Continuous semi-quantitative immunochromatography method

Abstract

The invention provides a continuous semi-quantitative immunochromatography method. The continuous semi-quantitative immunochromatography method comprises the following steps: carrying out immunochromatography by using immunochromatography test paper; and after the chromatography is completed, performing semi-quantitative reading on the result. The test paper comprises a sample pad, a marking pad, an immunochromatography membrane and a water absorption pad which are sequentially connected, a binding area is arranged on the immunochromatography membrane, the binding area is a reaction area continuously arranged in the chromatography direction, and the binding area is used for carrying out specific reaction with a target object in a sample to be detected and generating a color developing band; the binding area is provided with a scale line corresponding to the known concentration of a target object, and is used for realizing continuous semi-quantitative detection of the concentration of the target object by comparing the length of the color developing tape in the binding area with the scale line. According to the method, the result is directly subjected to semi-quantitative reading through naked eyes, and continuous semi-quantitative detection on the to-be-detected substance can be realized quickly, simply and efficiently.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, in particular to a continuous semi-quantitative immunochromatography method. Background Art

[0002] Immunochromatography is a simple, rapid, and convenient detection method, primarily used in medical diagnostic testing, agricultural production, animal quarantine, food safety, and other fields. Immunochromatography relies primarily on the specific binding between antigens, haptens, and antibodies to complete capture, enrichment, and detection. Currently, immunoassays are primarily used for qualitative diagnosis, i.e., determining whether the analyte in the sample has reached a certain level to distinguish between negative and positive results, such as pregnancy test strips and novel coronavirus antigen detection strips. However, immunochromatographic methods for quantitative detection, especially continuous semi-quantitative methods with the naked eye, are uncommon.

[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, quantitative immunochromatographic detection methods can be categorized as follows: 1) Using an immunochromatographic instrument to measure fluorescence, grayscale, absorbance, and other indicators in the sample, and then using a standard curve to quantitatively convert and calculate the content of the molecule being tested; 2) Using pre-printed color charts for colorimetric comparison to determine the content of the corresponding analyte. These methods require instrumentation or are difficult to achieve continuous semi-quantification (colorimetry can only determine pre-set concentrations, making it difficult to estimate the amount between bands). Currently, there is a lack of a rapid, continuous semi-quantitative immunochromatographic method that can be used with the naked eye. Summary of the Invention

[0011] In response to the shortcomings of the existing technology, the present invention provides a continuous semi-quantitative immunochromatography method for direct judgment with the naked eye, which can be used in multiple fields such as medical testing, food safety, and animal husbandry. Through the specific detection of antigens, haptens, and antibodies, rapid, simple, and convenient continuous semi-quantitative detection can be achieved. The entire process does not require the participation of instruments or equipment, nor does it require comparison with pre-made colorimetric cards.

[0012] Specifically, the present invention provides a continuous detection area (or binding zone) on the immunochromatographic test paper, which is uniformly coated with a specific binding partner (antigen, hapten, antibody, protein complex, etc.). When the specific analyte in the test sample flows through by capillary action, the specific binding partner captures the relevant analyte molecules and deposits them on the immunochromatographic membrane. Depending on the amount of analyte in the sample, the immunochromatographic test paper will present a continuous color-developing area. As the concentration of the analyte increases, the boundary of the color-developing area rises and the color-developing area increases.

[0013] In one aspect, the present invention provides a continuous semi-quantitative immunochromatographic test paper, comprising a sample pad, a labeling pad, an immunochromatographic membrane and a water-absorbing pad connected in sequence.

[0014] The immunochromatographic membrane is provided with a binding zone, which is a reaction area continuously arranged along the chromatography direction. The binding zone is used to react specifically with the target in the sample to be tested and produce a continuous color band in the binding zone;

[0015] A scale line corresponding to the known target concentration is provided on the corresponding area of ​​the binding zone, which is used to achieve continuous semi-quantitative detection of the target concentration by comparing the length of the color-developing band in the binding zone with the scale line.

[0016] According to an embodiment of the present invention, the concentration of the target substance corresponding to each scale line increases in sequence along the chromatography direction, and the number of scale lines may be 2 or more, for example, 2-10.

[0017] According to an embodiment of the present invention, the scale line is determined according to the length of the color band of the standard, which specifically includes the following steps:

[0018] (1) Perform chromatography detection using standard solutions with known concentration gradients and record the length of the color band at different concentrations;

[0019] (2) According to the length of the color band, scale lines of corresponding concentration values ​​are marked on the corresponding areas on both sides of the binding area.

[0020] According to an embodiment of the present invention, a specific binding partner is immobilized on the surface of the binding region.

[0021] According to an embodiment of the present invention, the specific binding partner is evenly distributed on the surface of the binding region.

[0022] According to an embodiment of the present invention, the concentration of the specific binding partner on the surface of the binding zone is distributed gradually decreasing along the chromatographic direction of the sample to be tested.

[0023] According to an embodiment of the present invention, the specific binding partner is immobilized on the surface of the binding region by chemical cross-linking, physical adsorption or bioaffinity binding.

[0024] According to an embodiment of the present invention, the sample pad, labeling pad, immunochromatographic membrane, and absorbent pad are connected in sequence along the chromatography direction; one end of the sample pad is pressed on the labeling pad, which contains a specific labeled partner labeled with signal microspheres; the other end of the labeling pad is pressed on the immunochromatographic membrane, which contains a quality control line, which contains a secondary antibody against the species of the specific labeled partner; the other end of the immunochromatographic membrane is pressed under the absorbent pad to form an immunochromatographic test paper.

[0025] According to an embodiment of the present invention, the specific labeling partner and the specific binding partner of the binding region are directed against different epitopes of the target.

[0026] 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.

[0027] 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, a surfactant, an antibacterial agent and an inorganic salt; preferably, the blocking solution is an aqueous solution, and the water used is pure water, deionized water, distilled water, etc.

[0028] 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%.

[0029] 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%.

[0030] 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%.

[0031] 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%.

[0032] 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; 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.

[0033] 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.

[0034] 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.

[0035] According to an embodiment of the present invention, the specific labeling partner includes small molecule organic compounds and biological macromolecules. The specific partner has the ability to specifically identify, bind, pair and capture the molecules to be detected, and can form a "double antibody sandwich" model with the specific binding partner and the molecules to be detected fixed on the immunochromatographic membrane to complete specific detection.

[0036] 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. Specifically, 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), and polyacrylic acid (hereinafter referred to as PAA); the surface functional groups of the signal microspheres can be common organic chemical functional groups such as amino, carboxyl, hydroxyl, thiol, amide, phenyl, alkyl, and alkenyl. The preferred surface functional groups are amino, carboxyl, hydroxyl, thiol, and phenyl, and more preferably amino, carboxyl, and phenyl; the particle size of the signal microspheres can be 5-200 nm, preferably 10-100 nm, and more preferably 25-50 nm.

[0037] 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.

[0038] 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 labeled partner labeled with the signal microspheres.

[0039] 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 a specific labeled partner for the labeled signal microspheres.

[0040] 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.

[0041] 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.

[0042] According to an embodiment of the present invention, the method for preparing the labeling pad includes the following steps: completely immersing the labeling pad material into a working solution containing a labeled specific labeling partner or spraying the working solution onto the labeling pad, allowing it to stand for a certain period of time, and drying the sample pad after the working solution is completely immersed in the labeling pad.

[0043] 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.

[0044] According to an embodiment of the present invention, the functional groups on the surface of the immunochromatographic membrane can be common organic chemical functional groups such as hydroxyl, amino, aldehyde, and carboxyl groups, and each type of functional group is adapted to different antibody binding modes.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The present invention also provides a continuous semi-quantitative immunochromatography method, comprising 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.

[0051] According to an embodiment of the present invention, the method comprises the following steps:

[0052] (a) performing chromatography detection on the sample to be tested;

[0053] (b) comparing the length of the color band formed by the sample to be tested in the binding zone with the concentration scale line to determine the semi-quantitative result of the concentration of the sample to be tested.

[0054] Specifically, the method further comprises:

[0055] ① Prepare the standard into multiple solutions of known concentrations to form a concentration gradient, and load them onto the sample pad of the immunochromatographic test paper respectively;

[0056] ② allowing the standard to pass through the label pad and the immunochromatographic membrane in sequence under chromatography, wherein on the label pad, the standard binds to the labeled specific binding molecule to form a complex; in the binding area of ​​the immunochromatographic membrane, the complex binds to the immobilized specific binding molecule;

[0057] ③ According to the length of the color band formed by the standard sample with different concentrations in the binding area, mark the corresponding concentration scale line;

[0058] ④ Load the sample to be tested onto the sample pad and pass through the labeling pad and immunochromatographic membrane in sequence under the action of chromatography;

[0059] ⑤ Compare the length of the color band formed by the sample to be tested in the binding area with the concentration scale line to determine the semi-quantitative result of the concentration of the sample to be tested.

[0060] According to the embodiment of the present invention, the signal change of the length of the color band in the binding area is continuous, which can realize a progressive estimation of the target substance in the sample to be tested within a certain concentration range.

[0061] According to an embodiment of the present invention, by comparing the length of the color-developing band with the relative position of preset concentration scale lines, the concentration range corresponding to two adjacent scale lines can be determined. Furthermore, by comparing the distances between the endpoint of the color-developing band and the two adjacent scale lines, it is determined which scale line represents the concentration value to which the target concentration in the sample is closer, thereby achieving continuous semi-quantitative analysis. Specifically, when the color-developing length is between two preset scale lines, the concentration of the sample to be tested is closer to the concentration value represented by the scale line closer to the color-developing length.

[0062] According to an embodiment of the present invention, the measurement of the length of the color-developed area is completed by naked eye observation or instrument analysis.

[0063] The present invention also provides a direct loading immunochromatography kit, which comprises the immunochromatography test paper of the present invention and a shell for accommodating the immunochromatography test paper; and the shell is provided with a loading port, which has 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.

[0064] 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.

[0065] According to an embodiment of the present invention, the housing is provided with an observation window.

[0066] According to an embodiment of the present invention, the scale lines are provided on the housing.

[0067] 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.

[0068] The present invention also provides a direct loading immunochromatography device, comprising a chromatography kit and a sampler; the chromatography kit 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, 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, and the sampler main body has an interface, and 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] According to an embodiment of the present invention, the interface above the sampling pad is a fan-shaped bayonet or a threaded bayonet.

[0073] 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.

[0074] According to an embodiment of the present invention, the sampler main body structure is a hollow structure or a solid structure.

[0075] According to an embodiment of the present invention, the housing is provided with an observation window.

[0076] 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.

[0077] According to an embodiment of the present invention, the sampling pad is made of flocking or sponge.

[0078] The present invention also provides a direct loading immunochromatography method, which includes using the direct loading immunochromatography kit or direct loading immunochromatography device described in the present invention, and includes the following steps: after sampling at the site 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 fluid, and the results are directly semi-quantitatively read.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] The present invention provides a continuous detection area on the test paper, on which a specific binding partner (antigen, hapten, antibody, protein complex, etc.) is fixed. 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, the color development length varies during the process of passing through the chromatography membrane due to the difference in the content of the analyte. Finally, the analyte in the sample can be semi-quantitatively detected by comparing the color development length with the scale line.

[0084] 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.

[0085] 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

[0086] Figure 1a A three-dimensional diagram of a direct loading chromatography kit with a fan-shaped interface according to the present invention;

[0087] Figure 1b A three-dimensional diagram of a direct loading immunochromatographic device with a fan-shaped interface according to the present invention;

[0088] Figure 1c This is a front view of the direct loading immunochromatographic device with a fan-shaped interface according to the present invention;

[0089] Figure 2a A three-dimensional diagram of a direct loading immunochromatographic device with a threaded interface according to the present invention;

[0090] Figure 2b This is a front view of the direct loading immunochromatographic device with a threaded interface according to the present invention;

[0091] Figure 3a A perspective view of a direct loading chromatography kit with a "siphon method" extended test strip outlet according to the present invention;

[0092] 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;

[0093] Figure 4 This is a structural diagram of the semi-quantitative immunochromatographic test paper of the present invention;

[0094] 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;

[0095] 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;

[0096] Figure 3a-3b Among them, 31. "Siphon method" extends the test paper outlet; 32. "Siphon method" extends the test paper;

[0097] Figure 4 In the figure, 41, bottom plate; 42, sample pad; 43, labeling pad; 44, immunochromatographic membrane; 45, absorbent pad; 46, blood filter pad; 47, detection area. DETAILED DESCRIPTION

[0098] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0099] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0100] Reagents used in the embodiments of the present invention:

[0101] 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).

[0102] 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.

[0103] Definition of terms

[0104] The "upper" and "lower" mentioned in the present invention are based on the direction of the test paper when it is used, that is, the sample absorbing end is the lower side and the water absorbing area is the upper side.

[0105] "Specific binding partner" refers to a molecule or substance that can specifically bind to an analyte (such as an antigen, protein or other target molecule), which is usually fixed to the detection area of ​​an immunochromatographic test paper to capture the analyte in the sample. In the context of the present invention, the specific binding partner is similar to a capture antibody, such as a monoclonal antibody, a polyclonal antibody, an aptamer or other binding molecules with high affinity, which forms a binding complex with the analyte in the binding area by specifically recognizing a specific epitope of the analyte. The specific binding partner is coated in the binding area for semi-quantitative analysis.

[0106] "Specifically labeled partner" refers to a molecule or substance that can specifically bind to the analyte in the binding zone and is detectably labeled for signal amplification and visual detection. In the context of the present invention, the specifically labeled partner is similar to a labeled antibody, such as an antibody or binding molecule labeled with colloidal gold, a fluorescent dye, an enzyme or other detectable marker. The specifically labeled partner forms a labeled complex with the analyte in the binding zone, flows with the sample to the detection zone, and further binds to the specific binding partner, thereby generating a detectable signal (such as a color change or a fluorescent signal) in the detection zone to indicate the presence and concentration of the analyte.

[0107] "Continuous semi-quantitative" refers to the preliminary estimation of the degree of proximity of the concentration of the sample to be tested by measuring the relative relationship between a certain continuously changing signal (such as the color development length) and a preset standard (such as a scale line or a reference value), rather than accurately determining a specific value. It is often used for rapid detection or on-site analysis, such as test paper detection in biomedical diagnosis, and is suitable for scenarios where the concentration range needs to be quickly determined without the need for precise values. The continuous semi-quantitative measurement results show continuous changes rather than discrete levels, thereby allowing progressive estimation within a certain range. The color development length can extend continuously with changes in concentration, rather than being limited to fixed points. By comparing with a preset scale or standard, the approximate range or degree of proximity of the sample concentration can be estimated. For example, when the color development length is close to a certain scale line, it can be judged that the sample concentration is closer to the concentration represented by the scale. In an embodiment of the present invention, the signal of the sample to be tested (such as the color development length) shows continuity with changes in concentration, and the concentration estimation is based on the relative distance or intensity comparison between the signal and the scale line, reflecting the continuous change trend of the concentration, rather than being fixed at a specific point. Example: On the test paper, the color development length gradually extends as the concentration of the analyte increases. When the length falls between two scale lines, the concentration is closer to the concentration value represented by the closer scale line.

[0108] "Progressive estimation" is a concentration assessment method based on continuous signal changes. Through the continuous change of the color band length, the concentration of the target in the sample is evaluated step by step and continuously, gradually approaching the possible range of the target concentration, realizing rapid and continuous semi-quantitative detection, rather than being limited to discrete fixed-point measurements. Specifically, the length of the color band changes continuously with the change of the target concentration, generating a continuous signal rather than a sudden or segmented signal. This continuity allows the capture of changes in concentration. The estimation process involves measuring the length of the color band and comparing it with the preset concentration scale line. It can determine the concentration range in which the target concentration is located and further evaluate which specific scale value it is close to. For example, if the length of the color band is between two scale lines, it can be inferred that the concentration value is closer to one of the scales. Progressive estimation combines the simplicity of qualitative detection with the accuracy of quantitative detection. It is suitable for rapid on-site detection or resource-limited environments, and can provide relatively accurate concentration range information within a continuous concentration range.

[0109] "Uniform distribution" means that the concentration of the specific binding partner per unit area on the surface of the binding zone remains basically consistent, that is, within the entire surface range of the binding zone, there is no obvious difference in the coverage density of the binding partner, ensuring that all parts of the binding zone have the same binding ability, thereby achieving stability and repeatability of the detection signal.

[0110] "Concentration distribution gradually decreases along the chromatographic direction of the sample" refers to the concentration of the specific binding partner on the binding zone surface following a gradient along the flow direction of the sample, starting with a higher concentration at the sample application end and gradually decreasing toward the water absorption end. This gradient distribution is designed to optimize the spatial distribution of the binding partner within the binding zone. By setting up a concentration gradient, binding efficiency can be enhanced, improving detection sensitivity and dynamic range, while reducing nonspecific binding and background signal caused by excess binding partner.

[0111] Detailed description

[0112] 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, wherein the marking pad contains a specific partner marked by a signal microsphere; the other end of the marking pad is tightly pressed on the immunochromatographic membrane, and the immunochromatographic membrane has a continuous binding area and a quality control line C, the binding area contains uniformly coated specific binding partners, scale lines are arranged in parallel on both sides of the binding area, the scale lines are marked with concentration values, and are used to achieve semi-quantitative detection by comparing the color development length of the binding area with the scale lines, the scale lines are determined according to the length of the color development area of ​​a standard, the number of scale lines can be 2 or more, and the spacing between the scale lines can be the same or different, and is established according to specific project requirements; the quality control line C contains a secondary antibody of the species of anti-specific binding partner; the other end of the immunochromatographic membrane is pressed under the water-absorbing pad to form the immunochromatographic test paper.

[0113] 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.

[0114] 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.

[0115] 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%.

[0116] 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%.

[0117] 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%.

[0118] 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%.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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:

[0125] (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.

[0126] (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.

[0127] 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.

[0128]

[0129]

[0130]

[0131]

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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 materials are NC membrane, CA membrane, MCE membrane, PVDF membrane One or more of the above, and further preferably one or more of the NC membrane and CA membrane; the functional groups on the surface of the immunochromatographic membrane can be common organic chemical functional groups such as hydroxyl, amino, aldehyde, and carboxyl, and each type of functional group is adapted to different antibody binding modes; the immunochromatographic membrane has a continuous binding area and a quality control line C, the continuous binding area is the area where the specific binding partner is fixed, and the quality control line is a secondary antibody derived from the species of the anti-specific labeled antibody. The width of the binding area is 1-100mm, the optimized binding area width is 1-50mm, and the further optimized binding area width is 10-30mm. The width of the quality control line C can be 1-5mm, preferably 1-3mm, and more preferably 1-2mm.

[0136] According to an embodiment of the present invention, the immunochromatographic membrane is immobilized with a specific binding partner and an immunoglobulin (such as sheep IgG) corresponding to the analyte, or is immobilized with a secondary antibody of the species of the specific labeled partner.

[0137] 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:

[0138] (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.

[0139] (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.

[0140] According to an embodiment of the present invention, the concentration of the specific binding partner on the immunochromatographic membrane is set within a range known to those skilled in the art. For example, it can be set according to the concentration range of the test sample. For low concentration test samples (pg / mL to ng / mL), a higher specific binding partner concentration (10 4 –10 8times) to ensure sufficient binding sites. For example, 0.1–2 mg / mL. For high concentration test samples (μg / mL or higher), a lower specific binding partner concentration (1–500 times) is used to avoid signal saturation and non-specific binding. For example, 0.5-5 mg / mL. Those skilled in the art can optimize the specific concentration to achieve the desired detection limit and linear range through routine experiments, such as by preparing standard samples of the test substance (such as 0.1-5 mg / mL) and testing the signal response of different specific binding partner concentrations (such as 5, 10, 50 mg / mL). According to an embodiment of the present invention, for high sensitivity detection, a homogenized high concentration distribution can be used to enhance the signal. For wide dynamic range detection, a gradient distribution can be used to optimize the signal resolution of different concentrations of the test substance.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] According to an embodiment of the present invention, the blood filter pad needs to be treated with a blocking solution of 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 it is left to stand for 5-15 minutes. After the blood filter pad material completely absorbs the blocking solution, the blood filter pad is taken out and air-dried at 50-60°C for 30 minutes. Then, the blood filter pad is placed in a cool and dry environment, the ambient temperature is controlled to be no higher than 30°C, and the relative humidity is no higher than 5%RH. It is left to stand until the blood filter pad is completely dry, and stored in a cool and dry environment with a relative humidity of no higher than 5%RH for use.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] According to an embodiment of the present invention, the housing is provided with an observation window.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] The direct sample loading immunochromatography device provided by the present invention comprises a chromatography kit and a sampler.

[0158] 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 detection area 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 detection area and the quality control line.

[0159] 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.

[0160] 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.

[0161] During operation:

[0162] 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.

[0163] 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.

[0164] Reference Figures 1a to 1cA 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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, which 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 a continuous binding area (detection area 47) and a control line C. The binding area is an area where evenly distributed specific binding partners are fixed. Scale lines are arranged parallel to the corresponding areas (housing 28) on both sides of the binding area. The control line C is an area 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.

[0169] Example 1 Continuous semi-quantitative immunochromatographic test paper for urine protein

[0170] This example explains the detection of urine protein. By using a urine protein monoclonal antibody, continuous semi-quantification of urine protein in urine samples can be achieved.

[0171] 1. Conjugation of Mouse Anti-Human Urinary Protein Monoclonal Antibody to Red Carboxyl Polystyrene Microspheres: Disperse 50 nm red carboxyl 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 end-over-end 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-human urinary protein monoclonal antibody at a concentration of 5 mg / mL. MES buffer is used. Cap the tube and incubate at 4°C for 12 hours. After centrifugation to concentrate the microspheres, add 1 mL of 5% BSA solution to block the microspheres. Cap the tube and incubate at room temperature for 2 hours. After centrifugation to enrich the microspheres, the microspheres were washed with PBS, and finally the labeled polystyrene microspheres were dispersed in 2 mL of PBS solution to prepare a 5 mg / mL mouse anti-human urine protein antibody-labeled polystyrene microsphere dispersion.

[0172] 2. Preparation of sample pad blocking solution: Prepare the blocking solution according to the standard solution preparation method. The specific formula is as follows: 0.5% PVA-8000, 1% BSA, 0.1% Tween 20, 0.005% sodium azide, 25 mM sodium chloride.

[0173] 3. 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.

[0174] The specific steps include: placing the blocking solution prepared in step 2 into a glass container with a lid, completely immersing the sample pad material in the blocking solution, and letting it stand for 5-15 minutes. After the sample pad material completely absorbs the blocking solution, remove the sample pad and air-dry it at 50-60°C for 30 minutes. Then, place the sample pad in a cool and dry environment, control the ambient temperature to be no higher than 30°C and the relative humidity to be no higher than 5% RH, let it stand until the sample pad is completely dry, and store it in a cool and dry environment with a relative humidity of no higher than 5% RH for future use.

[0175] 4. Preparation of labeling pad working solution: Prepare blocking solution according to the standard solution preparation method. The specific formula is as follows: 1 mg / mL mouse anti-human urine protein antibody-labeled polystyrene microspheres, 1% BSA, 3% sucrose, and 0.1% PEG-20000.

[0176] 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.

[0177] The specific steps include: immersing the marking pad in the working solution prepared in step 4 (or spraying the working solution on the marking pad by spraying), and after the working solution is completely absorbed into the marking pad, placing the marking pad in a cool and dry environment with a temperature no higher than 30°C and a relative humidity no higher than 5% RH, and allowing the marking pad to dry completely. The marking pad is then stored in a cool and dry environment with a relative humidity no higher than 5% RH until ready for use.

[0178] 6. Preparation of immunochromatographic membrane: In this example, NC membrane was selected as the immunochromatographic membrane material and prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent.

[0179] The specific steps include: dispersing mouse anti-human urine protein monoclonal antibody in PBS (pH = 7.4) aqueous solution to prepare a working solution, loading the prepared solution into the liquid pipeline of the film stripper, connecting the accessories, and using the film stripper to coat the corresponding position of the immunochromatographic membrane. The antibody concentration is 0.5 mg / mL and 10 μL / cm 2 The streaking speed (length 30 mm) was 0.5 mg / mL and 1 μL / cm 2 After completion, place the membrane in a cool, dry environment with a temperature no higher than 30°C and a relative humidity no higher than 5% RH until it is completely dry. Store the membrane in a cool, dry environment with a relative humidity no higher than 5% RH until it is ready for use.

[0180] 7. In this example, cellulose material is selected as the absorbent pad material.

[0181] 8. After assembling the above materials according to the immunochromatographic test paper assembly method described in this patent, cut them into 5mm test papers for standby use.

[0182] 9. Urine protein immunochromatographic test strips test results for urine protein standards of different concentrations:

[0183] 1) Dilute urine protein standards to the following concentrations: 0, 0.1, 0.5, 1, 2, and 5 mg / mL;

[0184] 2) Use a pipette to draw 60 μL of the above standard solution and drop it onto the sample pad of the urine protein immunochromatographic test paper prepared in this example, and wait for the chromatography reaction to end. The results are shown in Figure 2: The urine protein immunochromatographic test paper prepared in this example shows different results for different concentrations of urine protein standards: a) The quality control lines all show normal color; b) The 0 concentration standard shows a negative result, and the detection area does not show color; c) Under different concentrations of standard, as the concentration increases, the immunochromatographic test paper shows that the positive detection area from bottom to top (for the sample flow direction, extending from the sample addition end to the water absorption end) is getting larger and larger. According to the customized antigen, the detection area can be divided into areas of corresponding concentrations, and the corresponding concentration scale lines are marked on both sides of the detection area.

[0185] 10. Interference Test: Perform interference tests on the immunochromatographic test strips in this example using 10 mmol / mL hemoglobin, 3 μmol / mL heparin, and 15 mg / mL triglycerides. Use a pipette to drop 60 μL of each interfering solution onto the sample pad of the urine protein immunochromatographic test strip prepared in this example. Wait for the chromatographic reaction to complete.

[0186] 11. Actual sample testing: 8 different urine samples were collected through standardized collection and tested using the urine protein immunochromatographic test strips and urine protein ELISA test kit prepared in this example. The results showed that the immunochromatographic test strips prepared in this example had basically consistent test values ​​with ELISA in the analysis of actual samples. The specific results are shown in Table 1:

[0187] Table 1: Comparison of urine protein immunochromatographic test strips and urine protein ELISA test kit

[0188]

[0189] Example 2 Continuous semi-quantitative immunochromatographic test paper for cTnI troponin project

[0190] This example describes the detection of cTnI troponin. Mouse anti-human troponin monoclonal antibodies are used to prepare a continuous semi-quantitative blood troponin assay.

[0191] 1. Mouse anti-human troponin monoclonal antibody-coated colloidal gold: Disperse 40 nm colloidal gold in PBS at a colloidal gold concentration of 10 mg / mL in 0.5 mL. Add 0.5 mL mouse anti-human troponin 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 mouse anti-human troponin monoclonal antibody-labeled colloidal gold dispersion.

[0192] 2. Preparation of sample pad blocking solution: Prepare the blocking solution according to the standard solution preparation method. The specific formula is as follows: 0.5% PVP K30, 1% BSA, 0.02% Tween 20, 0.003% sodium azide, 25 mM sodium chloride.

[0193] 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.

[0194] 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 mouse anti-human troponin monoclonal antibody labeled with colloidal gold, 1% BSA, 2% sucrose, and 0.3% PEG-20000.

[0195] 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.

[0196] 6. Preparation of immunochromatographic membrane: CA membrane was selected as the immunochromatographic membrane material in this example. The membrane was prepared according to the general process of physical adsorption coating of immunochromatographic membrane described in this patent. Mouse anti-human troponin monoclonal antibody was dispersed in PBS (pH=7.4) aqueous solution to prepare a working solution. The prepared solution was loaded into the liquid pipeline of the membrane drawing machine. After connecting the accessories, the membrane drawing machine was used to coat the corresponding position of the immunochromatographic membrane. The antibody concentration was 5 mg / mL and 10 μL / cm 2 The quality control line was a goat anti-mouse IgG antibody with a concentration of 0.5 mg / mL and a streak rate of 1 μL / cm (30 mm length). After completion, store in a cool, dry environment with a temperature below 30°C and a relative humidity below 5% RH until the membrane is completely dry. Store in a cool, dry environment with a relative humidity below 5% RH until ready for use.

[0197] 7. In this example, polyester film is used as the material for the water-absorbing pad and blood-filtering pad.

[0198] 8. After assembling the above materials according to the immunochromatographic test strip assembly method described in this patent, cut them into 5mm test strips for later use. Optionally, use an immunochromatographic reagent card with a fan-shaped direct sample loading interface (with a solid sampler and siphon diffusion method) for testing.

[0199] 9. According to the scheme of Example 1, the numerical range of the immunochromatographic test paper of this example was calibrated, and the calibration concentration range was 0.5-10 ng / mL.

[0200] 10. Actual sample testing: Eight different serum samples were obtained through standardized collection and tested using the cTnI troponin continuous semi-quantitative immunochromatographic test strips and the troponin chemiluminescence detection kit prepared in this example. The results showed that the immunochromatographic test strips prepared in this example had basically consistent test values ​​with the chemiluminescence test kit in the analysis of actual samples.

[0201] The specific results are shown in Table 2:

[0202] Table 2: Comparison of cTnI troponin continuous semi-quantitative immunochromatographic test strips and chemiluminescence detection kits

[0203]

[0204]

[0205] Example 3 Continuous Semi-Quantitative Immunochromatographic Test Paper for Total Prostate Specific Antigen (tPSA)

[0206] This example describes the detection of total prostate-specific antigen (tPSA). Mouse anti-human total prostate monoclonal antibodies are used to prepare a continuous semi-quantitative blood tPSA assay.

[0207] 1. Mouse anti-human tPSA monoclonal antibody-coated blue polystyrene microspheres: Disperse 50 nm blue polystyrene microspheres in PBS at a concentration of 10 mg / mL in 0.5 mL. Add 0.5 mL of mouse anti-tPSA monoclonal antibody at a concentration of 5 mg / mL in PBS buffer. Seal the tube and incubate at room temperature for 2.5 hours. Wash the blue polystyrene microspheres by centrifugation, then rinse them in 1 mL of PBS. Finally, disperse the labeled blue polystyrene microspheres in 1 mL of PBS to prepare a 5 mg / mL dispersion of mouse anti-human tPSA monoclonal antibody-coated blue polystyrene microspheres.

[0208] 2. Prepare the labeling pad, blood filter pad, sample pad, and immunochromatographic pad according to the scheme of Example 1, and assemble the above materials according to the immunochromatographic test paper assembly method described in this patent, and cut them into 5mm test papers for standby use.

[0209] 3. tPSA immunochromatographic test strips test results for urine protein standards of different concentrations:

[0210] 1) Dilute the tPSA-specific antigen standard to the following concentrations: 0, 0.5, 1, 2, 5, and 10 ng / mL;

[0211] 2) Use a pipette to draw 60 μL of the above standard solution and dropwise add it to the sample pad of the tPSA immunochromatographic test strip prepared in this example, and wait for the chromatography reaction to complete. The results are shown in Figure 2: The tPSA immunochromatographic test strip prepared in this example exhibits different results for different concentrations of the tPSA-specific antigen standard: a) The quality control line displays normal color; b) The 0-concentration standard displays a negative result, with no color in the detection area; c) At different concentrations of the standard, the immunochromatographic test strip displays a positive detection area that increases in size from the bottom to the top as the concentration increases. Depending on the customized antigen, the detection area can be divided into zones corresponding to the concentration.

[0212] 4. Actual sample testing: 8 different serum samples were collected through standardized collection and tested using the tPSA continuous semi-quantitative immunochromatographic test strips and tPSA chemiluminescence detection kit prepared in this example. The results showed that the immunochromatographic test strips prepared in this example were basically consistent with the test values ​​of the chemiluminescence in the analysis of actual samples. The specific results are shown in Table 3:

[0213] Table 3: Comparison of tPSA continuous semi-quantitative immunochromatographic test strips and chemiluminescence detection kits

[0214]

[0215]

[0216] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A continuous semi-quantitative immunochromatographic test paper, comprising a sample pad, a labeling pad, an immunochromatographic membrane and a water-absorbing pad connected in sequence, characterized in that: The immunochromatographic membrane is provided with a binding area, which is a reaction area continuously arranged along the chromatography direction. The binding area is used to specifically react with the target in the sample to be tested and produce a color band in the binding area; A scale line corresponding to the known target concentration is provided on the corresponding area of ​​the binding zone, which is used to achieve continuous semi-quantitative detection of the target concentration by comparing the length of the color-developing band in the binding zone with the scale line.

2. A continuous semi-quantitative immunochromatographic test paper according to claim 1, characterized in that: The target concentration corresponding to each scale line increases in sequence along the chromatographic direction; or The scale marking is determined according to the length of the color band of the standard, and specifically includes the following steps: (1) Perform chromatography detection using standard solutions with known concentration gradients and record the length of the color band at different concentrations; (2) Marking a scale line corresponding to the concentration value in the corresponding area of ​​the binding area according to the length of the color band; or The number of the scale lines is at least 2, for example, 2-10; or A specific binding partner is immobilized on the surface of the binding region; or The specific binding partner is evenly distributed on the surface of the binding region; or The concentration of the specific binding partner on the surface of the binding area is gradually decreasing along the chromatographic direction of the sample to be tested; The specific binding partner is immobilized on the surface of the binding region by chemical cross-linking, physical adsorption or bioaffinity binding.

3. A continuous semi-quantitative immunochromatographic test paper according to claim 1, characterized in that: According to an embodiment of the present invention, the sample pad, labeling pad, immunochromatographic membrane, and absorbent pad are connected in sequence along the chromatography direction; one end of the sample pad is pressed on the labeling pad, which contains a specific labeled partner labeled with a signal microsphere; the other end of the labeling pad is pressed on the immunochromatographic membrane, which contains a quality control line containing a secondary antibody against the species of the specific labeled partner; the other end of the immunochromatographic membrane is pressed under the absorbent pad to form an immunochromatographic test paper; or The specific labeling partner and the specific binding partner of the binding region are directed to different epitopes of the sample to be tested.

4. A continuous semi-quantitative immunochromatographic test paper according to claim 1, characterized in that: 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, a surfactant, an antibacterial agent and an inorganic salt; preferably, the blocking solution is an aqueous solution, and the water used is pure water, deionized water or distilled water; 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.

5. A continuous semi-quantitative immunochromatographic test paper according to claim 1, characterized in that: 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 labeling partner includes a small molecule organic compound or a biomacromolecule. The specific partner 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 fixed on the immunochromatographic membrane and the analyte molecule 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; The preparation method of the marking pad comprises the following steps: completely immersing the marking pad material into a working solution containing a labeled specific marking partner or spraying the working solution onto the marking pad, leaving it to stand for a certain period of time, and drying the sample pad after the working solution is completely immersed in the marking pad.

6. A continuous semi-quantitative immunochromatographic test paper according to claim 1, characterized in that: 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 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.

7. A continuous semi-quantitative immunochromatographic method, characterized in that: The following steps are involved: (1) performing immunochromatography using the immunochromatographic test paper according to any one of claims 1 to 6; and (2) directly performing a semi-quantitative reading of the results after the immunochromatography is completed.

8. A continuous semi-quantitative immunochromatographic method according to claim 7, characterized in that: The method comprises the following steps: (a) performing chromatography detection on the sample to be tested; (b) comparing the length of the color band formed by the sample to be tested in the binding zone with the concentration scale line to determine the semi-quantitative result of the concentration of the sample to be tested; In step (b), the signal change of the length of the color band in the binding area is continuous, which can achieve a progressive estimation of the target substance in the sample to be tested within a certain concentration range; or By comparing the length of the color-developing band with the relative position between the preset concentration scale lines, the concentration range corresponding to the two adjacent scale lines of the target substance in the sample is determined; further, by comparing the distance between the end point of the color-developing band and the two adjacent scale lines, it is determined which scale line represents the concentration value to which the target substance concentration in the sample is closer, thereby achieving continuous semi-quantitative analysis.

9. A direct loading immunochromatography device, characterized in that: The chromatography device comprises the immunochromatographic test paper according to any one of claims 1 to 6, a housing for accommodating the immunochromatographic test paper, and a sampler. The housing is provided with a sample loading port, and the sample loading port has a sample loading port interface, and the sample loading port interface can match the interface of the sampler, so that the sampler can be installed on the sample loading port of the chromatography kit; The sampler includes a main body and a sampling pad. The sampling pad is provided at the bottom of the sampler. The sampler main body has an interface. The interface is located above the sampling pad and can be matched with the sample loading port interface of the chromatography kit, so that the sampler can be installed on the sample loading port of the chromatography kit.

10. A direct loading immunochromatography method, characterized in that: The method includes using the direct loading immunochromatography device described in claim 9, and includes the following steps: 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 fluid, and the results are directly semi-quantitatively read.

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