad material, the analyte in the serum sample—25-hydroxyvitamin D3—is carried forward along with the pre-prepared microsphere-antibody conjugate complex by the flow of liquid.

[0078] When this mixture flows through the T-line area where specific antibodies are immobilized, the 25-hydroxyvitamin D3 in the sample, acting as an antigen, undergoes a highly specific binding reaction with the antibodies immobilized on the T-line. This specific binding is based on the precise molecular structural complementarity between the antigen and antibody, much like the relationship between a key and a lock; only specific antigens can bind tightly to their corresponding antibodies.

[0079] During this process, the 25-hydroxyvitamin D3 content in the sample shows a positive correlation with the number of complexes accumulated on the test line. In other words, the more 25-hydroxyvitamin D3 there is in the sample, the more microsphere-antibody-coupled complexes can bind to and accumulate with the fixed antibody in the T-line area. These fluorescent antibodies accumulated on the test line emit a fluorescent signal of a specific intensity, and the signal intensity directly reflects the amount of captured 25-hydroxyvitamin D3. By accurately measuring and analyzing the intensity of this fluorescent signal with professional testing instruments, the content of 25-hydroxyvitamin D3 in the sample can be accurately determined, providing reliable data support for subsequent research.

[0080] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A fluorescent immunoassay kit for detecting canine vitamin D3, characterized in that: It comprises a dissociation agent and a reagent card, wherein the dissociation agent comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, EDTA-Na2, polyvinylpyrrolidone, sodium lauryl sulfate, sodium deoxycholate, sodium sulfosuccinate dioctyl ester and dithiothreitol; the reagent card comprises a shell and a fluorescent immunochromatographic test strip for detecting 25-hydroxyvitamin D3 built into the shell; the test strip comprises a PVC base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper sequentially arranged on the PVC base plate, wherein the nitrocellulose membrane is provided with a quality control area C line and a detection area T line spaced adjacent to each other, the detection area T line is coated with a 25-hydroxyvitamin D3 mouse monoclonal antibody, and the quality control area C line is coated with a rabbit IgG antibody; the conjugation pad is sprayed with an antibody solution labeled with fluorescent microspheres, and the fluorescent microsphere-labeled antibody solution specifically contains a mixture of a 25-hydroxyvitamin D3 mouse monoclonal antibody labeled with fluorescent microspheres and a quality control goat anti-rabbit antibody labeled with fluorescent microspheres.

2. A fluorescent immunoassay kit for detecting canine vitamin D3 according to claim 1, characterized in that: The fluorescent microspheres are XC050 fluorescent microspheres from Merck.

3. A fluorescent immunoassay kit for detecting canine vitamin D3 according to claim 1, characterized in that: The dissociation agent includes 0.05M 4-hydroxyethylpiperazineethanesulfonic acid buffer (pH 7.4), 0.3M sodium chloride, 0.005M EDTA-Na2, 1wt% polyvinylpyrrolidone, 1wt% sodium lauryl sulfate, 0.25wt% sodium deoxycholate, 0.25wt% sodium dioctyl sulfosuccinate and 0.2wt% dithiothreitol.

4. Use of the fluorescent immunoassay kit for detecting canine vitamin D3 according to claims 1-3 in detecting canine vitamin D3.

5. A method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3, characterized in that: The specific steps include: S1: Preparation of fluorescent microsphere-labeled antibodies Adding 25-hydroxyvitamin D3 mouse monoclonal antibody to the fluorescent microsphere solution, mixing and reacting to obtain fluorescent microsphere-labeled 25-hydroxyvitamin D3 mouse monoclonal antibody; Add the quality control goat anti-rabbit antibody to the fluorescent microsphere solution, and after mixed reaction, obtain the quality control goat anti-rabbit antibody labeled with fluorescent microspheres; S2: Preparation of conjugate pad Spraying the bonding pad pretreatment liquid on the glass fiber and forming a pretreated bonding pad after drying; Then spray the conjugate pad onto the pre-treated conjugate pad and form a conjugate pad after drying; The conjugate pad pretreatment solution comprises phosphate buffer, trehalose, bovine casein sodium salt, Tween-20, liquid biological preservative, hydroxypropyl methylcellulose and polyacrylic acid; The conjugate pad working solution includes trehalose, casein, mannitol, Tween, antibacterial preservative, phosphate buffer, 25-hydroxyvitamin D3 mouse monoclonal antibody labeled with fluorescent microspheres, and quality control goat anti-rabbit antibody labeled with fluorescent microspheres; S3: Preparation of nitrocellulose membrane (NC membrane) The 25-hydroxyvitamin D3 mouse monoclonal antibody was diluted with a coating solution containing trehalose and TRIS buffer and streaked on the NC membrane and dried to prepare the detection area T line; Rabbit IgG antibody was diluted with a coating solution containing trehalose and TRIS buffer and streaked on the NC membrane, and then dried to prepare the quality control area C line; S4: Preparation of sample pad The sample pad working solution is sprayed on the glass fiber and formed into a sample pad after drying; The sample pad working solution comprises trehalose, Tween 20, casein sodium salt, disodium hydrogen phosphate, anti-erythrocyte antibody and mouse IgG antibody; S5: Assembly of test strips A sample pad, a conjugate pad, an NC membrane, and absorbent paper were sequentially attached to a PVC bottom plate to obtain a fluorescent immunochromatographic test strip for detecting vitamin D3.

6. The method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3 according to claim 5, wherein: The fluorescent microspheres in step S1 are cleaned and activated before use.

7. The method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3 according to claim 5, wherein: In step S2, the spraying volume of the conjugate pad pretreatment solution and the conjugate pad working solution is 4 μL / cm.

8. The method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3 according to claim 5, wherein: In the step S3, the line concentrations of the detection area T line and the quality control area C line are both 1 μL / cm.

9. The method for preparing a fluorescent immunochromatographic test strip for detecting canine vitamin D3 according to claim 5, wherein: The spraying volume of the sample pad working solution in step S4 is 4 μL / cm.

10. A fluorescent immunochromatographic test strip for detecting canine vitamin D3, characterized in that: The fluorescent immunochromatographic test strip for detecting canine vitamin D3 is prepared by the preparation method of claims 5-9.

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