An immunochromatographic kit, a direct loading immunochromatographic device and a kit containing the same

Abstract

The utility model provides a kind of direct sample immunochromatography device, containing chromatography kit and sampler, the chromatography kit sample port has interface, the sampler has interface, can be matched with the interface of chromatography kit sample port.The direct sample immunochromatography device provided by the utility model compared with traditional immunochromatography device, qualitative, quantitative detection for low abundance sample can be realized, and the sensitivity and detection rate of detection are improved.

Description

Technical Field

[0001] This utility model relates to the field of diagnostic testing, specifically to a direct sample loading immunochromatographic apparatus. Background Technology

[0002] Immunochromatography is a simple, rapid, and convenient detection method, primarily used in medical diagnostics, agricultural production, animal quarantine, and food safety. It relies on the specific binding of antigens, haptens, and antibodies to capture, enrich, and detect the analytes. Currently, the immunochromatographic process involves collecting a sample, diluting it with a sample diluent or release solution, and then adding it to a sample pad for testing. This process often dilutes the analyte hundreds to tens of thousands of times, leading to poor accuracy, especially for low-abundance samples, particularly those collected via swabs. For example, when using antigen test kits for COVID-19 or influenza A, patients often only test positive 2-3 days after infection, after noticeable symptoms have appeared. This lag stems from two factors: firstly, the low antibody abundance in the early stages of infection, and secondly, the further dilution of the sample after collection with a diluent. Summary of the Invention

[0003] In view of this, the present invention provides a direct sample loading immunochromatographic device, which, compared with traditional immunochromatographic devices, enables qualitative and quantitative detection of low abundance samples.

[0004] This invention provides a direct sample loading immunochromatographic kit, which has a sample loading port with a sample loading interface that can be matched with the interface of a sampler, allowing the sampler to be installed into the sample loading port of the chromatography kit.

[0005] According to the embodiments of this utility model, the sample loading port interface of the chromatography kit can be a common engineering interface type, such as fan-shaped, threaded, enamel-type, or ground-glued; preferably, the sample loading port interface of the chromatography kit is a fan-shaped bayonet or a threaded interface.

[0006] According to an embodiment of the present invention, the chromatography kit includes a housing.

[0007] According to an embodiment of this utility model, the outer shell is provided with a sample loading port and an observation window, and the sample loading port has a sample loading port interface.

[0008] According to an embodiment of the present invention, the outer shell contains chromatography test paper.

[0009] According to an embodiment of the present invention, the chromatography test strip includes a sample pad, a labeling pad, an immunochromatographic membrane, and an absorbent pad.

[0010] According to an embodiment of this utility model, the immunochromatographic membrane is provided with a detection line and a quality control line.

[0011] According to an embodiment of the present invention, the chromatography kit has an extended test strip outlet, through which the chromatography test strip can extend to siphon diffusion liquid.

[0012] This utility model also provides a direct sample loading immunochromatographic apparatus, the apparatus comprising a chromatography kit and a sampler; the chromatography kit has a sample loading port, the sample loading port having a sample loading port interface; the sampler includes a main body and a sampling pad, the sampler having a sampling pad at its bottom end, the sampler main body having an interface located above the sampling pad, which can be matched with the sample loading port interface of the chromatography kit, so that the sampler can be installed into the sample loading port of the chromatography kit.

[0013] According to the embodiments of this utility model, the sample loading port interface of the chromatography kit can be a common engineering interface type, such as fan-shaped, threaded, enamel-coated, ground joint, etc.

[0014] According to the embodiments of this utility model, the sample loading port interface of the chromatography kit is a fan-shaped bayonet or a threaded interface.

[0015] According to the embodiments of this utility model, the interface above the sampling pad can be a common engineering interface type, such as fan-shaped, threaded, enamel-mouthed, ground-mouthed, etc.

[0016] According to the embodiments of this utility model, the interface above the sampling pad is a fan-shaped bayonet or a threaded bayonet.

[0017] According to an embodiment of the present invention, the sampler further includes a handheld rod, the handheld rod, the main body and the sampling pad are arranged sequentially along the longitudinal direction of the sampler, and the handheld rod is located at the top of the sampler for handheld operation.

[0018] According to the embodiments of this utility model, the main structure of the sampler is a hollow structure or a solid structure.

[0019] According to an embodiment of the present invention, the chromatography kit includes a housing.

[0020] According to an embodiment of this utility model, the outer shell is provided with a sample loading port and an observation window, and the sample loading port has a sample loading port interface.

[0021] According to an embodiment of the present invention, the outer shell contains chromatography test paper.

[0022] According to an embodiment of the present invention, the chromatography test strip includes a sample pad, a labeling pad, an immunochromatographic membrane, and an absorbent pad.

[0023] According to an embodiment of this utility model, the immunochromatographic membrane is provided with a detection line and a quality control line.

[0024] According to an embodiment of the present invention, the chromatography kit housing has an extended chromatography test strip outlet, through which the chromatography test strip can extend to siphon diffusion liquid.

[0025] According to the embodiments of this utility model, the sampling pad is made of flocked or sponge.

[0026] According to the embodiments of this utility model, the material of the sample pad is selected from one or more of cellulose materials, filter paper, polyester membrane, glass cellulose membrane, non-woven fabric or other common absorbent materials, preferably one or more of cellulose materials, polyester membrane or non-woven fabric, and more preferably one or more of cellulose materials and polyester membrane.

[0027] According to an embodiment of this utility model, the sample pad contains a sealing solution, the sealing solution comprising: a hydrophilic polymer material, a protective protein, an antibacterial agent, and an inorganic salt; preferably, the sealing solution is an aqueous solution.

[0028] According to the embodiments of this utility model, the preparation method of the sample pad includes the following steps: completely immersing the sample pad material in the sealing solution, letting it stand for a certain period of time, and after the sample pad material has completely absorbed the sealing solution, removing the sample pad and drying it.

[0029] According to the embodiments of this utility model, the material of the marking pad is selected from one or more of cellulose materials, filter paper, polyester film, glass cellulose film, non-woven fabric or other common absorbent materials, preferably one or more of cellulose materials, polyester film or non-woven fabric, and more preferably one or more of cellulose materials and polyester film.

[0030] According to an embodiment of the present invention, the labeling pad contains a specific companion labeled by signal microspheres.

[0031] According to the embodiments of this utility model, the specific partner includes small molecule organic compounds and biological macromolecules. The specific partner has the ability to specifically recognize, bind, pair, and capture analyte molecules. Preferably, the specific partner can form a "double antibody sandwich" model with the specific binding partner and analyte molecules immobilized on the immunochromatographic membrane to complete specific detection.

[0032] According to an embodiment of this utility model, the signal microsphere is a substance or particle carrying a recognition signal; preferably, the signal microsphere is selected from one or more of colloidal gold, gold nanoparticles, colloidal silver, silver nanoparticles, colored polymer microspheres, fluorescent polymer microspheres, and quantum dots; more preferably, the materials of the colored polymer microspheres and fluorescent polymer microspheres are selected from one or more of polystyrene, polyglycidyl methacrylate, and polyacrylic acid.

[0033] According to the embodiments of this utility model, the labeling method of the signal microspheres and the specific chaperone is selected from physical adsorption coating process or covalent coupling process.

[0034] According to the embodiments of this utility model, the immunochromatographic membrane material is selected from microporous filter membrane materials with large pore size; preferably, the immunochromatographic membrane material is selected from one or more of nitrocellulose membrane, polyethersulfone filter membrane, cellulose acetate filter membrane, mixed cellulose ester filter membrane, polyvinylidene fluoride filter membrane, nylon filter membrane, and polycarbonate core track etched filter membrane; more preferably, the immunochromatographic membrane material is selected from one or more of nitrocellulose membrane, cellulose acetate filter membrane, mixed cellulose ester filter membrane, and polyvinylidene fluoride filter membrane; more preferably, the immunochromatographic membrane material is selected from one or more of nitrocellulose membrane and cellulose acetate filter membrane.

[0035] According to an embodiment of the present invention, the detection line contains a region that specifically binds to a partner.

[0036] According to the embodiments of this utility model, the quality control line contains a corresponding immunoglobulin or a secondary antibody derived from an antibody species.

[0037] According to the embodiments of this utility model, the method of immobilizing the specific binding partner on the immunochromatographic membrane is selected from physical adsorption coating process or covalent coupling process.

[0038] According to the embodiments of this utility model, the number of detection lines T is 1-20, preferably 1-10, and more preferably 1-5.

[0039] According to the embodiments of this utility model, the material of the absorbent pad is selected from one or more of cellulose materials, filter paper, polyester membrane, glass cellulose membrane, non-woven fabric or other common absorbent materials, preferably one or more of cellulose materials, polyester membrane, and non-woven fabric, and more preferably one or more of cellulose materials and polyester membrane.

[0040] This invention also provides a reagent kit, which includes the direct sample loading immunochromatography kit or the direct sample loading immunochromatography device described in this invention.

[0041] The direct sample loading immunochromatographic kit or direct sample loading immunochromatographic device of this utility model is tested through the following steps: after the sampler is used to sample the site to be sampled, without dilution with diluent, the sampler is installed on the sample loading port of the chromatography kit, and chromatography is performed with chromatography diffusion solution.

[0042] According to the embodiment of this utility model, the sampler body is a hollow structure, and the chromatography diffusion liquid is dripped into the sample loading port by the dripping method to complete the chromatography.

[0043] According to the embodiments of this utility model, after the sampler is installed into the sample loading port of the chromatography kit, a part of the sampler is removed, such as the hand handle at the top of the sampler.

[0044] According to the embodiment of this utility model, the sampler body is a solid structure. After the sampler is installed on the sample port of the chromatography kit, chromatography is completed by siphoning the diffusion liquid through the extended chromatography test paper.

[0045] According to the embodiments of this utility model, the direct sample loading immunochromatographic kit or direct sample loading immunochromatographic device is used for sample testing in the fields of medical testing, food safety, agricultural planting or animal husbandry.

[0046] According to an embodiment of the present invention, the direct sample loading immunochromatographic kit or direct sample loading immunochromatographic device is used to detect one or more of C-reactive protein, influenza virus (e.g., influenza A virus, influenza B virus), follicle-stimulating hormone, luteinizing hormone, and estradiol.

[0047] This invention features a specially designed sampler for collecting samples from targets such as the nasal cavity, pharynx, eyes, skin, and environment. After collection, the sample collector is connected to an immunochromatographic test strip via a special interface, followed by chromatographic detection using either a dropper method or a siphon method. Compared to traditional immunochromatographic devices, this invention enables qualitative and quantitative detection of low-abundance samples.

[0048] Beneficial effects The direct-load immunochromatographic assay device provided by this invention solves the problem of detecting low-abundance samples that are difficult to detect with traditional immunochromatographic devices, thereby improving the sensitivity and detection rate. At the same time, the direct-load immunochromatographic assay device avoids secondary contamination caused by intermediate dilution, and improves the convenience and simplicity of operation.

[0049] The direct sample loading immunochromatographic device provided by this invention can be used in multiple fields such as medical testing, food safety, agricultural planting, and animal husbandry. By specifically detecting antigens, haptens, and antibodies, it can detect samples with lower abundance compared with traditional immunochromatographic devices, thus improving the performance of the immunochromatographic device. Attached Figure Description

[0050] Figure 1a This is a perspective view of the direct sample loading chromatography kit with a fan-shaped interface of this utility model; Figure 1b This is a perspective view of the direct sample loading immunochromatographic apparatus with a fan-shaped interface according to the present invention. Figure 1c This is a front view of the direct sample loading immunochromatographic apparatus with a fan-shaped interface according to the present invention. Figure 2a This is a perspective view of the direct sample loading immunochromatographic apparatus with a threaded interface according to the present invention. Figure 2b This is a front view of the direct sample loading immunochromatographic apparatus with a threaded interface according to this utility model. Figure 3a This is a perspective view of the direct sample loading chromatography kit of the present invention, which features a "siphon method" extended test strip outlet; Figure 3b This is a front view of the direct sample loading immunochromatographic apparatus of the present invention, which features a "siphon method" to extend the test strip outlet. Figures 1a-1c In the middle, 11. Chromatography kit; 12. Sampler; 13. Sample loading port; 14. Fan-shaped bayonet on the sample loading port; 15. Sampling pad; 16. Fan-shaped bayonet on the sampler; 17. Observation window; 18. Outer casing; 19. Chromatography test paper; Figures 2a-2b In the middle, 21. Chromatography kit; 22. Sampler; 23. Sample loading port; 24. Threaded interface of sample loading port; 25. Sampling pad; 26. Threaded interface on sampler; 27. Observation window; 28. Outer shell; 29. ​​Chromatography test paper; Figures 3a-3b In the middle, 31, "siphon method" extends the test strip outlet; 32, "siphon method" extends the test strip. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of this utility model clearer, the technical solutions of the embodiments of this utility model will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this utility model, not all of them. Therefore, the following detailed description of the embodiments of this utility model provided in the accompanying drawings is not intended to limit the scope of the claimed utility model, but merely to represent selected embodiments of this utility model. All other embodiments obtained by those skilled in the art based on the embodiments of this utility model without inventive effort are within the scope of protection of this utility model.

[0052] This invention provides a direct sample loading immunochromatographic apparatus, which includes a chromatography kit and a sampler.

[0053] The chromatography kit includes chromatography strips and a housing containing the chromatography strips. The housing has a sample loading port and an observation window. The sample loading port is located on the housing in an area corresponding to the sample pad of the chromatography strip, facilitating observation of the color development of the detection line and control line. The sample loading port allows the operator to accurately locate the optimal sample loading position. The sample loading port can be constructed in any shape, such as circular. The length of the observation window along the chromatography direction is determined by allowing convenient observation of the color development of the detection line and control line.

[0054] The sampler includes a handheld handle, a main body, and a sampling pad. The handheld handle, main body, and sampling pad are arranged longitudinally along the sampler. The sampler main body has an interface located above the sampling pad. The handheld handle is located at the top of the sampler for handheld operation. The sampler is made of hollow or solid material. The sampling pad, located at the bottom of the sampler, is made of flocked / sponge or similar material and is used to directly contact the sampling site (e.g., skin, object surface) to collect the sample (e.g., biological material, chemical substance) and release it into the sampling pad. The interface, located above the sampling pad, is used to connect with the sample loading port of the chromatography kit. Common engineering interface types include fan-shaped, threaded, enamel-coated, and ground glass types to achieve a tight connection between the sampler and the sample loading port of the chromatography kit, ensuring no loss or leakage of signal during sample transfer. Different interface types (e.g., threaded for tightening and ground glass for a tight fit) adapt to different usage scenarios, improving compatibility and stability. The main body connects the top handheld handle to the interface and supports the sampling pad. The sampler's main structure can be either hollow or solid. When the sampler is hollow, the chromatographic diffusion solution can be directly dripped into the sample loading port using a dropping method, pushing the sample towards the test strip for chromatographic detection. When the sampler is solid, the diffusion solution is drawn up by siphoning through the extended chromatographic test strip, completing the sample's chromatographic process. The sampler does not require dilution of the test sample; after collecting the sample through the sampling pad, it directly connects to the sample loading port of the chromatographic kit via the interface to complete sample transfer and chromatographic detection.

[0055] After the sampler is installed onto the sample loading port of the chromatography kit via the interface, the handle can be removed. Preferably, the handle is detachably connected to the upper end of the main body.

[0056] During operation: 1. When using a sampler for sampling, blood samples can be collected through a sampling pad or by inserting it into the throat or other parts of the body. The samples can be saliva / body fluid / serum / plasma / whole blood, etc., or environmental samples can be collected through swabbing or other operations, such as the surface and interior of instruments and equipment, ward facilities, ICU rooms, ambulances, etc. 2. After sampling the area to be sampled using the sampler, without dilution with diluent, insert the sampling pad into the sample loading port of the chromatography kit and secure it through the interface, ensuring contact between the sampling pad and the test strip. If the sampler has a hollow structure, the chromatography diffusion solution can be dripped into the sample loading port using the drop method to complete the chromatography; if the sampler has a solid structure, the chromatography solution can be siphoned off by extending the chromatography test strip to complete the chromatography.

[0057] Reference Figures 1a to 1c A specific embodiment of this utility model provides a direct sample loading immunochromatographic apparatus with a fan-shaped interface. The apparatus includes a chromatography kit 11 and a sampler 12. The chromatography kit 11 includes a chromatography test strip 19 and a housing 18 containing the chromatography test strip. The housing 18 has a sample loading port 13 and an observation window 17. The sample loading port 13 is located on the housing 18 in an area corresponding to the sample pad of the chromatography test strip 19, and the sample loading port 13 has a fan-shaped bayonet 14. The sampler 12 includes a main body and a sampling pad 15. The sampling pad 15 is located at the bottom of the sampler 12, and the sampler main body has a fan-shaped bayonet 16 above the sampling pad 15, which matches the fan-shaped bayonet 14 of the sample loading port of the chromatography kit 11. In use, after the sampler 12 is used to sample the area to be sampled, it does not need to be diluted with diluent. The sampler is then installed into the sample loading port 13 of the chromatography kit 11, and chromatography is performed using a chromatography diffusion buffer.

[0058] Reference Figures 2a to 2b Another specific embodiment of this utility model provides a direct sample loading immunochromatographic apparatus with a threaded interface. The apparatus includes a chromatography kit 21 and a sampler 22. The chromatography kit includes a chromatography test strip 29 and a housing 28 containing the chromatography test strip. The housing 28 has a sample loading port 23 and an observation window 27. The sample loading port 23 is located on the housing in an area corresponding to the sample pad of the chromatography test strip, and the sample loading port 23 has a threaded interface 24. The sampler 22 includes a main body and a sampling pad 25. The sampling pad 25 is located at the bottom of the sampler 22, and the sampler main body has a threaded interface 26 above the sampling pad 25, which matches the threaded interface 24 of the sample loading port of the chromatography kit 21. In use, after the sampler 22 is used to sample the site to be sampled, it is not necessary to dilute it with diluent. The sampler 22 is then installed into the sample loading port 23 of the chromatography kit 21, and chromatography is performed using a chromatography diffusion buffer.

[0059] Reference Figures 3a to 3b Another specific embodiment of this utility model provides a direct sample loading immunochromatographic kit and a direct sample loading immunochromatographic device with a "siphon method" extended test strip outlet 31. The sampler is a solid structure. After the sampler is installed into the sample loading port of the chromatography kit through a specific interface, the chromatography test strip 32 extends through the outlet 31 and contacts the chromatography diffusion liquid, siphoning the diffusion liquid to complete the chromatography process of the sample.

[0060] It should be noted that when the sampler body is a hollow structure, a direct sample loading immunochromatographic kit or a direct sample loading immunochromatographic device with an extended test strip outlet can also be used for sample detection. After the sampler is installed into the sample loading port of the chromatography kit through a specific interface, the chromatography test strip 32 extends through the outlet 31 to contact the chromatography diffusion liquid, siphons the diffusion liquid, and completes the chromatography process of the sample.

[0061] According to the embodiments of this utility model, the sample pad material can be cellulose material, filter paper, polyester membrane, glass cellulose membrane, non-woven fabric, or common absorbent materials, etc., preferably cellulose material, polyester membrane, or non-woven fabric, and more preferably cellulose material and polyester membrane.

[0062] Furthermore, the sample pad contains a sealing solution, which comprises: hydrophilic polymers such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylamide (PAM), polyacrylic acid (PAA), polymethacrylic acid (PMAA), and polyethylene glycol (PEG or PEO); protective proteins such as bovine serum albumin (BSA), casein, oligopeptides, and polypeptides; surfactants such as Tween 20, Tween 40, Tween 80, Tween 60, and Triton; antibacterial agents such as sodium azide, potassium sorbate, potassium sorbate, and Proclin-300; and inorganic salts such as sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, and tris-HCl. The sealing solution is an aqueous solution, and the water used is pure water, deionized water, or distilled water.

[0063] Furthermore, the hydrophilic polymeric material contained in the sealing solution can be an oligomeric polymer, a polymeric polymer, a cross-linked polymer, or other hydrophilic materials. Specifically, it can be one or more common hydrophilic polymeric materials such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylamide (PAM), polyacrylic acid (PAA), polymethacrylic acid (PMAA), and polyethylene glycol (PEG or PEO). Preferred polymeric materials are PEO, PVA, PVP, and PAM, and more preferably PVA and PVP. The degree of polymerization of the hydrophilic polymeric material contained in the sealing solution can be 100-20000, preferably 4000-20000, and more preferably 8000-12000. The concentration of the hydrophilic polymeric material contained in the sealing solution is 0.05-2%, preferably 0.05-1%, and more preferably 0.1-0.5%.

[0064] Furthermore, the protective protein contained in the blocking solution can be purified protein, polypeptide, or oligopeptide. The protein source can be obtained through processes such as purification of natural substances, chemical synthesis, polypeptide synthesis, or recombinant protein. Specifically, it can be one or more protective proteins such as bovine serum albumin (hereinafter referred to as BSA), casein, oligopeptides, and polypeptides. The preferred protective proteins are BSA and casein, and more preferably BSA. The concentration of the protective protein contained in the blocking solution is 0.01-5%, preferably 0.01-1%, and more preferably 0.1-0.5%.

[0065] Furthermore, the surfactant in the sealing liquid can be a neutral surfactant or anionic surfactant. Specifically, it can be one or more of surfactants such as Tween 20, Tween 40, Tween 80, Tween 60, Triton, and sodium dodecyl sulfate (hereinafter referred to as SDS). The preferred surfactants are Tween 20, Tween 40, Triton, and SDS, and more preferably Tween 20, Tween 40, and SDS. The concentration of the surfactant in the sealing liquid is 0.01-5%, preferably 0.01-1%, and more preferably 0.05-0.5%.

[0066] Furthermore, the antibacterial agent in the blocking solution can be an inorganic or organic compound with antibacterial activity, specifically sodium azide, potassium sorbate, potassium sorbitol, sodium phenylpropionate, isothiazolinone, methylchloroisothiazolinone, methylisothiazolinone, Proclin-300, etc., with preferred antibacterial agents being sodium azide, methylchloroisothiazolinone, methylisothiazolinone, and Proclin-300, and more preferably sodium azide and Proclin-300; the concentration of the antibacterial agent in the blocking solution is 0.001-1%, preferably 0.005-0.5%, and more preferably 0.005-0.05%.

[0067] Furthermore, the inorganic salt in the sealing solution can be one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, tris-HCl, etc., preferably potassium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate, and more preferably disodium hydrogen phosphate and sodium dihydrogen phosphate; the concentration of the inorganic salt in the sealing solution is 10 μM - 100 mM, preferably 10-100 μM, and more preferably 25-100 μM.

[0068] Furthermore, the general preparation method for the sample pad is as follows: Place the prepared sealing solution in a covered glass container, completely immerse the sample pad material in the sealing solution, let it stand for 5-15 minutes, and after the sample pad material has completely absorbed the sealing solution, remove the sample pad and air dry it at 50-60℃ for 30 minutes. Then place the sample pad in a cool and dry environment, controlling the ambient temperature not to exceed 30℃ and the relative humidity not to exceed 5% RH, and let it stand until the sample pad is completely dry. Store it in a cool and dry environment with a relative humidity not exceeding 5% RH for later use.

[0069] According to the embodiments of this utility model, the material of the marking pad can be cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric, or common absorbent materials, etc., preferably cellulose material, polyester film, non-woven fabric, and more preferably cellulose material and polyester film.

[0070] Furthermore, the labeling pad contains a specific partner labeled by the signal microspheres. The specific partner includes small molecule organic compounds and biological macromolecules (such as nucleic acids, antibodies, antigens, haptens, etc.). This molecule has the ability to specifically recognize, bind, pair, and capture another molecular substance (such as the analyte molecule). It can form a "double antibody sandwich" model with the specific binding partner and the analyte molecule contained on the immunochromatographic membrane mentioned in this patent to complete specific detection.

[0071] Furthermore, the signal microspheres are substances or particles carrying recognition signals, specifically, they can be one or more of common substances such as colloidal gold, gold nanoparticles, colloidal silver, silver nanoparticles, colored polymer microspheres, fluorescent polymer microspheres, and quantum dots; even further, the materials of the colored polymer microspheres and fluorescent polymer microspheres can be one or more of common polymer materials such as polystyrene (hereinafter referred to as PS), polyglycidyl methacrylate (hereinafter referred to as PGMA), and polyacrylic acid (hereinafter referred to as PAA); the surface functional groups of the signal microspheres can be common organic chemical functional groups such as amino, carboxyl, hydroxyl, mercapto, amide, phenyl, alkyl, and alkenyl, with preferred surface functional groups being amino, carboxyl, hydroxyl, mercapto, and phenyl, and more preferably amino, carboxyl, and phenyl; the particle size of the signal microspheres can be 5-200 nm, preferably 10-100 nm, and even more preferably 25-50 nm.

[0072] Furthermore, the labeling methods for the signal microspheres and specific chaperones include two processes: physical adsorption coating and covalent coupling. Specifically, the two processes are as follows: (1) Taking colloidal gold as an example, the general physical adsorption coating process is as follows: the colloidal gold solution and the specific companion are mixed in a glass container with a lid, and after sealing, the container is placed in a mixer or shaker to mix for 2-6 h. After the colloidal gold and the specific companion are in full contact, the container is left to stand for 30-60 min, and the specific companion labeled with colloidal gold is separated by centrifugation.

[0073] (2) Taking polystyrene colored microspheres on carboxyl surfaces as an example, the general covalent coupling process is as follows: PS microspheres are dispersed in MES solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDCI) and N-hydroxysuccinimide (hereinafter referred to as NHS) are added, the mixture is capped and mixed in a mixer or shaker for 1-2 h, the supernatant is removed by centrifugation, a solution containing a specific chaperone is added, and the mixture is mixed in a mixer or shaker for 4-24 h. The specific chaperone labeled with colloidal gold is separated by centrifugation.

[0074] For specific labeling methods, please refer to LJ Krichka, Ligand-Conjugate Determination, Marcel Dekker, New York, 1985; TH Ji, Bifunctional Reagents, Enzymatic Methods, 1983, 91, 580, and other relevant publications.

[0075] Furthermore, the general preparation method of the labeling pad is as follows: a working solution is prepared by mixing a solution of the specific companion labeled with signal microspheres (0.3-1 mg / mL), BSA (0.05-3%), sucrose (5-10%), and PEG20000 (0.05-1%). The labeling pad is immersed in the working solution (or the working solution is sprayed onto the labeling pad using a spraying method). After the working solution has completely immersed the labeling pad, the labeling pad is placed in a cool and dry environment, with the ambient temperature controlled not higher than 30°C and the relative humidity not higher than 5% RH. The pad is allowed to stand until it is completely dry and then stored in a cool and dry environment with a relative humidity not higher than 5% RH for later use.

[0076] According to the embodiments of this utility model, the immunochromatographic membrane material is mainly a large-pore microporous filter membrane material, specifically, it can be a nitrocellulose membrane (hereinafter referred to as NC membrane), a polyethersulfone membrane (hereinafter referred to as PES membrane), a cellulose acetate membrane (hereinafter referred to as CA membrane), a mixed cellulose ester membrane (hereinafter referred to as MCE membrane), a polyvinylidene fluoride membrane (hereinafter referred to as PVDF membrane), a nylon membrane (hereinafter referred to as NY membrane), a polycarbonate nuclear track etched membrane (hereinafter referred to as PCTE membrane), etc. Preferred immunochromatographic membrane materials are NC membrane, CA membrane, MCE membrane, and PVDF membrane, and more preferably NC membrane and CA membrane; the functional groups on the surface of the immunochromatographic membrane can be hydroxyl, amino, etc. The immunochromatographic membrane contains common organic chemical functional groups such as aldehyde and carboxyl groups, each of which is adapted to different antibody binding modes. It has one or more detection lines T (from T1 to Tn) and one control line C. Detection lines T are regions containing specific binding partners, and the control line is a region containing the corresponding immunoglobulin (such as sheep IgG) or contains a secondary antibody of species origin. The number of detection lines T can be 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1-10, and more preferably 1-5. The width of the detection lines T and control line C can be 1-5 mm, preferably 1-3 mm, and more preferably 1-2 mm.

[0077] According to the embodiments of this utility model, there are two methods for immobilizing specific binding partners on the immunochromatographic membrane: physical adsorption coating and covalent coupling. Specifically, the two methods are as follows: (1) Taking the specific binding partner as an example, the general process of physical adsorption coating is as follows: the specific binding partner is dispersed in the aqueous solution of PBS (pH = 7.4) to prepare a working solution. The prepared solution is loaded into the liquid line of the membrane scribing machine. After connecting the accessories, the membrane scribing machine is used to scribing the corresponding position on the immunochromatographic membrane (the scribing can also be completed by a manual pipette or other pipette device). After completion, the membrane is placed in a cool and dry environment with the ambient temperature not exceeding 30°C and the relative humidity not exceeding 5% RH until the membrane is completely dry. It is then stored in a cool and dry environment with a relative humidity not exceeding 5% RH for later use.

[0078] (2) Taking the NC membrane with a carboxyl surface as an example, the general process for covalent coupling is as follows: Immerse the NC membrane in 5-10% EDCI and 5-10% NHS solution, seal it, and mix it by shaking or on a shaker for 2-4 h. Then remove the supernatant and place it in a cool, dry environment, controlling the ambient temperature not to exceed 30℃ and the relative humidity not to exceed 5% RH, until the chromatography membrane is completely dry. Disperse the specific binding partner in an aqueous solution of PBS (pH = 7.4) to prepare a working solution. Put the prepared solution into the liquid line of the membrane scribing machine, connect the accessories, and use the membrane scribing machine to scrib the corresponding position on the immunochromatographic membrane for coupling (a manual pipetting device such as a pipette can also be used to complete the scribing coupling). After standing for 1 h, immerse the NC membrane in an aqueous solution of 1-3% ethanolamine and 3-5% BSA, seal it, and mix it by shaking or on a shaker for 30 min. After completion, place it in a cool, dry environment, controlling the ambient temperature not to exceed 30℃ and the relative humidity not to exceed 5%. Allow the chromatographic membrane to stand until it is completely dry (RH), then store it in a cool, dry environment with a relative humidity not exceeding 5% RH for later use.

[0079] According to the embodiments of this utility model, the absorbent pad material can be cellulose material, filter paper, polyester membrane, glass cellulose membrane, non-woven fabric, or common absorbent materials, etc., preferably cellulose material, polyester membrane, non-woven fabric, and more preferably cellulose material and polyester membrane.

[0080] According to the embodiments of this utility model, the sample pad, labeling pad, immunochromatographic membrane, and absorbent pad can be cut by a cutting machine. The cutting size varies depending on the usage requirements. After cutting, the above materials are tightly glued to the transparent base plate in sequence. After all the materials are completely glued, they are cut into immunochromatographic test strips with a width of 3-5 mm using a high-speed cutting machine and stored in a cool and dry environment with a relative humidity of no more than 5% RH for later use.

[0081] According to the embodiments of this utility model, when the immunochromatographic test strip is used for blood samples, a blood filtration pad needs to be added between the sample pad and the labeling pad. Specifically, the blood filtration pad can be made of cellulose material, filter paper, polyester membrane, glass cellulose membrane, non-woven fabric, or common absorbent materials, etc., preferably cellulose material, polyester membrane, non-woven fabric, and more preferably cellulose material and glass cellulose membrane.

[0082] According to the embodiments of this utility model, the width of the immunochromatographic test strip can be 1-10 mm, preferably 1-5 mm, and more preferably 3-5 mm.

[0083] The technical solution of this utility model will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only illustrative and explanatory of this utility model, and should not be construed as limiting the scope of protection of this utility model. All technologies implemented based on the above content of this utility model are covered within the scope of protection intended by this utility model.

[0084] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0085] Example 1: Preparation and performance evaluation of C-reactive protein-based immunochromatographic test strips This example illustrates the detection of C-reactive protein (CRP), using an anti-CRP monoclonal antibody to qualitatively detect the CRP content in a blood sample.

[0086] Conjugation of mouse anti-C-reactive protein monoclonal antibody with red carboxylated polystyrene microspheres: 50 nm red carboxylated polystyrene microspheres were dispersed in 2-morpholine ethanesulfonic acid (MES) solution (pH = 5.6) at a concentration of 10 mg / mL. 200 mg of EDCI and 200 mg of NHS were added to 1 mL of the microsphere dispersion. After capping, the mixture was vortexed until the EDCI and NHS were completely dissolved. The mixture was then incubated at room temperature for 2 h using an inverting mixer. After the incubation period, the microspheres were enriched by centrifugation, and the supernatant was removed. Mouse anti-C-reactive protein monoclonal antibody (5 mg / mL) was added to 1 mL of the mixture, with MES as the buffer solution. The mixture was capped and incubated at 4°C for 12 h using an inverting mixer. After centrifugation to enrich the microspheres, 1 mL of PBS was added to wash the microspheres. Finally, the labeled polystyrene microspheres were dispersed in 2 mL of PBS solution to prepare a 5 mg / mL dispersion of mouse anti-C-reactive protein antibody-labeled polystyrene microspheres.

[0087] MES solution: 2-morpholinoethanesulfonic acid solution. Dissolve 19.52 g of MES in 80 mL of pure water, adjust the pH to 6.3 with 5 mol / L KOH, and finally dilute to 100 mL with pure water. Filter for sterilization using a pre-treated Nalgene membrane (0.45 μm).

[0088] PBS solution: Weigh out 35.814 g of Na2HPO4·12H2O, 2.4496 g of KH2PO4, 80.0669 g of NaCl, and 2.0129 g of KCl using an analytical balance. Dissolve in 800 mL of pure water. Adjust the pH to 7.4 after dissolution. Transfer the solution to a 1 L volumetric flask and finally dilute to 1 L with pure water.

[0089] Preparation method of mouse anti-goat IgG antibody-coated colloidal gold: 40 nm colloidal gold was dispersed in PBS solution at a concentration of 10 mg / mL. 0.5 mL of this solution was filled with mouse anti-goat IgG antibody at a concentration of 5 mg / mL. The buffer solution was PBS. After capping, the solution was incubated at room temperature and inverted for 2.5 h. After centrifugation to enrich the colloidal gold, 1 mL of PBS was added to wash the gold. Finally, the labeled colloidal gold was dispersed in 1 mL of PBS solution to prepare a 5 mg / mL mouse anti-goat IgG antibody-labeled colloidal gold dispersion.

[0090] Preparation of the sample pad blocking solution: Prepare the blocking solution according to the standard solution preparation method. The specific formula is as follows: 0.5% PVA-8000, 0.5% BSA, 0.05% Tween 20, 0.005% sodium azide, 25 mM sodium chloride.

[0091] Sample pad preparation: In this example, polyester film is selected as the sample pad material, and the sample pad is prepared according to the general preparation scheme of the sample pad described in this patent.

[0092] Preparation of labeling pad working solution: Prepare the blocking solution according to the standard solution preparation method. The specific formula is as follows: 1 mg / mL of mouse anti-C-reactive protein antibody-labeled polystyrene microspheres, 1 mg / mL of mouse anti-goat IgG monoclonal antibody-coated colloidal gold dispersion, 0.5% BSA, 5% sucrose, and 0.1% PEG-20000.

[0093] Preparation of the marking pad: In this example, polyester film is selected as the marking pad material, and the marking pad is prepared according to the general preparation scheme of the marking pad described in this patent.

[0094] Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 1 detection line and 1 control line were prepared. The detection antibody concentration was 50 mg / L and the streak rate was 2 μL / cm. The control line used goat IgG antibody with an antibody concentration of 0.5 mg / mL and a streak rate of 1 μL / cm.

[0095] In this example, cellulose material was selected as the material for the absorbent pad and the blood filtration pad.

[0096] After assembling the above materials according to the immunochromatographic test strip assembly method described in this patent, the strips are cut into 3 mm diameter pieces for later use.

[0097] Actual sample testing: Eight different blood samples were obtained through standardized collection and tested using the C-reactive protein assay kit prepared in this example, which features a fan-shaped direct sample loading interface (used with a solid sampler, siphon diffusion method), traditional immunochromatography, and chemiluminescence detection kits. Specific results are shown in Table 1. Table 1: Comparison of different immunochromatographic methods for C-reactive protein assay

[0098] The results showed that, using chemiluminescence as a reference method, the immunochromatographic assay designed by the traditional dilution method and the method using the immunochromatographic assay device of this invention yielded results that were basically consistent with those of the chemiluminescence method. The immunochromatographic test strip prepared in this example showed consistent results with the chemiluminescence method in the analysis of actual samples, whether in determining positive or negative results or identifying high-risk patients. In contrast, the traditional immunochromatographic assay had the risk of false negatives, for example, different conclusions were obtained for samples 1 and 4.

[0099] Example 2: Preparation and performance evaluation of immunochromatographic test strips for influenza B virus detection. This example illustrates the detection of influenza B virus, using a monoclonal antibody against influenza B virus to qualitatively detect the content of influenza B virus antigen in throat swabs.

[0100] Influenza B virus monoclonal antibody adsorption and coating with colloidal gold: Colloidal gold with a 40 nm carboxyl group surface was dispersed in PBS solution at a concentration of 10 mg / mL. 0.5 mL of the colloidal gold dispersion was then added to 0.5 mL of the dispersion containing influenza B virus monoclonal antibody at a concentration of 5 mg / mL. The buffer solution was PBS. After capping, the mixture was incubated at room temperature and inverted for 2.5 h. After centrifugation to enrich the colloidal gold, 1 mL of PBS was added to wash the gold. Finally, the labeled colloidal gold was dispersed in 1 mL of PBS solution to prepare a 5 mg / mL influenza B virus antibody-labeled colloidal gold dispersion.

[0101] In this example, the marking pad, sample pad, and absorbent pad are all made of polyester film.

[0102] The label pad, sample pad, and absorbent pad in this example are prepared according to the method in Example 1.

[0103] Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 1 detection line and 1 control line were prepared. The concentration of the influenza B virus monoclonal antibody labeling was 0.5 mg / mL, and the streak rate was 2 μL / cm. The control line used goat IgG antibody with an antibody concentration of 0.5 mg / mL and a streak rate of 1 μL / cm.

[0104] After assembling the above materials according to the immunochromatographic test strip assembly method described in this patent, the strips are cut into 3 mm diameter pieces for later use.

[0105] Actual sample testing: Eight different pharyngeal swab samples were obtained through standardized collection and tested using the influenza B immunochromatographic reagent card with a threaded interface (used with a hollow sampler) prepared according to the present invention and the influenza B real-time PCR kit. Specific results are shown in Table 2. Table 2: Comparison of Influenza B Immunochromatographic Test Strips and Influenza B Quantitative PCR Kits

[0106] The results showed that the immunochromatographic test strip prepared in this example yielded consistent results with the PCR detection method in terms of both positive and negative results and patient status assessment in the analysis of actual samples.

[0107] The embodiments of the present utility model have been described above by way of example. It should be understood that the protection scope of the present utility model is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present utility model should be included within the protection scope of the claims of this application.

Claims

1. A direct sample loading immunochromatographic kit, the kit having a sample loading port, the sample loading port having a sample loading port interface, the sample loading port interface being compatible with an interface of a sampler, such that the sampler is installed into the sample loading port of the chromatography kit.

2. The immunochromatographic kit according to claim 1, characterized in that, The sample loading port of the chromatography kit is of the fan-shaped, threaded, enamel-type, or ground-plate type.

3. The immunochromatographic kit according to claim 1 or 2, characterized in that, The chromatography kit includes a housing that contains chromatography strips, the chromatography strips including a sample pad, a labeling pad, an immunochromatographic membrane, and an absorbent pad.

4. The immunochromatographic kit according to claim 3, characterized in that, The chromatography kit housing has an extended chromatography strip outlet, through which the chromatography strip can extend to siphon diffusion liquid.

5. A direct sample loading immunochromatographic assay device, characterized in that, The device includes a chromatography kit and a sampler; the chromatography kit has a sample loading port with a sample loading port interface; the sampler includes a main body and a sampling pad, the sampler has a sampling pad at its bottom end, the sampler main body has an interface located above the sampling pad, which can be matched with the sample loading port interface of the chromatography kit, so that the sampler can be installed into the sample loading port of the chromatography kit.

6. The immunochromatographic apparatus according to claim 5, characterized in that, The sample loading port of the chromatography kit is fan-shaped, threaded, enamel-coated, or ground-glass type; the interface above the sampling pad is fan-shaped, threaded, enamel-coated, or ground-glass type.

7. The immunochromatographic apparatus according to claim 6, characterized in that, The chromatography kit includes a housing that contains chromatography strips, the chromatography strips including a sample pad, a labeling pad, an immunochromatographic membrane, and an absorbent pad.

8. The immunochromatographic apparatus according to claim 7, characterized in that, The chromatography kit housing has an extended chromatography strip outlet, through which the chromatography strip can extend to siphon diffusion liquid.

9. The immunochromatographic apparatus according to any one of claims 5-8, characterized in that, The main structure of the sampler is either hollow or solid.

10. A reagent kit, characterized in that, The kit includes the direct loading immunochromatographic assay kit according to any one of claims 1-4, or the direct loading immunochromatographic assay device according to any one of claims 5-9.

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