Semi-quantitative immunochromatography test paper and semi-quantitative immunochromatography method

Abstract

The invention provides semi-quantitative immunochromatography test paper, a direct loading immunochromatography device comprising the test paper, and an immunochromatography method for directly performing semi-quantitative reading on a result through naked eyes. A plurality of detection lines are arranged on the semi-quantitative immunochromatography test paper, and when an object to be detected in a sample is combined with a specific partner marked by signal microspheres in the marking pad, in the process of passing through a chromatography film, different numbers of detection lines can be presented due to the content difference of the object to be detected; and semi-quantitative detection can be carried out on the to-be-detected substance in the sample according to the number of the detection lines.

Description

Technical Field

[0001] The present invention relates to the field of diagnostic testing, and in particular to a semi-quantitative immunochromatographic test paper, a preparation method thereof, and a semi-quantitative immunochromatographic method using the test paper. The semi-quantitative immunochromatographic method can directly perform semi-quantitative readings of the results with the naked eye. Background Art

[0002] Quantitative testing is a very important part of the diagnostic testing industry. Current quantitative diagnostic testing methods often require the intervention of large-scale instruments and equipment and the training of professionals, and the testing process takes a relatively long time. For example, fluorescent quantitative PCR testing takes about 60-80 minutes to complete the test and output the results after sample collection, and ELISA testing also takes about 40-60 minutes. Such stringent testing requirements are difficult to accept for patients with sudden illnesses. For example, for patients with acute myocardial infarction, a long wait for results will result in missing the best time for rescue. Immunochromatography (Lateral flow immunoassay) is one of the commonly used diagnostic testing methods. It has the advantages of simple operation, rapid detection, small sample requirement, easy storage, and portability. However, immunochromatography methods are most commonly used for qualitative testing (distinguishing between negative and positive), and there are relatively few reports on immunochromatography products and technologies for quantitative testing.

[0003] Zhang's team (Daohong Zhang, Peiwu Li, Qi Zhang, etc., Analytica Chimica Acta, 2012, 740, 74-79) reported a method for capturing and detecting the same substance by marking three test lines on the same immunochromatographic test paper. Specifically, the method uses a competitive method. When the sample does not contain the molecule to be tested, the colloidal gold-labeled antibody will be captured by the test line, forming three lines. As the amount of analyte in the sample increases, the colloidal gold content that can be captured by the test line decreases, so the corresponding test line will not appear. However, compared with the double antibody sandwich method, the competitive method has poor specificity and is easily affected by external interference.

[0004] Paek's team (Joung-Hwan Cho, Se-Hwan Paek, Biotech. and Bioengin., 2001, 75, 6) reported a competitive quantitative immunochromatographic assay. Specifically, the method utilizes a bifunctional biotinylated colloidal gold-labeled antibody to compete with a biotin-labeled antibody for binding to the analyte in the sample, thereby achieving quantitative detection.

[0005] CN109900890A discloses an immunochromatographic test paper method for detecting small molecules using a black phosphorus-gold nanoparticle complex. After the immunochromatographic operation is completed, the sample is irradiated with an 808nm laser, and then an infrared imager receives the relevant signal. The capture temperature is calculated using an infrared thermometer, thereby calculating the content of the molecule to be tested.

[0006] CN110596376A discloses a method for detecting immunochromatographic signals using a resistance sensor. Specifically, a discontinuous conductive coating material is printed on a nitrocellulose membrane. The material directly releases electrical signals during the antigen-antibody reaction without relying on other non-immune reaction signals.

[0007] CN115754275A discloses a method of combining magnetic particles as a new type of missing substance with immunochromatography technology. Specifically, magnetic particles are used instead of traditional colloidal gold or fluorescent microspheres. After being labeled with corresponding specific antibodies, they are added to the labeling pad to complete relevant detection. Finally, the quantitative work of the sample to be tested can be completed by a magnetic quantitative detector.

[0008] CN110988356A discloses a method for rapid quantitative immunochromatographic analysis of C-reactive protein. This patent utilizes a method for covalently binding carboxylated colloidal gold to a specific antibody to prepare a test strip. After completing conventional immunochromatographic analysis, the corresponding strips are read using an immunochromatographic analyzer, and the sample to be tested is quantified by plotting a standard curve.

[0009] CN204330777U discloses an instrument-free immunochromatographic colloidal gold technology solution that can quickly detect the MPO concentration in the blood. Specifically, the test paper uses a color card comparison method (or a colloidal gold quantitative reader) to compare the colors of the test lines after testing to achieve quantitative detection of the sample to be tested. However, the color card is obtained by printing, so when comparing colors, color difference often leads to misinterpretation of the results.

[0010] In summary, traditional immunochromatographic quantitative testing relies primarily on external instruments to read the grayscale, brightness, and fluorescence values ​​of the test strips, or on pre-printed color charts for color comparison, thereby achieving quantitative detection of the desired indicator. Currently, there is a lack of immunochromatographic methods that can directly read the test results semi-quantitatively with the naked eye, enabling rapid, simple, instrument-free, and color chart-free semi-quantitative testing. Summary of the Invention

[0011] The first object of the present invention is to provide a semi-quantitative immunochromatographic test paper, the second object of the present invention is to provide a direct-loading immunochromatographic kit comprising the semi-quantitative immunochromatographic test paper, the third object of the present invention is to provide a direct-loading immunochromatographic device comprising the semi-quantitative immunochromatographic test paper, and the fourth object of the present invention is to provide an immunochromatographic method using the test paper, the immunochromatographic kit, or the immunochromatographic device, in which the results can be directly read semi-quantitatively by the naked eye.

[0012] Specifically, the principle of the semi-quantitative immunochromatography method of the present invention is to set multiple detection lines on the test paper, and each detection line is fixed with a quantitative specific binding partner (antigen, hapten, antibody, protein complex, etc.). When the analyte in the sample binds to the specific partner labeled with signal microspheres (colloidal gold, colloidal silver, colored latex microspheres, carbon nanospheres, etc.) in the labeling pad, different numbers of detection lines will appear due to the difference in the content of the analyte in the process of passing through the chromatography membrane. Finally, the analyte in the sample can be semi-quantitatively detected by the number of detection lines.

[0013] The technical solutions of the present invention are as follows:

[0014] A semi-quantitative immunochromatographic test paper comprises a base plate, a sample pad, a marking pad, an immunochromatographic membrane and a water-absorbing pad. On the base plate, the sample pad, the marking pad, the immunochromatographic membrane and the water-absorbing pad are arranged in order along the chromatography direction. One end of the sample pad is adhered to the base plate, and the other end is tightly pressed against the marking pad, which contains a specific partner marked by a signal microsphere. One end of the marking pad is tightly pressed against the immunochromatographic membrane, which has multiple detection lines T and a quality control line C. The detection line T contains a specific binding partner, and the quality control line contains a corresponding immunoglobulin, or the quality control line C contains a secondary antibody of a quantitative antibody species. The other end of the immunochromatographic membrane is pressed under the water-absorbing pad to form the immunochromatographic test paper.

[0015] According to an embodiment of the present invention, the material of the sample pad is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, and more preferably one or more of cellulose material and polyester film.

[0016] According to an embodiment of the present invention, the sample pad contains a blocking solution, and the blocking solution comprises: a hydrophilic polymer material, a protective protein, an antibacterial agent and an inorganic salt; preferably, the blocking solution is an aqueous solution.

[0017] According to an embodiment of the present invention, the method for preparing the sample pad includes the following steps: completely immersing the sample pad material in a blocking solution, leaving it to stand for a certain period of time, and after the sample pad material completely absorbs the blocking solution, taking out the sample pad and drying it.

[0018] According to an embodiment of the present invention, the material of the marking pad is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, and further preferably one or more of cellulose material and polyester film.

[0019] According to an embodiment of the present invention, the specific partner includes small molecule organic compounds and biological macromolecules. The specific partner has the ability to specifically identify, bind, pair and capture the analyte molecules, and can form a "double antibody sandwich" model with the specific binding partner and the analyte molecules fixed on the immunochromatographic membrane to complete specific detection.

[0020] According to an embodiment of the present invention, the signal microspheres are a substance or particle with an identification signal. Preferably, the signal microspheres are selected from one or more of colloidal gold, gold nanoparticles, colloidal silver, silver nanoparticles, colored polymer microspheres, fluorescent polymer microspheres, and quantum dots.

[0021] According to an embodiment of the present invention, the labeling method of the signal microspheres and the specific partner is selected from a physical adsorption coating process or a covalent coupling process.

[0022] According to an embodiment of the present invention, the physical adsorption coating process of the signal microspheres and the specific partner comprises the following steps: mixing the signal microsphere solution with the specific partner to obtain the specific partner labeled with the signal microspheres.

[0023] According to an embodiment of the present invention, the covalent coupling process of the signal microspheres and the specific partner includes the following steps: dispersing the signal microspheres in a solution, adding a condensing agent, reacting for a period of time, adding a solution containing a specific partner, and continuing the reaction for a period of time to obtain the specific partner that marks the signal microspheres.

[0024] According to an embodiment of the present invention, the covalent coupling reaction includes an amidation condensation reaction, a sulfonamidation condensation reaction, a urea-forming reaction, an ester-forming reaction, a guanidine-forming reaction, an imine-forming reaction, a triazole-forming reaction, a biotin-streptavidin-forming reaction, a disulfide bond-forming reaction, a thioester-forming reaction, a reaction.

[0025] According to an embodiment of the present invention, the condensing agent for the amidation condensation reaction may be a carbodiimide condensing agent such as dicyclohexylcarbodiimide (hereinafter referred to as DCC), diisopropylcarbodiimide (hereinafter referred to as DIC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (hereinafter referred to as EDCI), or may be a heterocyclic condensing agent such as 4-dimethylaminopyridine (hereinafter referred to as DMAP), 1-hydroxybenzotriazole (hereinafter referred to as HOBT), N-hydroxy-7-azabenzotriazole (hereinafter referred to as HOAt), N-hydroxysuccinimide (hereinafter referred to as NHS), or may be a combination of one or more types of onium salt condensing agents such as HATU, HCTU, HAPyU, TBTU, preferably one or more of EDCI, NHS, HOBT, HATU, HCTU, and more preferably one or more of EDCI, NHS, and HATU.

[0026] According to an embodiment of the present invention, the material of the immunochromatography membrane is selected from a microporous filter membrane material with a large pore size; the preferred immunochromatography 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 nuclear track etched filter membrane; further preferred immunochromatography 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; and further preferred immunochromatography membrane material is selected from one or more of nitrocellulose membrane and cellulose acetate filter membrane.

[0027] According to an embodiment of the present invention, the number of the detection lines T is 2-20, preferably 2-10, more preferably 2-5, and even more preferably 2, 3 or 4.

[0028] According to an embodiment of the present invention, the method of immobilizing the specific binding partner on the immunochromatographic membrane is selected from a physical adsorption coating process or a covalent coupling process.

[0029] According to an embodiment of the present invention, the physical adsorption coating process for fixing a specific binding partner on an immunochromatographic membrane includes the following steps: dispersing the specific binding partner in an aqueous solution of PBS to prepare a working solution, loading the prepared solution into the liquid pipeline of a film marking machine, and using the film marking machine to mark and coat the corresponding positions on the immunochromatographic membrane.

[0030] According to an embodiment of the present invention, the covalent coupling process for fixing a specific binding partner on an immunochromatographic membrane includes the following steps: immersing the immunochromatographic membrane in a solution containing a condensing agent (such as one or more of EDCI and NHS), reacting for a period of time, and drying the immunochromatographic membrane; dispersing the specific binding partner in an aqueous solution of PBS to prepare a working solution, loading the prepared solution into the liquid pipeline of a film marking machine, and using the film marking machine to mark the corresponding positions on the immunochromatographic membrane for coupling.

[0031] According to an embodiment of the present invention, the definition of the condensing agent used in the covalent coupling process of fixing the specific binding partner on the immunochromatographic membrane refers to the condensing agent used in the covalent coupling process of the signal microspheres and the specific partner of the present invention.

[0032] According to an embodiment of the present invention, the absorbent pad material is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbent materials, preferably one or more of cellulose material, polyester film, non-woven fabric, and further preferably one or more of cellulose material and polyester film.

[0033] A semi-quantitative immunochromatography method comprises the following steps: (1) performing immunochromatography using the immunochromatography test paper of the present invention; and (2) directly performing a semi-quantitative reading of the result after the immunochromatography is completed.

[0034] A direct loading immunochromatography kit, comprising the immunochromatography test paper of the present invention and a shell for accommodating the immunochromatography test paper; the shell is provided with a loading port, the loading port having a loading port interface, the loading port interface being compatible with an interface of a sampler, so that the sampler can be installed at the loading port of the chromatography kit.

[0035] According to an embodiment of the present invention, the chromatography kit sample loading port interface can be a common engineering interface type, such as a fan-shaped, threaded, enamel-shaped, or ground-shaped interface; preferably, the chromatography kit sample loading port interface is a fan-shaped bayonet or a threaded interface.

[0036] According to an embodiment of the present invention, the housing is provided with an observation window.

[0037] According to an embodiment of the present invention, the chromatography kit has an extended test paper outlet, and the chromatography test paper can extend through the outlet to siphon the diffusion liquid.

[0038] A direct loading immunochromatographic device comprises a chromatography kit and a sampler; the chromatography kit comprises the immunochromatographic test paper of the present invention and a shell for accommodating the immunochromatographic test paper; the shell is provided with a loading port, and the loading port has a loading port interface; the sampler comprises a main body and a sampling pad, the bottom end of the sampler is provided with a sampling pad, the sampler main body has an interface, the interface is located above the sampling pad and can be matched with the loading port interface of the chromatography kit, so that the sampler can be installed on the loading port of the chromatography kit.

[0039] According to an embodiment of the present invention, the sample loading port interface of the chromatography kit can be a common engineering interface type, such as a fan-shaped type, a threaded type, an enamel-shaped type, a ground-shaped type, etc.

[0040] According to an embodiment of the present invention, the sample loading port interface of the chromatography kit is a fan-shaped bayonet interface or a threaded interface.

[0041] According to an embodiment of the present invention, the interface above the sampling pad can be a common engineering interface type, such as a fan-shaped type, a threaded type, an enamel-shaped type, a ground-shaped type, etc.

[0042] According to an embodiment of the present invention, the interface above the sampling pad is a fan-shaped bayonet or a threaded bayonet.

[0043] According to an embodiment of the present invention, the sampler further comprises a handheld rod, wherein the handheld rod, the main body and the sampling pad are arranged in sequence along the longitudinal direction of the sampler, and the handheld rod is located at the top of the sampler for handheld operation.

[0044] According to an embodiment of the present invention, the sampler main body structure is a hollow structure or a solid structure.

[0045] According to an embodiment of the present invention, the housing is provided with an observation window.

[0046] According to an embodiment of the present invention, the chromatography kit housing has an extended chromatography test paper outlet, and the chromatography test paper can extend through the outlet to siphon the diffusion liquid.

[0047] According to an embodiment of the present invention, the sampling pad is made of flocking or sponge.

[0048] A direct loading immunochromatography method comprises using the direct loading immunochromatography kit or the direct loading immunochromatography device of the present invention, and comprises the following steps: after sampling at a site to be sampled with a sampler, the sampler is installed on the loading port of the chromatography kit without dilution with a diluent, immunochromatography is performed with a chromatographic diffusion fluid, and the results are directly semi-quantitatively read.

[0049] According to an embodiment of the present invention, the direct loading immunochromatography method is applied to sample detection in medical diagnosis, medical testing, food, agriculture or animal husbandry fields.

[0050] According to an embodiment of the present invention, the direct loading immunochromatography method targets samples selected from one or more of medical samples, environmental swab samples, feed and plant tissue homogenates.

[0051] According to an embodiment of the present invention, the medical sample is selected from one or more of whole blood, plasma, serum, urine, tears, sweat, saliva, oral swab, anal test paper, alveolar lavage fluid and cerebrospinal fluid.

[0052] Beneficial effects

[0053] The present invention provides multiple detection lines on the test paper, and each detection line is fixed with a quantitative specific binding partner (antigen, hapten, antibody, protein complex, etc.). When the analyte in the sample binds to the specific partner labeled by signal microspheres (colloidal gold, colloidal silver, colored latex microspheres, carbon nanospheres, etc.) in the labeling pad, different numbers of detection lines will appear due to the difference in the content of the analyte in the process of passing through the chromatography membrane. Finally, the analyte in the sample can be semi-quantitatively detected by the number of detection lines.

[0054] The immunochromatographic test strips described in the present invention can be applied to testing scenarios in multiple fields, including medical diagnosis, food, animal husbandry, and agriculture. They can be used for a variety of common medical samples, including whole blood, plasma, serum, urine, tears, sweat, saliva, oral swabs, anal test strips, alveolar lavage fluid, and cerebrospinal fluid, as well as common samples from other fields, such as environmental wipe samples, feed, and plant tissue homogenates. The collection methods for various samples can refer to standard collection methods in various fields. After collection, the samples do not require excessive purification, enabling rapid, simple, instrument-free, and color card-free semi-quantitative testing.

[0055] The immunochromatographic test paper and the immunochromatographic method for directly reading the results semi-quantitatively by the naked eye described in the present invention can be applied to multiple fields such as medical testing, food safety, agricultural planting, and animal husbandry, achieving rapid, simple, and efficient semi-quantitative detection of test substances. The entire process does not require the intervention of large-scale instruments or personnel with complex professional training. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1a A three-dimensional diagram of a direct loading chromatography kit with a fan-shaped interface according to the present invention;

[0057] Figure 1b A three-dimensional diagram of a direct loading immunochromatographic device with a fan-shaped interface according to the present invention;

[0058] Figure 1c This is a front view of the direct loading immunochromatographic device with a fan-shaped interface according to the present invention;

[0059] Figure 2a A three-dimensional diagram of a direct loading immunochromatographic device with a threaded interface according to the present invention;

[0060] Figure 2b This is a front view of the direct loading immunochromatographic device with a threaded interface according to the present invention;

[0061] Figure 3a A perspective view of a direct loading chromatography kit with a "siphon method" extended test strip outlet according to the present invention;

[0062] Figure 3b This is a front view of the direct loading immunochromatographic device with a "siphon method" extended test strip outlet according to the present invention;

[0063] Figure 4 This is a structural diagram of the semi-quantitative immunochromatographic test paper of the present invention;

[0064] Figures 1a-1c Among them, 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, housing; 19, chromatography test paper;

[0065] Figure 2a-2b Among them, 21, chromatography kit; 22, sampler; 23, sample loading port; 24, sample loading port threaded interface; 25, sampling pad; 26, threaded interface on sampler; 27, observation window; 28, housing; 29, chromatography test paper;

[0066] Figure 3a-3b Among them, 31. "Siphon method" extends the test paper outlet; 32. "Siphon method" extends the test paper;

[0067] Figure 4 In the figure, 41, bottom plate; 42, sample pad; 43, labeling pad; 44, immunochromatographic membrane; 45, absorbent pad; 46, blood filter pad. DETAILED DESCRIPTION

[0068] The invention provides a semi-quantitative immunochromatographic test paper, comprising a base plate, a sample pad, a marking pad, an immunochromatographic membrane and a water-absorbing pad. On the base plate, the sample pad, the marking pad, the immunochromatographic membrane and the water-absorbing pad are arranged in sequence along the chromatography direction; one end of the sample pad is adhered to the base plate, and the other end is tightly pressed on the marking pad, the marking pad contains a specific partner marked by a signal microsphere; one end of the marking pad is tightly pressed on the immunochromatographic membrane, and the immunochromatographic membrane is provided with multiple detection lines T (from T1 to Tn) and a quality control line C, the detection line T contains a quantitative specific binding partner, the quality control line contains a corresponding immunoglobulin (such as sheep IgG), or the detection line T contains a quantitative secondary antibody derived from an antibody species; the other end of the immunochromatographic membrane is pressed under the water-absorbing pad to form the immunochromatographic test paper.

[0069] According to an embodiment of the present invention, the material of the sample pad can be one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, and more preferably one or more of cellulose material and polyester film.

[0070] According to an embodiment of the present invention, the sample pad contains a blocking solution, which comprises: hydrophilic polymer materials, such as polyvinyl alcohol (hereinafter referred to as PVA), polyvinyl pyrrolidone (hereinafter referred to as PVP), polyacrylamide (hereinafter referred to as PAM), polyacrylic acid (hereinafter referred to as PAA), polymethacrylic acid (hereinafter referred to as PMAA), polyethylene glycol (hereinafter referred to as PEG or PEO), and other common hydrophilic polymer materials; protective proteins, such as bovine serum albumin (hereinafter referred to as BSA), casein, oligopeptides, polypeptides, and the like; surfactants, such as Tween 20, Tween 40, Tween 80, Tween 60, and Triton, and the like; 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, and the like. The blocking solution is an aqueous solution, and the water used can be purified water, deionized water, distilled water, and the like.

[0071] According to an embodiment of the present invention, the hydrophilic polymer material contained in the blocking solution may be a hydrophilic material such as an oligomeric polymer, a polymeric polymer, or a cross-linked polymer. Specifically, it may be a mixture of common hydrophilic polymer materials such as polyvinyl alcohol (hereinafter referred to as PVA), polyvinyl pyrrolidone (hereinafter referred to as PVP), polyacrylamide (hereinafter referred to as PAM), polyacrylic acid (hereinafter referred to as PAA), polymethacrylic acid (hereinafter referred to as PMAA), polyethylene glycol (hereinafter referred to as PEG or PEO), etc. The preferred polymer materials are PEO, PVA, PVP, and PAM, and more preferably PVA and PVP. The degree of polymerization of the hydrophilic polymer material contained in the blocking solution may be 100-20,000, preferably the degree of polymerization of the polymer material is 4,000-20,000, and more preferably the degree of polymerization is 8,000-12,000. The concentration of the hydrophilic polymer material contained in the blocking solution is 0.05-2%, preferably the concentration of the polymer material is 0.05-1%, and more preferably the concentration is 0.1-0.5%.

[0072] According to an embodiment of the present invention, the protective protein contained in the blocking solution can be a purified protein, polypeptide, or oligopeptide. The protein source can be obtained by processes such as purification of natural substances, chemical synthesis, synthesis by a polypeptide synthesizer, and recombinant protein. Specifically, it can be a mixture of 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 the concentration of the protective protein is 0.01-1%, and further preferably 0.1-0.5%.

[0073] According to an embodiment of the present invention, the surfactant in the blocking solution can be a neutral surfactant and an anionic surfactant, specifically, a mixture of one or more surfactants such as Tween 20, Tween 40, Tween 80, Tween 60, Triton, sodium dodecyl sulfate (hereinafter referred to as SDS), etc. 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 blocking solution is 0.01-5%, preferably 0.01-1%, and more preferably 0.05-0.5%.

[0074] According to an embodiment of the present invention, the antibacterial agent in the sealing solution can be an inorganic or organic compound with antibacterial activity, specifically sodium azide, potassium sorbate, potassium sorbate, sodium phenylpropionate, isothiazolinone, methylchloroisothiazolinone, methylisothiazolinone, Proclin-300, etc. The preferred antibacterial agents are sodium azide, methylchloroisothiazolinone, methylisothiazolinone, Proclin-300, and more preferably sodium azide and Proclin-300; the concentration of the antibacterial agent in the sealing solution is 0.001-1%, preferably the antibacterial agent concentration is 0.005-0.5%, and more preferably the concentration is 0.005-0.05%.

[0075] According to an embodiment of the present invention, the inorganic salt in the blocking solution can be a mixture of 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, sodium dihydrogen phosphate, and more preferably disodium hydrogen phosphate and sodium dihydrogen phosphate; optionally, the concentration of the inorganic salt in the blocking solution is 10 μM-100 mM, preferably 10-100 μM, and more preferably 25-100 μM.

[0076] According to an embodiment of the present invention, the general preparation scheme of the sample pad is as follows: the prepared blocking solution is placed in a glass container with a lid, the sample pad material is completely immersed in the blocking solution, and it is allowed to stand for 5-15 minutes. After the sample pad material completely absorbs the blocking solution, the sample pad is removed and air-dried at 50-60°C for 30 minutes. The sample pad is then placed in a cool and dry environment with an ambient temperature of no more than 30°C and a relative humidity of no more than 5% RH. The sample pad is allowed to stand until it is completely dry and stored in a cool and dry environment with a relative humidity of no more than 5% RH until it is ready for use.

[0077] According to an embodiment of the present invention, the material of the marking pad can be one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, and further preferably one or more of cellulose material and polyester film.

[0078] According to an embodiment of the present invention, the labeling pad contains a specific partner labeled with a signal microsphere. The specific partner includes a small molecule organic compound or a biomacromolecule (such as a nucleic acid, an antibody, an antigen, a hapten, etc.). The specific partner has the ability to specifically recognize, bind, pair, and capture another molecular substance (such as a molecule to be detected). It can form a "double antibody sandwich" model with the specific binding partner and the molecule to be detected fixed on the immunochromatographic membrane mentioned in this patent to complete specific detection. Preferably, the specific partner is selected from one or more of mouse anti-C-reactive protein monoclonal antibody, mouse anti-sheep IgG monoclonal antibody, FSH monoclonal antibody, luteinizing hormone (LH) monoclonal antibody, estradiol (E3G) monoclonal antibody, and influenza B virus monoclonal antibody.

[0079] According to an embodiment of the present invention, the signal microspheres are a substance or particle with an identification signal, specifically, they can be a mixture of one or more common substances such as colloidal gold, gold nanoparticles, colloidal silver, silver nanoparticles, colored polymer microspheres, fluorescent polymer microspheres, quantum dots, etc.; further, the materials of the colored polymer microspheres and fluorescent polymer microspheres can be a mixture of one or more common polymer materials such as polystyrene (hereinafter referred to as PS), poly(glycidyl methacrylate) (hereinafter referred to as PGMA), polyacrylic acid (hereinafter referred to as PAA), etc.; the surface functional groups of the signal microspheres can be common organic chemical functional groups such as amino, carboxyl, hydroxyl, thiol, amide, phenyl, alkyl, alkenyl, etc., and the preferred surface functional groups are amino, carboxyl, hydroxyl, thiol, phenyl, and more preferably amino, carboxyl, and phenyl; the particle size of the signal microspheres can be 5-200nm, preferably 10-100nm, and more preferably 25-50nm.

[0080] According to an embodiment of the present invention, the signal microspheres and the specific partner are labeled by physical adsorption coating and covalent coupling. Specifically, the two processes are as follows:

[0081] (1) Taking colloidal gold as an example, the general physical adsorption coating process is as follows: the colloidal gold solution is mixed with the specific partner in a covered glass container, which is sealed and placed on a mixer or shaker for 2-6 hours. After the colloidal gold and the specific partner are fully in contact, the container is allowed to stand for 30-60 minutes and the specific partner labeled with colloidal gold is separated by centrifugation.

[0082] (2) Taking the polystyrene colored microspheres with carboxyl surfaces as an example, the general covalent coupling process is as follows: the PS microspheres are dispersed in a 2-morpholineethanesulfonic acid (hereinafter referred to as MES) solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDCI) and N-hydroxysuccinimide (hereinafter referred to as NHS) are added, and after capping, the mixture is mixed on a mixer or shaker for 1-2 hours, and the supernatant is removed by centrifugation. After adding a solution containing a specific partner, the mixture is continued to be mixed on a mixer or shaker for 4-24 hours, and the specific partner labeled with the polystyrene colored microspheres is separated by centrifugation.

[0083] Furthermore, the covalent coupling labeling process can refer to "amidation condensation reaction", "bioorthogonal reaction" and commonly used biocoupling methods. In addition to the amidation reaction combination shown in the above example, labeling can also be performed using methods including but not limited to the following combinations, or labeling methods commonly used and easily thought of by people in the industry.

[0084]

[0085]

[0086]

[0087] According to an embodiment of the present invention, the amidation condensation reaction condensing agent may be a carbodiimide condensing agent such as dicyclohexylcarbodiimide (hereinafter referred to as DCC), diisopropylcarbodiimide (hereinafter referred to as DIC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (hereinafter referred to as EDCI), or may be a heterocyclic condensing agent such as 4-dimethylaminopyridine (hereinafter referred to as DMAP), 1-hydroxybenzotriazole (hereinafter referred to as HOBT), N-hydroxy-7-azabenzotriazole (hereinafter referred to as HOAt), N-hydroxysuccinimide (hereinafter referred to as NHS), or may be a combination of one or more types of onium salt condensing agents such as HATU, HCTU, HAPyU, TBTU, preferably EDCI, NHS, HOBT, HATU, HCTU, and more preferably EDCI, NHS, HATU.

[0088] For specific labeling methods, please refer to LJ Krichka, Ligand-Binder Assays, published by Marcel Dekker, New York, 1985; TH Ji, Bifunctional Reagents, Methods in Enzymology, 1983, 91, 580 and other related works.

[0089] According to an embodiment of the present invention, the general preparation scheme of the labeling pad is as follows: a solution of a specific partner labeled with signaling microspheres (0.3-1 mg / mL), BSA (0.05-3%), sucrose (5-10%), and PEG20000 (0.05-1%) are mixed to form a working solution, the labeling pad is immersed in the working solution (or the working solution is sprayed onto the labeling pad by a spraying method), and after the working solution is completely absorbed into the labeling pad, the labeling pad is placed in a cool and dry environment with a temperature controlled not to exceed 30°C and a relative humidity not to exceed 5% RH. The labeling pad is allowed to stand until it is completely dry, and then stored in a cool and dry environment with a relative humidity not to exceed 5% RH until it is ready for use.

[0090] According to an embodiment of the present invention, the material of the immunochromatographic membrane is mainly a large-pore microporous membrane material, specifically, it can be one or more of nitrocellulose membrane (hereinafter referred to as NC membrane), polyethersulfone membrane (hereinafter referred to as PES membrane), cellulose acetate membrane (hereinafter referred to as CA membrane), mixed cellulose ester membrane (hereinafter referred to as MCE membrane), polyvinylidene fluoride membrane (hereinafter referred to as PVDF membrane), nylon membrane (hereinafter referred to as NY membrane), polycarbonate nuclear track etched membrane (hereinafter referred to as PCTE membrane), etc. The preferred immunochromatographic membrane material is one or more of NC membrane, CA membrane, MCE membrane, and PVDF membrane, and the further preferred material is one or more of NC membrane and CA membrane; the functional groups on the surface of the immunochromatographic membrane can be hydroxyl, amino, aldehyde, carboxyl, etc. Common organic chemical functional groups, each type of functional group is adapted to different antibody binding modes; the immunochromatographic membrane has multiple detection lines T (from T1 to Tn) and a quality control line C, the detection line T is an area fixed with a specific binding partner, the quality control line is the corresponding immunoglobulin (such as sheep IgG), or the quality control line is fixed with a secondary antibody of the antibody species, the number of the detection lines T can be 1-20, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, preferably 2-10, more preferably 2-5, further preferably 2, 3 or 4; the width of the detection line T and the quality control line C can be 1-5 mm, preferably 1-3 mm, and further preferably 1-2 mm.

[0091] According to an embodiment of the present invention, the immunochromatographic membrane is immobilized with a specific binding partner and an immunoglobulin (such as goat IgG) corresponding to the analyte, or the quality control line is immobilized with a secondary antibody of the same species.

[0092] According to an embodiment of the present invention, the specific binding partner includes one or more of anti-C-reactive protein antibodies, FSH antibodies, LH antibodies, E3G antibodies, and influenza virus monoclonal antibodies (including influenza A virus monoclonal antibodies and influenza B virus monoclonal antibodies).

[0093] According to an embodiment of the present invention, there are two methods for immobilizing the specific binding partner on the immunochromatographic membrane: physical adsorption coating and covalent coupling. Specifically, the two methods are as follows:

[0094] (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 a PBS (pH = 7.4) aqueous solution to prepare a working solution, the prepared solution is loaded into the liquid pipeline of the film stripper, and after connecting the accessories, the film stripper is used to mark the corresponding position of the immunochromatographic membrane for coating (the marking coating can also be completed using a manual pipetting device such as a pipette). After completion, the ambient temperature is controlled to be no higher than 30°C and the relative humidity is no higher than 5% RH in a cool and dry environment, and the membrane is allowed to stand until it is completely dry. The membrane is then stored in a cool and dry environment with a relative humidity of no more than 5% RH for future use.

[0095] (2) Taking the NC membrane with carboxyl surface as an example, the general process of covalent coupling is as follows: immerse the NC membrane in 5-10% EDCI and 5-10% NHS solution, seal it and shake or shake it for 2-4 hours, then remove the supernatant and place it in a cool and dry environment, control the ambient temperature to be no higher than 30℃ and the relative humidity to be no higher than 5% RH, and let it stand until the chromatographic membrane is completely dry, disperse the specific binding partner in PBS (pH=7.4) aqueous solution to make a working solution, load the prepared solution into the liquid pipeline of the membrane stripper, connect the accessories, use the membrane stripper to draw lines on the corresponding position of the immunochromatographic membrane for coupling (line coupling can also be completed using a manual pipetting device such as a pipette), let it stand for 1 hour, immerse the NC membrane in 1-3% ethanolamine and 3-5% BSA aqueous solution, seal it and shake or shake it for 30 minutes, and then place it in a cool and dry environment, control the ambient temperature to be no higher than 30℃ and the relative humidity to be no higher than 5% RH. RH, let it stand until the chromatography membrane is completely dry, and store it in a cool and dry environment with a relative humidity not higher than 5% RH until use.

[0096] According to an embodiment of the present invention, the material of the absorbent pad can be one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common absorbent materials, preferably one or more of cellulose material, polyester film, non-woven fabric, and further preferably one or more of cellulose material and polyester film.

[0097] According to an embodiment of the present invention, the sample pad, marking pad, immunochromatographic membrane, and absorbent pad can be cut by a cutting machine. The cutting size varies according to different usage requirements. After cutting, the above materials are tightly adhered to the transparent bottom plate in sequence. After all materials are completely adhered, they are cut into 3-5 mm wide immunochromatographic test strips 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 use.

[0098] According to an embodiment of the present invention, the width of the immunochromatographic test paper may be 1-10 mm, preferably 1-5 mm, and more preferably 3-5 mm.

[0099] According to an embodiment of the present invention, when the immunochromatographic test strip is used for blood samples, a blood filter pad is added between the sample pad and the labeling pad. Specifically, the blood filter pad can be made of one or more of cellulose, filter paper, polyester film, glass cellulose film, non-woven fabric, or other common absorbent materials, preferably one or more of cellulose, polyester, and non-woven fabric, and more preferably one or more of cellulose and glass cellulose.

[0100] According to an embodiment of the present invention, the blood filter pad needs to be treated with a blocking solution for the sample pad. The general preparation scheme of the blood filter pad is as follows: the prepared blocking solution is placed in a glass container with a lid, the blood filter pad material is completely immersed in the blocking solution, and the blood filter pad is allowed to stand for 5-15 minutes. After the blood filter pad material completely absorbs the blocking solution, the blood filter pad is removed and air-dried at 50-60°C for 30 minutes. The blood filter pad is then placed in a cool and dry environment, with the ambient temperature controlled to be no higher than 30°C and the relative humidity no higher than 5% RH. The blood filter pad is allowed to stand until it is completely dry and stored in a cool and dry environment with a relative humidity no higher than 5% RH for future use.

[0101] According to an embodiment of the present invention, the immunochromatographic test strip is an immunochromatographic test strip based on C-reactive protein. Specifically, the labeling pad of the test strip contains a mouse anti-C-reactive protein monoclonal antibody labeled with carboxyl polystyrene microspheres and a mouse anti-goat IgG antibody labeled with colloidal gold. The test line of the test strip contains anti-C-reactive protein antibodies. The test strip contains three test lines. Optionally, the concentration of anti-C-reactive protein antibodies in each test line from the first to the third test line is 50, 50, and 200 mg / L, respectively.

[0102] According to an embodiment of the present invention, the immunochromatographic test paper is an immunochromatographic test paper based on follicle-stimulating hormone (FSH). Specifically, the labeling pad of the test paper contains a colloidal gold-labeled FSH monoclonal antibody (FSH monoclonal antibody and colloidal gold physical adsorption coating). The test line of the test paper contains FSH antibody. The quality control line of the test paper contains sheep IgG antibody. The test paper contains 4 test lines. Optionally, the FSH antibody concentration of each test line from the 1st to the 4th test line is 1, 1, 0.5, and 1.5 IU / mL, respectively.

[0103] According to an embodiment of the present invention, the immunochromatographic test paper is a multiple-detection immunochromatographic test paper based on luteinizing hormone (LH) and estradiol (E3G) hormones. Specifically, the labeling pad of the test paper contains anti-LH monoclonal antibodies labeled with carboxyl polystyrene microspheres and E3G monoclonal antibodies labeled with azide polystyrene microspheres. The detection lines of the test paper contain LH antibodies and E3G antibodies, respectively. The test paper contains 2 LH detection lines and 2 E3G detection lines, respectively. Optionally, the concentration of LH antibodies in each detection line from the first to the second LH detection line is 2.5 and 1.5 U / mL, respectively. Optionally, the concentration of E3G antibodies in each detection line from the first to the second LH detection line is 2.5 and 1.5 μg / mL, respectively.

[0104] According to an embodiment of the present invention, the immunochromatographic test strip is an immunochromatographic test strip for detecting influenza B virus. Specifically, the labeling pad of the test strip contains influenza B virus antibodies labeled with carboxyl polystyrene microspheres. The test line of the test strip contains influenza B virus antibodies. The test strip contains two test lines. Optionally, the concentration of the influenza B monoclonal antibody marker in each test line from the first to the second test line is 0.2 and 1.5 mg / mL, respectively.

[0105] The present invention also provides a semi-quantitative immunochromatography method, which comprises the following steps: (1) performing immunochromatography using the immunochromatography test paper of the present invention; and (2) directly performing a semi-quantitative reading of the result by naked eyes after immunochromatography on the test paper.

[0106] According to an embodiment of the present invention, the semi-quantitative immunochromatography method of the present invention is applied to the fields of medical diagnosis, medical testing, food, agriculture, and animal husbandry.

[0107] According to an embodiment of the present invention, the semi-quantitative immunochromatographic method of the present invention targets samples including medical samples such as whole blood, plasma, serum, urine, tears, sweat, saliva, oral swabs, anal test strips, alveolar lavage fluid, cerebrospinal fluid, as well as common samples in other fields such as environmental wipe samples, feed, and plant tissue homogenate.

[0108] The present invention also provides a direct loading immunochromatography kit, which comprises the immunochromatography test paper described in the present invention and a shell for accommodating the immunochromatography test paper; and the shell is provided with a loading port, the loading port having a loading port interface, and the loading port interface can be matched with the interface of the sampler, so that the sampler can be installed on the loading port of the chromatography kit.

[0109] According to an embodiment of the present invention, the chromatography kit sample loading port interface can be a common engineering interface type, such as a fan-shaped, threaded, enamel-shaped, or ground-shaped interface; preferably, the chromatography kit sample loading port interface is a fan-shaped bayonet or a threaded interface.

[0110] According to an embodiment of the present invention, the housing is provided with an observation window.

[0111] According to an embodiment of the present invention, the chromatography kit has an extended test paper outlet, and the chromatography test paper can extend through the outlet to siphon the diffusion liquid.

[0112] The present invention also provides a direct loading immunochromatography device, comprising a chromatography kit and a sampler; the chromatography kit has a loading port, and the loading port has a loading port interface; the chromatography kit includes the immunochromatography test paper described in the present invention and a shell for accommodating the immunochromatography test paper described in the present invention; the sampler includes a main body and a sampling pad, the bottom end of the sampler is provided with a sampling pad, the sampler main body has an interface, the interface is located above the sampling pad, and can be matched with the loading port interface of the chromatography kit, so that the sampler can be installed on the loading port of the chromatography kit.

[0113] The present invention also provides a direct loading immunochromatography method, which uses the direct loading immunochromatography device described in the present invention. The method of use includes: after the sampler is used to sample the part to be sampled, the sampler is installed on the loading port of the chromatography kit without dilution with a diluent, immunochromatography is performed with a chromatographic diffusion liquid, and the results are directly semi-quantitatively read by the naked eye.

[0114] According to an embodiment of the present invention, the direct loading immunochromatography method of the present invention is applied to the fields of medical diagnosis, medical testing, food, agriculture, and animal husbandry.

[0115] According to the embodiment of the present invention, the direct loading immunochromatography method described in the present invention targets samples including medical samples such as whole blood, plasma, serum, urine, tears, sweat, saliva, oral swabs, anal test strips, alveolar lavage fluid, cerebrospinal fluid, as well as common samples in other fields such as environmental wipe samples, feed, and plant tissue homogenate.

[0116] The direct sample loading immunochromatography device provided by the present invention comprises a chromatography kit and a sampler.

[0117] The chromatography kit includes a chromatography test paper and a shell for accommodating the chromatography test paper. The shell is provided with a sample loading port and an observation window. The sample loading port is provided in an area on the shell corresponding to the sample pad of the chromatography test paper to facilitate observation of the color development of the test line and the quality control line. The opening of the sample loading port is conducive to the operator accurately finding the best sample loading position. The sample loading port can be constructed in any shape, such as a circle. The length of the observation window along the chromatography direction is based on the ability to conveniently observe the color development of the test line and the quality control line.

[0118] The sampler includes a hand-held rod, a main body and a sampling pad. The hand-held rod, the main body and the sampling pad are arranged in sequence along the longitudinal direction of the sampler. The sampler main body has an interface, the interface is located above the sampling pad, and the hand-held rod is located at the top of the sampler for hand-held operation. The sampler is made of hollow or solid materials. The sampling pad is arranged at the bottom of the sampler and is made of materials such as flocking / sponge. It is used to directly contact the part to be sampled (such as skin, surface of an object, etc.), collect the sample to be tested (such as biological substances, chemicals, etc.), and release the sample to the sample pad. The interface is arranged above the sampling pad and is used to match and connect with the sample loading port of the chromatography kit. The interface types include common engineering interfaces such as fan-blade type, threaded type, enamel-mouth type, and ground-mouth type, which are used to achieve a close connection between the sampler and the sample loading port of the chromatography kit, ensuring that the sample is not missed, the signal is not lost, and there is no leakage or contamination during the sample transfer process. Different interface types (such as threaded type provides tightening fixation, and ground-mouth type provides a tight fit) adapt to different usage scenarios and improve compatibility and stability. The main body connects the top hand-held rod and the interface, and supports the sampling pad. The main structure of the sampler can be a hollow or solid design. When the sampler is a hollow structure, it allows the chromatography diffusion liquid to be dripped directly into the sample loading port by the drip method, pushing the sample to diffuse to the test paper and perform chromatography detection. When the sampler is a solid structure, the chromatography test paper is extended and the diffusion liquid is absorbed by the siphon effect to complete the chromatography process of the sample. When in use, the sampler does not need a diluent to dilute the sample to be tested. After collecting the sample through the sampling pad, the sample is directly transferred and chromatographically detected through the interface and the sample loading port of the chromatography kit.

[0119] After the sampler is mounted on the sample loading port of the chromatography kit via the interface, the hand-held rod can be removed. Preferably, the hand-held rod is detachably connected to the upper end of the main body.

[0120] During operation:

[0121] 1. When using a sampler for sampling, blood samples can be collected through a sampling pad or inserted into the throat or other parts for sampling. The sample can be saliva / body fluid / serum / plasma / whole blood sample, etc.; or environmental samples can be collected by wiping and other operations, such as the surface of instruments and equipment, inner cavity, ward facilities, ICU room, ambulance and other environments.

[0122] 2. After placing the sampler at the site to be sampled, insert the sampling pad into the sample port of the chromatography kit without diluting with a diluent and secure it through the interface so that the sampling pad and the test strip are in contact. If the sampler is hollow, the chromatography diffusion liquid can be dripped into the sample port to complete the chromatography. If the sampler is solid, the chromatography can be completed by extending the chromatography test strip to siphon the diffusion liquid.

[0123] Reference Figures 1a to 1c A specific embodiment of the present invention provides a direct loading immunochromatographic device with a fan-shaped interface, the device comprising a chromatography kit 11 and a sampler 12; the chromatography kit 11 comprises a chromatography test paper 19 and a housing 18 comprising the chromatography test paper, the housing 18 being provided with a loading port 13 and an observation window 17, the loading port 13 being provided in an area of ​​the housing 18 corresponding to the sample pad of the chromatography test paper 19, the loading port 13 having a fan-shaped bayonet 14; the sampler 12 comprising a main body and a sampling pad 15, the sampling pad 15 being provided at the bottom end of the sampler 12, the sampler main body having a fan-shaped bayonet 16 above the sampling pad 15, which matches the fan-shaped bayonet 14 of the loading port of the chromatography kit 11. When in use, after the sampler 12 is sampled at the site to be sampled, the sampler is installed in the loading port 13 of the chromatography kit 11 without dilution with a diluent, and chromatography is performed with a chromatographic diffusion fluid.

[0124] Reference Figure 2a to Figure 2b Another embodiment of the present invention provides a direct-loading immunochromatographic device with a threaded interface, comprising a chromatography kit 21 and a sampler 22. The chromatography kit comprises a chromatography test strip 29 and a housing 28 comprising the chromatography test strip. The housing 28 is provided with a sample loading port 23 and an observation window 27. The sample loading port 23 is located in an area of ​​the housing corresponding to the sample pad of the chromatography test strip and has a threaded interface 24. The sampler 22 comprises a main body and a sampling pad 25. The sampling pad 25 is provided at the bottom end of the sampler 22. The main body of the sampler has a threaded interface 26 above the sampling pad 25 that matches the threaded interface 24 of the sample loading port of the chromatography kit 21. During use, after the sampler 22 is applied to the area to be sampled, it is installed in the sample loading port 23 of the chromatography kit 21 without dilution with a diluent, and chromatography is performed using a chromatographic diffusion fluid.

[0125] Reference Figure 3a to Figure 3b Another specific embodiment of the present invention provides a direct loading immunochromatography kit and a direct loading immunochromatography device having a "siphon method" extended test paper outlet 31, wherein the sampler is a solid structure. After the sampler is installed to the loading port of the chromatography kit through a specific interface, the chromatography test paper 32 is extended through the outlet 31 to contact the chromatography diffusion liquid, siphoning the diffusion liquid to complete the chromatography process of the sample.

[0126] It should be pointed out that when the sampler body is a hollow structure, a direct loading immunochromatography kit or a direct loading immunochromatography device with an extended test paper outlet can also be used for sample detection. After the sampler is installed to the loading port of the chromatography kit through a specific interface, the chromatography test paper 32 is extended through the outlet 31 to contact the chromatography diffusion liquid, siphoning the diffusion liquid to complete the chromatography process of the sample.

[0127] The structure of the semi-quantitative immunochromatographic test paper of the present invention is as shown in the attached Figure 4 As shown: On a base plate (plastic plate) 41, from left to right are a sample pad 42, a blood filter pad 46, a labeling pad 43, an immunochromatographic membrane 44, and an absorbent pad 45. One end of the sample pad 42 is adhered to the plastic plate 41, while the other end is firmly pressed against the blood filter pad 46. The other end of the blood filter pad 46 is firmly pressed against the labeling pad 43. The labeling pad 43 contains a specific binding partner labeled with signal microspheres. The other end of the labeling pad 43 is firmly pressed against the immunochromatographic membrane 44. The immunochromatographic membrane has multiple test lines T (from T1 to Tn) and a control line C. The test lines T are regions where specific binding partners are fixed, while the control lines are regions where immunoglobulins corresponding to the analyte (e.g., sheep IgG) are fixed, or a secondary antibody of the same species is fixed to the control line. The other end of the immunochromatographic membrane is pressed under the absorbent pad 45, forming an immunochromatographic test paper.

[0128] In order to better illustrate the content of the present invention, the following is a description of the present invention in conjunction with specific embodiments. The specific embodiments in this section are only used to explain the content of the present invention and are not used to limit the present invention.

[0129] Example 1: Preparation of C-reactive protein-based immunochromatographic test paper and evaluation of its detection performance

[0130] This example describes the detection of C-reactive protein (CRP). Anti-CRP monoclonal antibodies are used to semi-quantitatively detect the C-reactive protein content in blood samples.

[0131] 1. Conjugation of Mouse Anti-CRP Monoclonal Antibody to Red Carboxylated Polystyrene Microspheres: Disperse 50 nm red carboxylated polystyrene microspheres in MES solution (pH 5.6) at a concentration of 10 mg / mL. Add 200 mg of EDCI and 200 mg of NHS. Cap the tube and vortex to mix thoroughly. Once the EDCI and NHS are completely dissolved, incubate on an endover mixer at room temperature for 2 hours. After this time, concentrate the microspheres by centrifugation, remove the supernatant, and add 1 mL of mouse anti-CRP monoclonal antibody at a concentration of 5 mg / mL. MES buffer is used. Cap the tube and incubate at 4°C with endover mixer for 12 hours. After centrifugation to concentrate the microspheres, wash them with 1 mL of PBS. Finally, disperse the labeled polystyrene microspheres in 2 mL of PBS to create a 5 mg / mL polystyrene microsphere-labeled mouse anti-CRP antibody dispersion.

[0132] MES solution: 2-Morpholineethanesulfonic 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 dissolve it in 100 mL of pure water. Filter and sterilize with a pre-treated Nalgene filter membrane (0.45 μm).

[0133] PBS solution: Weigh 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 and dissolve them in 800 mL of pure water. Adjust the pH to 7.4 after dissolution and transfer to a 1 L volumetric flask. Finally, add pure water to make up to 1 L.

[0134] 2. Preparation of mouse anti-goat IgG monoclonal antibody-coated colloidal gold: Disperse 40 nm colloidal gold in PBS at a concentration of 10 mg / mL in 0.5 mL. Add 0.5 mL mouse anti-goat IgG monoclonal antibody at a concentration of 5 mg / mL in PBS buffer. Seal the tube and incubate at room temperature for 2.5 hours. After centrifugation to concentrate the colloidal gold, add 1 mL of PBS to wash the colloidal gold. Finally, disperse the labeled colloidal gold in 1 mL of PBS to prepare a 5 mg / mL colloidal gold-labeled mouse anti-goat IgG antibody dispersion.

[0135] 3. Preparation of sample pad blocking solution: Prepare 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.

[0136] 4. Preparation of sample pad: In this example, polyester film was selected as the sample pad material, and the sample pad was prepared according to the general sample pad preparation protocol described in this patent.

[0137] 5. 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 polystyrene microspheres labeled with mouse anti-C-reactive protein antibody, 1 mg / mL mouse anti-goat IgG monoclonal antibody coated with colloidal gold dispersion, 0.5% BSA, 5% sucrose, and 0.1% PEG-20000.

[0138] 6. Preparation of 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.

[0139] 7. Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 3 test lines and 1 quality control line were prepared. The concentrations of anti-C-reactive protein antibodies in each test line from the first to the third test line in order from near to far from the sample loading port were 50, 50, and 200 mg / L, respectively, with a streaking rate of 2 μL / cm. The quality control line selected sheep IgG antibody with an antibody concentration of 0.5 mg / mL and a streaking rate of 1 μL / cm.

[0140] 8. In this example, cellulose material is selected as the water absorbent pad and blood filter pad material.

[0141] 9. After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, cut them into 3mm test papers for standby use.

[0142] 10. C-reactive protein immunochromatographic test strips test results for different concentrations of C-reactive protein antigen standards:

[0143] 1) Dilute the C-reactive protein antigen standard to the following concentrations: 0, 0.5, 1, and 3 mg / L;

[0144] 2) Use a pipette to draw 60 μL of the above standard solution and drop it onto the sample pad of the C-reactive protein immunochromatographic test paper prepared in this example, and wait for the chromatography reaction to end.

[0145] The results showed that the C-reactive protein immunochromatographic test strips prepared in this example displayed different results for different concentrations of the C-reactive protein antigen standard: a) the control lines all displayed normal color; b) the 0-concentration standard showed a negative result, with no color development on the test lines; c) at different standard concentrations, the number of positive test lines on the immunochromatographic test strips increased with increasing concentration. Combining these results with the recommended guidelines for cardiovascular disease diagnosis, each test line on the immunochromatographic test strips in this example can be classified as: one positive test line indicates a low risk of coronary artery disease, two positive test lines indicate an intermediate risk of coronary artery disease, and three positive test lines indicate a high risk of coronary artery disease.

[0146] 11. Interference Test: The immunochromatographic test strips prepared in this example were subjected to interference tests using 10 mmol / mL hemoglobin, 3 μmol / mL heparin, and 15 mg / mL triglycerides. 60 μL of each interfering solution was pipetted onto the sample pad of the C-reactive protein immunochromatographic test strip prepared in this example, and the chromatographic reaction was allowed to complete. The results showed that the interfering substances had no effect on the immunochromatographic test strips prepared in this example.

[0147] 12. Actual sample testing: 8 different blood samples were collected through standardized methods and tested using the C-reactive protein immunochromatographic test strips and chemiluminescence detection kit prepared in this example. The specific results are shown in Table 1:

[0148] Table 1: Comparison of C-reactive protein immunochromatographic test strips and chemiluminescence detection kits

[0149]

[0150] Table Note: In the immunochromatographic results, (-) represents 0 detection line, normal range, (+) represents 1 detection line, (++) represents

[0151] Table 2 test lines, (+++) represents 3 test lines

[0152] The results showed that the immunochromatographic test paper prepared in this example showed consistent results with the chemiluminescence detection method in the analysis of actual samples, whether it was positive or negative judgment or judgment of high-risk patients.

[0153] Example 2: Preparation of Immunochromatographic Test Paper for Follicle-Stimulating Hormone (FSH) and Evaluation of Its Detection Performance

[0154] This example focuses on follicle-stimulating hormone (FSH). FSH monoclonal antibodies are used to semi-quantitatively detect the FSH antigen content in urine samples.

[0155] 1. Physical adsorption coating of FSH monoclonal antibody and colloidal gold: Disperse 40nm of colloidal gold with a carboxyl surface in PBS solution at a colloidal gold concentration of 10mg / mL in 0.5mL. Add FSH monoclonal antibody at a concentration of 5mg / mL in 0.5mL. Use PBS as the buffer system. Seal the tube and place it at room temperature, mixing by inversion for 2.5 hours. After centrifugation to enrich the colloidal gold, add 1mL of PBS to wash the colloidal gold. Finally, disperse the labeled colloidal gold in 1mL of PBS solution to prepare a 5mg / mL FSH antibody-labeled colloidal gold dispersion.

[0156] 2. Preparation of sample pad blocking solution: Prepare the blocking solution according to the standard solution preparation method. The specific formula is as follows: 0.8% PVP K30, 1% BSA, 0.03% Tween 20, 0.005% sodium azide, 30 mM sodium chloride.

[0157] 3. Preparation of sample pad: In this example, cellulose membrane was selected as the sample pad material, and the sample pad was prepared according to the general sample pad preparation protocol described in this patent.

[0158] 4. 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 FSH antibody labeled with colloidal gold, 1 mg / mL mouse anti-goat IgG monoclonal antibody coated with colloidal gold dispersion, 0.5% BSA, 3% sucrose, and 0.5% PEG-20000.

[0159] 5. Preparation of 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.

[0160] 6. Preparation of immunochromatographic membrane: CA membrane was selected as the immunochromatographic membrane material in this example and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. A total of 4 test lines and 1 quality control line were prepared in this example. The concentrations of FSH antibody markers of each test line from the 1st to the 4th test line, in order from near to far from the sample loading port, were 1, 1, 0.5, and 1.5 IU / mL, respectively, with a streaking rate of 2 μL / cm. The quality control line used sheep IgG antibody with an antibody concentration of 0.5 mg / mL and a streaking rate of 1 μL / cm. Combined with the diagnostic recommendation guidelines, each test line of the immunochromatographic test strip in this example can be classified, where 1 positive test line indicates diminished ovarian reserve function, 2 positive test lines indicate the latent stage of premature ovarian failure, 3 positive test lines indicate premature ovarian insufficiency, and 4 positive test lines indicate amenorrhea or premature ovarian failure.

[0161] 7. In this example, cellulose material is selected as the absorbent pad material.

[0162] 8. After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, cut them into 3mm test papers for standby use.

[0163] 9. Actual sample testing: 8 different urine samples were collected through standardized collection and tested using the FSH immunochromatographic test strips and chemiluminescence detection kit prepared in this example. The specific results are shown in Table 2:

[0164] Table 2: Comparison of FSH immunochromatographic test strips and chemiluminescence detection kits

[0165]

[0166] Table Note: In the immunochromatographic results, (-) represents 0 detection line, which is within the normal range, (+) represents 1 detection line, and (++) represents 2

[0167] Test lines, (+++) represents 3 test lines

[0168] The results showed that the immunochromatographic test paper prepared in this example showed consistent results with the chemiluminescence detection method in the analysis of actual samples, both in terms of positive and negative judgment and patient status judgment.

[0169] Example 3: Preparation of a Multiple-Test Immunochromatographic Test Strip Based on Luteinizing Hormone (LH) and Estradiol (E3G) Hormones and Evaluation of Its Detection Performance

[0170] This example describes the combined test for luteinizing hormone (LH) and estradiol (E3G). Monoclonal antibodies against LH and E3G are used to semi-quantitatively detect the levels of LH and E3G antigens in urine samples.

[0171] 1. Conjugation of LH Monoclonal Antibody to Red Carboxylated Polystyrene Microspheres: Disperse 50 nm red carboxylated polystyrene microspheres in MES solution (pH 5.6) at a concentration of 10 mg / mL. Add 150 mg of HATU and 150 mg of DMAP to the microspheres in 1 mL of MES solution (pH 5.6). Cover the tube and vortex to mix thoroughly. Once the HATU and DMAP are completely dissolved, incubate on an endover-end mixer at room temperature for 2.5 hours. After this time, concentrate the microspheres by centrifugation, remove the supernatant, and add 1 mL of LH monoclonal antibody at a concentration of 3 mg / mL in MES buffer. Cover the tube and incubate at room temperature with endover-end mixing for 4 hours. After centrifugation to concentrate the microspheres, wash the microspheres with 1 mL of PBS. Finally, disperse the labeled polystyrene microspheres in 2 mL of PBS to produce a 5 mg / mL dispersion of red carboxylated polystyrene microspheres-labeled anti-LH monoclonal antibody.

[0172] 2. Referring to the labeling method of LJ Krichka, Ligand-Binder Assay, published by Marcel Dekker, New York, 1985, the monoclonal antibody against E3G was labeled with 3-bromopropyne (CAS: 106-96-7), and the propyne-modified E3G monoclonal antibody was obtained after purification by dialysis.

[0173] 3. Conjugation of E3G monoclonal antibody to blue azido polystyrene microspheres: Disperse 50 nm blue azido polystyrene microspheres in PBS (pH 5.6) at a concentration of 10 mg / mL. Add 1 mL of propyne-modified E3G monoclonal antibody at a concentration of 3 mg / mL. Then, add 20 μL of 50 mM copper sulfate solution, followed by 500 μL of 200 mM sodium ascorbate solution. Seal the tube and incubate at room temperature for 6 hours, mixing by inversion. After centrifugation to enrich the microspheres, wash them with 1 mL of PBS. Finally, disperse the labeled polystyrene microspheres in 2 mL of PBS to prepare a 5 mg / mL blue polystyrene microsphere-labeled E3G monoclonal antibody dispersion.

[0174] 4. 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 dispersion of red polystyrene microspheres labeled with LH antibody and blue polystyrene microspheres labeled with E3G monoclonal antibody, 1 mg / mL dispersion of colloidal gold coated with mouse anti-goat IgG monoclonal antibody, 0.5% BSA, 5% sucrose, and 0.5% PEG-20000.

[0175] 5. Preparation of 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.

[0176] 6. The sample pad and absorbent pad of this example were prepared according to the method of Example 1.

[0177] 7. Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 4 test lines (corresponding to 2 LH test lines and 2 E3G test lines) and 1 quality control line were prepared. The LH antibody labeling concentrations of each test line from the first to the second LH test line in order from near to far from the sample loading port were 2.5 and 1.5 U / mL, respectively, and the streaking rate was 2 μL / cm. The E3G antibody labeling concentrations of each test line from the first to the second E3G test line in order from near to far from the sample loading port were 2.5 and 1.5 μg / mL, respectively, and the streaking rate was 2 μL / cm. The quality control line selected sheep IgG, the antibody concentration was 0.5 mg / mL, and the streaking rate was 1 μL / cm. After completion, place the test in a cool, dry environment with a temperature no higher than 30°C and a relative humidity no higher than 5% RH until the membrane is completely dry. Store in a cool, dry environment with a relative humidity no higher than 5% RH until ready for use. Based on the diagnostic recommendation guidelines, each test line of the immunochromatographic test strip in this example can be classified. The specific classifications are shown in the following table:

[0178]

[0179] 8. Actual sample testing: 8 different urine samples were collected through standardized collection and tested using the LH and E3G multiplex immunochromatographic test strips and chemiluminescence detection kit prepared in this example. The specific results are shown in Table 3:

[0180] Table 3: Comparison of LH and E3G multiplex immunochromatographic test strips and chemiluminescence detection kits

[0181]

[0182] Table Notes: In the immunochromatographic results, (-)(-) represents 0 detection line, (-)(+) represents 0 detection line of LH + 1 detection line of E3G,

[0183] (+)(+) represents 1 detection line of LH + 1 detection line of E3G, (+)(++) represents 1 detection line of LH + 2 detection lines of E3G

[0184] Detection lines, (++)(++) represents 2 detection lines of LH + 2 detection lines of E3G.

[0185] The results showed that the immunochromatographic test paper prepared in this example showed consistent results with the chemiluminescence detection method in the analysis of actual samples, both in terms of positive and negative judgment and patient status judgment.

[0186] Example 4: Preparation of immunochromatographic test paper for influenza B virus detection and evaluation of its detection performance

[0187] This example explains the detection of influenza B virus. Monoclonal antibodies against influenza B virus are used to semi-quantitatively detect the influenza B antigen content in throat swab samples.

[0188] 1. Conjugation of Influenza B Monoclonal Antibodies to Red Carboxylated Polystyrene Microspheres: Disperse 50 nm red carboxylated polystyrene microspheres in MES solution (pH 5.6) at a concentration of 10 mg / mL. Add 200 mg of EDCI and 200 mg of NHS. Cap the tube and vortex to mix thoroughly. Once the EDCI and NHS are completely dissolved, incubate on an endover mixer at room temperature for 2 hours. After this time, concentrate the microspheres by centrifugation, remove the supernatant, and add 1 mL of influenza B monoclonal antibody at a concentration of 5 mg / mL. MES buffer is used. Cap the tube and incubate at 4°C with endover mixer for 12 hours. After centrifugation to concentrate the microspheres, wash them with 1 mL of PBS. Finally, disperse the labeled polystyrene microspheres in 2 mL of PBS to create a 5 mg / mL dispersion of polystyrene microspheres labeled with influenza B antibodies.

[0189] 2. The marking pad, sample pad and absorbent pad in this example are all made of polyester film.

[0190] 3. The marking pad, sample pad and absorbent pad of this example were prepared according to the method of Example 1.

[0191] 4. Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 2 test lines and 1 quality control line were prepared. The concentrations of influenza B virus monoclonal antibody markers in each test line from the first to the second test line in order from near to far from the sample loading port were 0.2 and 1.5 mg / mL, respectively, with a streaking rate of 2 μL / cm. The quality control line used sheep IgG antibody with an antibody concentration of 0.5 mg / mL and a streaking rate of 1 μL / cm.

[0192] 5. After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, cut them into 3mm test papers for standby use.

[0193] 6. Actual sample testing: 8 different throat swab samples were collected through standardized collection and tested using the influenza B virus immunochromatographic test strips and influenza B fluorescent quantitative PCR kit prepared in this example. The specific results are shown in Table 4:

[0194] Table 4: Comparison of influenza B virus immunochromatographic test strips and influenza B virus fluorescent quantitative PCR kit detection results

[0195] Sample serial number Ct value PCR test results The test results of this example 1 32.87 Positive (+) 2 40.32 Negative (-) 3 28.55 Positive (++) 4 14.87 Positive (++) 5 NoCt Negative (-) 6 NoCt Negative (-) 7 29.33 Positive (+) 8 NoCt Negative (-)

[0196] Table Note: In the immunochromatographic results, (-) represents 0 detection line, which is within the normal range, (+) represents 1 detection line, and (++) represents 2

[0197] Detection lines.

[0198] The results showed that the immunochromatographic test paper prepared in this example showed consistent results with the influenza B virus fluorescent quantitative PCR kit in the analysis of actual samples, both in terms of positive and negative judgment and patient status judgment.

[0199] Example 5: Preparation of C-reactive protein-based immunochromatographic test paper and evaluation of its detection performance

[0200] This example explains the detection of C-reactive protein (CRP). Anti-CRP monoclonal antibodies are used to qualitatively detect the C-reactive protein content in blood samples.

[0201] To conjugate mouse anti-CRP monoclonal antibody to red carboxyl polystyrene microspheres: 50 nm red carboxyl polystyrene microspheres were dispersed in 2-morpholinoethanesulfonic acid (MES) solution (pH 5.6) at a concentration of 10 mg / mL. To 1 mL of the microsphere dispersion, 200 mg of EDCI and 200 mg of NHS were added. The suspension was capped and vortexed to mix thoroughly. Once the EDCI and NHS were completely dissolved, the suspension was incubated on an endover-end mixer at room temperature for 2 hours. After this time, the microspheres were enriched by centrifugation, and the supernatant was removed. 1 mL of mouse anti-CRP monoclonal antibody was added at a concentration of 5 mg / mL. The suspension was capped and incubated at 4°C with endover-end mixing for 12 hours. 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 to produce a 5 mg / mL mouse anti-CRP antibody-labeled polystyrene microsphere dispersion.

[0202] MES solution: 2-Morpholineethanesulfonic 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 dissolve it in 100 mL of pure water. Filter and sterilize with a pre-treated Nalgene filter membrane (0.45 μm).

[0203] PBS solution: Weigh 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 and dissolve them in 800 mL of pure water. Adjust the pH to 7.4 after dissolution and transfer to a 1 L volumetric flask. Finally, add pure water to make up to 1 L.

[0204] Preparation of mouse anti-goat IgG antibody-coated colloidal gold: Disperse 40 nm colloidal gold in 0.5 mL of PBS at a colloidal gold concentration of 10 mg / mL. Add 0.5 mL of mouse anti-goat IgG antibody at a concentration of 5 mg / mL. Use PBS as the buffer. Seal the tube and incubate at room temperature for 2.5 hours. After centrifugation to concentrate the colloidal gold, add 1 mL of PBS to wash the colloidal gold. Finally, disperse the labeled colloidal gold in 1 mL of PBS to prepare a 5 mg / mL mouse anti-goat IgG antibody-labeled colloidal gold dispersion.

[0205] Preparation of sample pad blocking solution: Prepare 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.

[0206] Preparation of sample pad: In this example, polyester film was selected as the sample pad material, and the sample pad was prepared according to the general sample pad preparation protocol described in this patent.

[0207] 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 mouse anti-C-reactive protein antibody-labeled polystyrene microspheres, 1 mg / mL mouse anti-goat IgG monoclonal antibody-coated colloidal gold dispersion, 0.5% BSA, 5% sucrose, and 0.1% PEG-20000.

[0208] Preparation of the marking pad: In this example, polyester film was selected as the marking pad material, and the marking pad was prepared according to the general preparation scheme of the marking pad described in this patent.

[0209] Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 1 test line and 1 quality control line were prepared. The detection antibody concentration was 50 mg / L and the streaking rate was 2 μL / cm. The quality control line selected sheep IgG antibody with an antibody concentration of 0.5 mg / mL and a streaking rate of 1 μL / cm.

[0210] In this example, cellulose material is selected as the water absorbent pad and blood filter pad material.

[0211] After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, they are cut into 3mm test papers for standby use.

[0212] Actual sample testing: Eight different blood samples were collected through standardized collection methods and tested using the C-reactive protein test using the immunochromatographic reagent card with a fan-shaped direct sample loading interface (used with a solid sampler and siphon diffusion method), traditional immunochromatographic methods, and a chemiluminescence detection kit. The specific results are shown in Table 5:

[0213] Table 5: Comparison of different immunochromatographic methods for C-reactive protein

[0214]

[0215] The results showed that, using chemiluminescence as the reference method, by comparing the immunochromatography designed by the traditional dilution method with the method of the present invention, the results of the method of the present invention were basically consistent with those of the chemiluminescence method. The immunochromatography test paper prepared in this example showed consistent results with the chemiluminescence method in the analysis of actual samples, whether it was positive or negative judgment or judgment of high-risk patients. The traditional immunochromatography method had the risk of missed detection, for example, different conclusions were obtained for sample 1 and sample 4.

[0216] Example 6: Preparation of immunochromatographic test paper for influenza B virus detection and evaluation of its detection performance

[0217] This example explains the detection of influenza B virus. Monoclonal antibodies against influenza B virus are used to qualitatively detect the influenza B virus antigen content in throat swabs.

[0218] Adsorption and coating of influenza B virus monoclonal antibodies with colloidal gold: Disperse 40 nm of carboxyl-coated colloidal gold in PBS at a colloidal gold concentration of 10 mg / mL. Add 0.5 mL of colloidal gold dispersion to a 5 mg / mL concentration of influenza B virus monoclonal antibody in PBS buffer. Seal the tube and incubate at room temperature for 2.5 hours, mixing by inversion. After centrifugation to concentrate the colloidal gold, add 1 mL of PBS to wash the colloidal gold. Finally, disperse the labeled colloidal gold in 1 mL of PBS to prepare a 5 mg / mL influenza B virus antibody-labeled colloidal gold dispersion.

[0219] The marking pad, sample pad and absorbent pad in this example are all made of polyester film.

[0220] The marking pad, sample pad and absorbent pad of this example were prepared according to the method of Example 5.

[0221] Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. In this example, a total of 1 test line and 1 quality control line were prepared. The concentration of the influenza B virus monoclonal antibody marker was 0.5 mg / mL, and the streaking rate was 2 μL / cm. The quality control line selected sheep IgG antibody, the antibody concentration was 0.5 mg / mL, and the streaking rate was 1 μL / cm.

[0222] After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, they are cut into 3mm test papers for standby use.

[0223] Actual sample testing: Eight different throat swab samples were collected through standardized collection and tested using the influenza B immunochromatographic reagent card with a threaded interface (used with a hollow sampler) and the influenza B fluorescent quantitative PCR kit prepared according to the embodiments of the present invention. The specific results are shown in Table 6:

[0224] Table 6: Comparison of influenza B immunochromatographic test strips and influenza B fluorescent quantitative PCR kits

[0225] Sample serial number Ct value PCR test results The test results of this example 1 32.87 Positive Positive 2 40.32 Negative Negative 3 28.55 Positive Positive 4 14.87 Positive Positive 5 NoCt Negative Negative 6 NoCt Negative Negative 7 29.33 Positive Positive 8 NoCt Negative Negative

[0226] The results showed that the immunochromatographic test paper prepared in this example showed consistent results with the PCR detection method in the analysis of actual samples, both in terms of positive and negative judgment and patient status judgment.

[0227] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. A semi-quantitative immunochromatographic test paper, comprising a base plate, a sample pad, a labeling pad, an immunochromatographic membrane, and a water-absorbing pad. The base plate is provided with the sample pad, the labeling pad, the immunochromatographic membrane, and the water-absorbing pad in the order of the chromatography direction. One end of the sample pad is adhered to the base plate, and the other end is tightly pressed against the labeling pad, wherein the labeling pad contains a specific partner labeled with a signal microsphere. One end of the labeling pad is tightly pressed against the immunochromatographic membrane, and the immunochromatographic membrane has multiple detection lines T and a quality control line C. The detection line T contains a specific binding partner, and the quality control line contains a corresponding immunoglobulin, or the quality control line C contains a secondary antibody derived from a quantitative antibody species. The other end of the immunochromatographic membrane is pressed under the water-absorbing pad to form the immunochromatographic test paper.

2. The semi-quantitative immunochromatographic test paper according to claim 1, wherein The material of the sample pad is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, more preferably one or more of cellulose material and polyester film; or The sample pad contains a blocking solution, which comprises: a hydrophilic polymer material, a protective protein, an antibacterial agent and an inorganic salt; preferably, the blocking solution is an aqueous solution; or The preparation method of the sample pad comprises the following steps: completely immersing the sample pad material in a blocking solution, leaving it to stand for a certain period of time, and after the sample pad material completely absorbs the blocking solution, taking out the sample pad and drying it.

3. The semi-quantitative immunochromatographic test paper according to claim 1, wherein The material of the marking pad is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common water-absorbing materials, preferably one or more of cellulose material, polyester film or non-woven fabric, more preferably one or more of cellulose material and polyester film; or The specific partner includes a small organic compound or a biomacromolecule, which has the ability to specifically recognize, bind, pair, and capture the analyte molecule, and can form a "double antibody sandwich" model with the specific binding partner and the analyte molecule fixed on the immunochromatographic membrane to complete specific detection; or The signal microspheres are a substance or particle with an identification signal. Preferably, the signal microspheres are selected from one or more of colloidal gold, gold nanoparticles, colloidal silver, silver nanoparticles, colored polymer microspheres, fluorescent polymer microspheres, and quantum dots; or The labeling method of the signal microspheres and the specific partner is selected from a physical adsorption coating process or a covalent coupling process.

4. The semi-quantitative immunochromatographic test paper according to claim 1, wherein The immunochromatographic membrane material is selected from a microporous filter membrane material with a large pore size; the preferred 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, polycarbonate nuclear track etched filter membrane; further preferred immunochromatographic membrane material is selected from one or more of nitrocellulose membrane, cellulose acetate filter membrane, mixed cellulose ester filter membrane, polyvinylidene fluoride filter membrane; further preferred immunochromatographic membrane material is selected from one or more of nitrocellulose membrane and cellulose acetate filter membrane; or The number of the detection lines T is 2-20, preferably 2-10, more preferably 2-5, and even more preferably 2, 3 or 4; or The method of fixing the specific binding partner on the immunochromatographic membrane is selected from a physical adsorption coating process or a covalent coupling process; or The absorbent pad material is selected from one or more of cellulose material, filter paper, polyester film, glass cellulose film, non-woven fabric or other common absorbent materials, preferably one or more of cellulose material, polyester film, non-woven fabric, and more preferably one or more of cellulose material and polyester film.

5. A semi-quantitative immunochromatographic method comprising the following steps: (1) performing immunochromatography using the immunochromatographic test paper according to any one of claims 1 to 5; and (2) performing a semi-quantitative reading of the results directly after immunochromatography is completed.

6. A direct loading immunochromatography kit, comprising the immunochromatography test paper according to any one of claims 1 to 4 and a shell for accommodating the immunochromatography test paper; and the shell is provided with a loading port, the loading port having a loading port interface, the loading port interface being compatible with an interface of a sampler, so that the sampler can be installed on the loading port of the chromatography kit.

7. The chromatography kit according to claim 6, characterized in that The sample loading port interface of the chromatography kit can be a common engineering interface type, such as a fan-shaped, threaded, enamel-shaped, or ground-shaped interface; preferably, the sample loading port interface of the chromatography kit is a fan-shaped bayonet or a threaded interface; or The housing is provided with an observation window; or The chromatography kit has an extended test paper outlet, and the chromatography test paper can extend through the outlet to siphon the diffusion liquid.

8. A direct loading immunochromatography device, comprising a chromatography kit and a sampler; the chromatography kit comprises the immunochromatography test paper according to any one of claims 1 to 4 and a shell for accommodating the immunochromatography test paper; and the shell is provided with a loading port, the loading port having a loading port interface; the sampler comprises a main body and a sampling pad, the bottom end of the sampler is provided with a sampling pad, the sampler main body has an interface, the interface is located above the sampling pad and can be matched with the loading port interface of the chromatography kit, so that the sampler can be installed on the loading port of the chromatography kit.

9. The immunochromatographic device according to claim 8, characterized in that The sample loading port interface of the chromatography kit can be a common engineering interface type, such as a fan-shaped, threaded, enamel-shaped, ground-shaped, etc.; preferably, the sample loading port interface of the chromatography kit is a fan-shaped bayonet or a threaded interface; or The interface above the sampling pad can be a common engineering interface type, such as fan-shaped, threaded, enamel-shaped, ground-shaped, etc.; preferably, the sampler interface is a fan-shaped bayonet or a threaded bayonet; or The sampler further comprises a hand-held rod, wherein the hand-held rod, the main body and the sampling pad are arranged in sequence along the longitudinal direction of the sampler, and the hand-held rod is located at the top of the sampler for hand-held operation; or The main structure of the sampler is a hollow structure or a solid structure; or The housing is provided with an observation window; or The chromatography kit housing has an extended chromatography test paper outlet, and the chromatography test paper can extend through the outlet to siphon the diffusion liquid; or The sampling pad is made of flocking or sponge.

10. A direct loading immunochromatography method, comprising using the direct loading immunochromatography kit according to claim 6 or 7 or the direct loading immunochromatography device according to claim 8 or 9, and comprising the following steps: After the sampler is placed at the site to be sampled, it is installed on the sample loading port of the chromatography kit without dilution with a diluent, and immunochromatography is performed with a chromatographic diffusion fluid, and the results are directly read semi-quantitatively. Preferably, the direct loading immunochromatographic method is used for sample detection in medical diagnosis, medical testing, food, agriculture or animal husbandry fields; Preferably, the direct loading immunochromatographic method targets samples selected from one or more of medical samples, environmental swab samples, feed and plant tissue homogenates; Preferably, the medical sample is selected from one or more of whole blood, plasma, serum, urine, tears, sweat, saliva, oral swab, anal test paper, alveolar lavage fluid and cerebrospinal fluid.

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