TW202127031A - Test kit for detecting a plurality of analytes - Google Patents

Abstract

The present disclosure provides a test kit for simultaneously detecting a plurality of analytes, in which the test kit includes a lysis solution and a test strip. The lysis solution includes a salt, a surfactant, a stabilizer, and a buffer solution. The test strip includes a sample pad, a conjugation pad, a cellulose membrane, and a water-absorbing pad sequentially arranged on a support plate. The conjugation pad includes a conjugation pad solution and a plurality of antibody-conjugated microspheres, and the antibody-conjugated microspheres recognize a plurality of analytes. A method for preparing the antibody- conjugated microspheres includes performing an activation reaction between 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and microspheres, and an activation reaction time is from 16 to 24 hours.

Description

Inspection kits for detecting a variety of analytes

The present disclosure relates to inspection kits and methods, and in particular, to inspection kits and methods that can simultaneously detect multiple analytes.

Clinically, although many diseases are derived from different pathogens or viruses, they have similar clinical symptoms (such as respiratory diseases caused by respiratory viruses), which poses a high risk of doctors' misjudgment of medication. Although medical institutions have gradually introduced procedures for rapid pathogen screening using test kits (such as immunochromatography kits) before treatment, to prescribe the right medicine to improve the treatment effect. However, since most of the current test kits are in one sample, they can only screen for a single test object. If you want to test multiple objects to be tested at the same time, it is often because of the need to collect samples repeatedly, which causes discomfort to the patient and affects the willingness of the patient to receive the test. Therefore, often most medical hospitals can only perform quick screening of a single test substance, but cannot further know whether there are still other pathogenic bacteria or viruses lurking in the body, which affects the effectiveness of the treatment.

The immunochromatography kit is a rapid diagnostic technique that has emerged in recent years, which has the characteristics of accuracy, speed and simple operation. The principle is to pre-treat the sample with a lysis solution to improve the binding ability of the test substance or its specific antigen. The test piece has an absorption area, an identification area, and a color development area in sequence. The concept of detection is as follows. The absorption area in the test strip can absorb the test substance or its specific antigen in the sample, and the absorbed test substance or its specific antigen will gradually move in the test strip with capillary action. Then, the analyte or its specific antigen will bind to the color-forming molecule with specific antibody (for example, the microsphere with the color-forming group) in the identification area. Then, when the analyte or its specific antigen moves to the coloring area, another specific antibody fixed on the coloring area can identify the analyte or its specific antigen, and then show a color band in the coloring area In this way, it can be distinguished whether there is an object to be tested in the specimen.

However, because the test strips in the immunochromatography kit have an upper limit on the maximum liquid capacity, if you want to detect multiple analytes on the same test strip at the same time, when you need to configure multiple specific antibodies, it will inevitably lead to A specific antibody is lower than the concentration of a single antibody used in the determination of a single analyte. When there are more kinds of analytes, the upper limit of the loadable antibody concentration in the test piece will be lower, resulting in weaker signal and difficult to interpret.

Therefore, it is necessary to improve the current inspection kits and methods, and propose inspection kits and methods that can detect multiple objects to be tested at the same time.

In some embodiments of the present disclosure, a lysis solution is provided. The lysis solution is used for the pretreatment of quick screening test. The weight percentage of the lysis solution is calculated as 100%. The lysis solution includes salts, surfactants, stabilizers, and buffer solutions. . The weight percentage of the salt is between about 0.5% and 5%. The weight percentage of the surfactant is between about 0.5% to about 5%. The weight percentage of the stabilizer is between about 0.5% to about 5%.

In some embodiments, the weight percentage of the salt is between about 1% and 2%, the weight percentage of the surfactant is between about 1% and about 3%, and the weight percentage of the stabilizer is between about 1. % To about 3%.

In some embodiments, the lysis solution separates the antigen of the respiratory virus.

In some embodiments, the respiratory virus includes adenovirus (Adenovirus), influenza A virus (Influenza A, Flu A), influenza B virus (Influenza B, Flu B), influenza C virus (Influenza C, Flu C) , Respiratory syncytial virus (RSV), Rhinovirus (RhV), or Coronavirus (CoV).

In some embodiments, the salt comprises sodium chloride, potassium chloride, or a combination of the foregoing.

In some embodiments, the surfactant includes sodium dodecyl sulfate (SDS), polysorbate (Tween) series, Triton series, nonylphenol (NP) series Or a combination of the foregoing.

In some embodiments, the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing.

In some embodiments, the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In some embodiments, the lysis solution contains sodium azide.

In some embodiments of the present disclosure, a method for preparing antibody-coupled microspheres is provided, which includes the following steps: providing microspheres; providing rinsing liquid; mixing the microspheres and the rinsing liquid to obtain a first mixed liquid; mixing the first mixed liquid with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC), 1-ethyl-3-(3-di (Methylaminopropyl) carbodiimide and the microspheres in the first mixed solution undergo an activation reaction, and the activation reaction time is between 16 to 24 hours; the microspheres after the activation reaction are mixed with the coupling solution to obtain the first Two mixed solutions; and mixing the first antibody and the second mixed solution, so that the first antibody and the microspheres in the second mixed solution are coupled at room temperature to obtain antibody-coupled microspheres.

In some embodiments, the rinse solution includes 2-(N-morpholino)ethanesulfonic acid (MES).

In some embodiments, the microsphere is a latex particle, a gold particle, a chemiluminescent group, an enzyme group or biotin.

In some embodiments, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the first mixed solution is between 0.1M and 0.5M.

In some embodiments, the step of mixing the first mixed solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide further comprises mixing 1-ethyl-3-(3-diimide). Methylaminopropyl) carbodiimide, the first mixture and N-Hydroxysuccinimide (N-Hydroxysuccinimide; NHS), or mixed 1-ethyl-3-(3-dimethylamino) Propyl)carbodiimide, the first mixed liquid and sulfonated N-hydroxysuccinimide.

In some embodiments, the coupling solution is a buffer solution, including phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, trimethylolaminomethane, trimethylolmethylaminopropanesulfonic acid, N,N-bis(2- Hydroxyethyl)glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine ) Ethanesulfonic acid or a combination of the foregoing.

In some embodiments, in the step of mixing the first antibody and the second mixed solution, the weight ratio of the first antibody and the microspheres in the second mixed solution is about 1:40 to 1:120.

In some embodiments of the present disclosure, a sample pad solution in an immunochromatographic test kit is provided. The sample pad solution has a weight percentage of 100% and includes a surfactant, a stabilizer, a dispersant, and a buffer solution. Surfactant, the weight percentage is between about 0.5% to 5%. Stabilizer, the weight percentage is between about 0.05% to about 10%. Dispersant, the weight percentage is between about 0.5% to about 5%.

In some embodiments, the weight percentage of the surfactant is between about 0.5% and 3%. The weight percentage of the stabilizer is between about 0.1% to about 0.5%. The weight percentage of the dispersant is between about 0.5% to about 1.5%.

In some embodiments, the surfactant comprises sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series, or a combination of the foregoing.

In some embodiments, the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing.

In some embodiments, the dispersant comprises polyvinylpyrrolidone (PVP), polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropyl cellulose, or a combination of the foregoing.

In some embodiments, the buffer solution comprises phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propane sulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In some embodiments, the sample pad solution further contains sodium azide.

In some embodiments of the present disclosure, there is provided a test kit for simultaneously detecting multiple analytes, including a lysis solution and a test piece. The test piece includes a sample pad, a bonding pad, a fiber membrane, and a water-absorbing pad sequentially lapped on the support plate. The binding pad includes a binding pad solution and a plurality of antibody-coupled microspheres. These antibody-coupled microspheres identify a variety of test objects, and these antibody-coupled microspheres are prepared by the aforementioned method.

In some embodiments, the sample pad contains a sample pad solution. One of the antibody-coupled microspheres recognizes one of the analytes, any one of the antibody-coupled microspheres has a first antibody on the surface, and any one of the antibody-coupled microspheres recognizes a specific one of the analytes through the first antibody . When the first antibodies of a group of these antibody-coupled microspheres recognize the same test object, these antibody-coupled microspheres have the same color. When the first antibodies of the antibody-coupled microspheres of different groups recognize different test objects, the antibody-coupled microspheres have different colors. The fiber membrane contains a plurality of strips, these strips do not overlap each other, any one of these strips contains a second antibody, the second antibody recognizes the test substance, and the second antibody on different strips recognizes different test substances. The second antibody of each band and one of these first antibodies respectively recognize different sites of the same antigen of the test substance.

In some embodiments, these test substances are respiratory viruses, including adenovirus, influenza A virus, influenza B virus, influenza C virus, respiratory fusion virus, rhinovirus, coronavirus, or a combination thereof.

In some embodiments, the lysis solution is used for the pre-treatment of the test, the weight percentage of the lysis solution is 100%, and the lysis solution includes salts, surfactants, stabilizers, and buffer solutions. Salts, the weight percentage is between about 0.5% to 5%. Surfactant, the weight percentage is between about 0.5% to about 5%. Stabilizer, the weight percentage is between about 0.5% to about 5%.

In some embodiments, the salt comprises sodium chloride, potassium chloride, or a combination of the foregoing.

In some embodiments, the surfactant comprises sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series, or a combination of the foregoing.

In some embodiments, the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing.

In some embodiments, the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In some embodiments, the lysis solution further contains sodium azide.

In some embodiments, the weight percentage of the sample pad solution is 100%, and the sample pad solution includes a surfactant, a stabilizer, a dispersant, and a buffer solution. Surfactant, the weight percentage is between about 0.5% to 5%. Stabilizer, the weight percentage is between about 0.05% to about 10%. Dispersant, the weight percentage is between about 0.5% to about 5%.

In some embodiments, the surfactant comprises sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series, or a combination of the foregoing.

In some embodiments, the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing.

In some embodiments, the dispersant comprises polyvinylpyrrolidone, polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropylcellulose, or a combination of the foregoing.

In some embodiments, the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In some embodiments, the sample pad solution further contains sodium azide.

In some embodiments, the bonding pad solution further contains stabilizers, dispersants, emulsifiers, and buffer solutions.

In some embodiments, the weight percentage of the bonding pad solution is 100%, the weight percentage of the stabilizer is between about 0.05% and 2%, the weight percentage of the dispersant is between about 0.05% and 10%, and The weight percentage of emulsifier is between about 0.1% to about 5%.

In some embodiments, the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing.

In some embodiments, the dispersant comprises polyvinylpyrrolidone, polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropylcellulose, or a combination of the foregoing.

In some embodiments, the emulsifier includes a non-ionic emulsifier, including polyoxyethylene 35 castor oil (Polyoxyl 35 Castor Oil), polyoxyethylene 40 hydrogenated castor oil (Polyoxyl 40 Hydrogenerated Castor Oil), polysorbate 20 (Polysorbate 20). 20), or a combination thereof.

In some embodiments, the total concentration of the first antibody is no higher than 600 μg/ml, and the total concentration of the second antibody is no higher than 7.2 mg/ml.

In some embodiments of the present disclosure, a method for simultaneously detecting multiple test objects is provided, which includes the following steps: providing a sample; providing a test kit, which includes a test piece and a lysis solution. The test piece includes a sample pad, a bonding pad, a fiber membrane, and a water-absorbing pad sequentially lapped on the support plate; the sample pad includes a sample pad solution. The binding pad includes a binding pad solution and a plurality of antibody-coupled microspheres. These antibody-coupled microspheres identify a variety of test objects. Any of these antibody-coupled microspheres has a specific color and a first antibody; the fiber membrane contains a plurality of strips. , The positions of these bands do not overlap with each other, any one of these bands contains a second antibody, the second antibody recognizes the analyte, the second antibody on different bands recognizes different analytes; the second antibody of each band Recognize different sites of the same test substance antigen with one of these primary antibodies; add the specimen to the lysis solution; immerse the test piece in the lysis solution containing the specimen with the sample pad facing downward, and The bonding pad will not come into contact with the lysis solution; it is judged whether the strips of the test piece are colored, and when one or more of these strips are colored, the relative one or more test objects are detected.

In some embodiments, the weight percentage of the lysis solution is 100%, and the lysis solution includes salts, surfactants, stabilizers, and buffer solutions. Salts, the weight percentage is between about 0.5% to 5%. Surfactant, the weight percentage is between about 0.5% to about 5%. Stabilizer, the weight percentage is between about 0.5% to about 5%.

In some embodiments, the salt includes sodium chloride, potassium chloride or a combination of the foregoing, the surfactant includes a nonylphenol series, the stabilizer includes serum protein, and the buffer solution includes a phosphate buffer solution.

In some embodiments, the lysis solution further contains sodium azide.

In some embodiments, the antibody-coupled microspheres are prepared by the following steps: providing microspheres; providing a washing solution; mixing the microspheres and the washing solution to obtain a first mixed solution; mixing 1-ethyl-3-(3- Dimethylaminopropyl) carbodiimide and the first mixed liquid; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the microspheres in the first mixed liquid Activation reaction, the activation reaction time is between 16 to 24 hours; mix the microspheres after the activation reaction and the coupling solution to obtain a second mixed solution; mix the first antibody and the second mixed solution to make the first antibody and the second The microspheres in the mixed solution are coupled at room temperature to obtain antibody-coupled microspheres.

In some embodiments, the rinse solution includes 2-(N-morpholino)ethanesulfonic acid.

In some embodiments, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the first mixed solution is between 0.1M and 0.5M.

In some embodiments, the step of mixing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the first mixed solution further comprises mixing 1-ethyl-3-(3-diimide (Methylaminopropyl)carbodiimide, the first mixed solution and N-hydroxybutanediimide, or mixed 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Amine, the first mixed liquid and sulfonated N-hydroxysuccinimide.

In some embodiments, the coupling fluid is a phosphate buffer solution.

In some embodiments, when the first antibodies of a group of the antibody-coupled microspheres recognize the same analyte, the antibody-coupled microspheres have the same color, and when the first antibodies of the different groups of the antibody-coupled microspheres When antibodies recognize different test objects, these antibody-coupled microspheres have different colors.

In some embodiments, the weight percentage of the sample pad solution is 100%, and the sample pad solution includes a surfactant, a stabilizer, a dispersant, and a buffer solution. The weight percentage of the surfactant is between about 0.5% and 5%. The weight percentage of the stabilizer is between about 0.05% and about 10%. The weight percentage of the dispersant is between about 0.5% to about 5%.

In some embodiments, the surfactant includes nonylphenol-40 (NP-40), the stabilizer includes casein, the dispersant includes polyvinylpyrrolidone, and the buffer solution includes tris.

In some embodiments, the sample pad solution further contains sodium azide.

In summary, the present disclosure achieves good applicability for lysing a variety of analytes through the preparation of a lysis solution with appropriate components, and improves the preparation method of microspheres, improves the efficiency of microsphere coupling antibodies, and thus achieves Under the limited capacity of the antibody in the binding pad of the test strip, the effects of more than four test objects can be detected simultaneously, and the detection of each test object has an ideal detection limit.

In order to make the description of the present invention more detailed and complete, the following describes the embodiments and specific examples of the present invention in detail; but this is not the only way to implement or use the specific embodiments of the present invention. The embodiments disclosed below can be combined or substituted with each other under beneficial circumstances, and other embodiments can also be added to an embodiment without further description or description. In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, the embodiments of the present invention may be practiced without these specific details.

In this article, unless there are special restrictions on the article in the text, "一" and "the" can generally refer to one or more. It will be further understood that the words "include", "include", "have" and similar words used in this text indicate the recorded features, regions, integers, steps, operations, elements and/or components, but do not Exclude other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

Although a series of operations or steps are used in the following to illustrate the method disclosed herein, the sequence of these operations or steps should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in a different order and/or simultaneously with other steps. In addition, not all operations, steps, and/or features must be performed to implement the embodiments of the present invention. Furthermore, each operation or step described herein may include several sub-steps or actions.

Please refer to FIG. 1 and FIG. 2. Some embodiments of the present disclosure provide a test kit 1 for detecting multiple analytes at the same time, including a lysis reagent kit 2 and a test piece 3. The lysis reagent set 2 includes at least a lysis solution 210, which is used for the quick screening test pretreatment of the specimen, and improves the detection sensitivity of the test strip 3 to the test object in the specimen and the fluidity of the specimen. The test strip 3 is based on the principle of immunochromatography to detect whether the sample processed by the lysis solution 210 contains the test substance antigen, so as to determine whether the test substance is present in the sample.

Generally speaking, if you want to detect multiple test objects on the same test strip at the same time, it is limited by the maximum capacity of the antibody The color rendering is weak and even undetectable. The present disclosure improves the detection sensitivity of the test kit 1 for the antigen of the test object through the improvement of the lysis solution 210 and the soaking solution of the test piece 3, and can detect a variety of test objects (such as 2, 3, 4) at the same time. Kind or more). The test kit 1 is helpful for real-time clinical interpretation of the results by the color of the test piece, and can detect multiple test objects at the same time, which can shorten the test time and labor and material costs.

In one embodiment, the specimen is derived from the respiratory tract, such as the oral cavity, nasal cavity, trachea, or bronchi. In an embodiment, the specimen may be saliva, oral mucosa, nasal mucosa, nasal mucosa, tracheal secretions, or/and bronchial secretions.

In one embodiment, the test substance contains a respiratory virus, such as adenovirus, influenza A virus, influenza B virus, influenza C virus, respiratory syncytial virus, rhinovirus, or coronavirus.

In some embodiments of the present disclosure, there is provided a lysis solution 210 comprising salts, surfactants, stabilizers, and buffer solutions. In one embodiment, the lysis solution 210 may be suitable for separating antigens of various respiratory viruses.

In one embodiment, the salt can cause the lysis solution 210 to be in a hypertonic state, which helps to destroy the ionic bonds in the specimen's mucus and reduce the viscosity of the specimen. Since the movement of the specimen on the test piece 3 is through capillary action, reducing the viscosity of the specimen can improve the smoothness of the movement of the specimen on the test piece 3. If the concentration is too high, the identification result of the test piece 3 will appear false positive. In some embodiments, the salt includes an alkali metal halide (e.g., potassium chloride), an alkaline earth metal halide (e.g., sodium chloride), or a combination of the foregoing. In some embodiments, the weight percentage of the lysis solution 210 is 100%, and the weight percentage of the salt is between about 0.5% and 5%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%. %, 3.5%, 4%, 4.5%, 5% or any value in any interval above. In one embodiment, the weight percentage of the salt is between about 1% and 2%.

In one embodiment, the surfactant can be used to destroy the membrane-like structure on the surface of the test object (for example, the membrane protein of a virus), so as to increase the exposure of the test object antigen and improve the detectability of the antigen. In some embodiments, the surfactant includes sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series, or a combination of the foregoing. In some embodiments, the weight percentage of the lysis solution 210 is 100%, and the weight percentage of the surfactant is between about 0.5% to about 5%, such as 0.5%, 1%, 1.5%, 2%, 2.5% , 3%, 3.5%, 4%, 4.5%, 5% or any value in any interval above. In one embodiment, the weight percentage of the surfactant is between about 1% and 3%.

In one embodiment, the stabilizer can be used to prevent protein decomposition and non-specific identification of the antibody to the analyte. If the concentration is too high, the identification result of the test strip 3 will be falsely positive. In some embodiments, the stabilizer includes serum protein, casein, artificial polymer, or a combination of the foregoing. In some embodiments, the weight percentage of the lysis solution 210 is 100%, and the weight percentage of the stabilizer is between about 0.5% and about 5%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value in any interval above. In one embodiment, the weight percentage of the stabilizer is between about 1% and 3%.

In one embodiment, the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In one embodiment, the lysis solution 210 may include sodium azide.

In one embodiment, the lysis reagent set 2 may optionally include a collection tube 220, a sampling rod 230, or both. The sampling rod 230 is used to collect the specimen, for example, the specimen is collected from the nasal cavity or oral mucosa. The collection tube 220 can be used to store the lysis solution 210 and serve as a reaction area for sample pretreatment.

Next, please refer to FIG. 3, the test piece 3 includes a sample pad 310, a bonding pad 320, a fiber membrane 330, and a water absorbing pad 340 which are sequentially overlapped on the support plate 350.

In one embodiment, the sample pad 310 includes a sample pad solution, including a surfactant, a stabilizer, a dispersant, and a buffer solution.

In one embodiment, the surfactant can be used to destroy the membrane-like structure on the surface of the analyte, so as to increase the exposure of the analyte antigen and improve the detectability of the antigen. In some embodiments, the surfactant of the sample pad solution includes sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series, or a combination of the foregoing. In some embodiments, the weight percentage of the sample pad solution is 100%, and the weight percentage of the surfactant is between about 0.5% and about 5%, such as 0.5%, 1%, 1.5%, 2%, 2.5% , 3%, 3.5%, 4%, 4.5%, 5% or any value in any interval above. In one embodiment, the weight percentage of the surfactant is between about 0.5% and 3%.

In one embodiment, the stabilizer can be used to prevent the decomposition of protein and the non-specific identification of the antibody to the analyte. In some embodiments, the stabilizer of the sample pad solution includes serum protein (for example, bovine serum albumin), casein (for example, derived from cow milk), amino acid (for example, glycine), artificial polymer, or a combination of the foregoing . In some embodiments, the weight percentage of the sample pad solution is 100%, and the weight percentage of the stabilizer is between about 0.05% and about 10%, such as 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any value in any interval above. It is worth mentioning that when the weight percentage of the stabilizer is between 0.1 and 0.5% and/or casein is used as the stabilizer, the sample pad solution can have better fluidity.

In one embodiment, the dispersant can prevent the solution from settling or agglomerating, so as to improve the storage stability and maintain the storage concentration. In some embodiments, the dispersant of the sample pad solution includes polyvinylpyrrolidone (e.g., polyvinylpyrrolidone-10), polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropylcellulose, or a combination of the foregoing. In some embodiments, the weight percentage of the sample pad solution is 100%, and the weight percentage of the dispersant is between about 0.5% and about 5%, such as 0.5%, 1%, 2%, 3%, 4%, 5% or any value in any interval above. In one embodiment, the weight percentage of the dispersant is between about 0.5% and 1.5%.

In one embodiment, the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2-hydroxyethyl) Glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine)ethanesulfonic acid Or a combination of the foregoing.

In one embodiment, the sample pad solution contains sodium azide.

In one embodiment, the binding pad 320 includes a binding pad solution and a plurality of antibody-coupled microspheres, and these antibody-coupled microspheres can identify a variety of test objects. In one embodiment, any one of the antibody-coupled microspheres has a first antibody on the surface, and the antibody-coupled microspheres can be divided into different groups according to the type of antigen recognized by the first antibody on the surface of the antibody-coupled microspheres. Therefore, a specific group of antibody-coupled microspheres can identify a specific one of a variety of test objects. In one embodiment, the first antibody detects viral antigens, such as respiratory viruses, including, for example, adenovirus, influenza A virus, influenza B virus, influenza C virus, respiratory syncytial virus, rhinovirus, or coronavirus .

In one embodiment, the microsphere itself can be directly colored (for example, colored latex particles or gold particles), can be a color-producing group (for example, a chemiluminescent group), or be present after adding a suitable reaction solution. The color, for example, contains an enzyme group (such as horseradish peroxidase). In addition, it may optionally contain other functional groups, such as biotin or streptavidin.

In one embodiment, referring to Figs. 3 and 4, the material of the fiber membrane 330 may be nitrocellulose. The fiber membrane 330 includes a plurality of strips 331, the positions of the strips 331 do not overlap each other, and any one of the strips 331 contains a second antibody, which can identify the test object. In one embodiment, the second antibody of each strip 331 and the specific first antibody respectively identify different sites of the same analyte antigen. Therefore, the second antibody on different strips 331 can identify different analytes. Things. That is, when there are multiple types of test objects in the specimen, different groups of antibody-coupled microspheres will respectively identify the specific test objects. Then, after the test objects are respectively combined with the strips 331 on the fiber membrane 330, the color of the antibody-coupled microspheres is transmitted through the color of the fiber membrane 330. The inspector can distinguish the presence or absence of a specific object to be tested according to the color position of the strip 331.

In one embodiment, the total concentration of the first antibody is not higher than 600 μg/ml, such as 100, 200, 300, 400, 500, 600 μg/ml, or any value in any interval above, and the second antibody The total concentration of is not higher than 7.2 mg/ml, such as 1, 2, 3, 4, 5, 6, 6.5, 7.2 mg/ml, or any value in any interval above.

In one embodiment, please also see Figures 3 and 4. When the first antibody on a group of these antibody-coupled microspheres can identify the same test substance, the group of antibody-coupled microspheres has the same color at the same time . In one embodiment, when the first antibodies of different groups of antibody-coupled microspheres recognize different test objects, the different groups of antibody-coupled microspheres have different colors. According to this, the examiner can distinguish the type of the test object contained in the sample according to the color. In some embodiments, when there is respiratory fusion virus (RSV), influenza B virus (Flu B), influenza A virus (Flu A), adenovirus (Adeno) or any combination of the above viruses in the specimen, the test piece 3 With antibody-coupled microspheres that can identify these four viruses. For example, in the fiber membrane 330 in Figure 4, from top to bottom, there are control lines, RSV, Flu B, Flu A, and Adeno colored strips. The order of the strips 331 can be adjusted according to requirements and is not limited to the above examples. Accordingly, different bands 331 can reflect the presence or absence of a specific virus. In addition, in order to simplify the interpretation, the different strips 331 are presented in different colors.

In some embodiments, the method for preparing antibody-coupled microspheres includes the following steps: providing microspheres and providing a washing solution; mixing the microspheres with the washing solution to obtain a first mixed solution; mixing the first mixed solution with 1-ethyl-3 -(3-Dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the microspheres in the first mixture are activated Reaction; mixing the activated reaction of the microspheres and the coupling solution to obtain a second mixed solution; and mixing the first antibody and the second mixed solution so that the first antibody and the microspheres in the second mixed solution are coupled at room temperature Function to obtain antibody-coupled microspheres.

In one embodiment, the rinse solution includes 2-(N-morpholino)ethanesulfonic acid.

In one embodiment, the first mixed solution is mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) Base) carbodiimide and the microspheres in the first mixed solution are subjected to an activation reaction step, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the first mixed solution The concentration is between 0.1M and 0.5M, such as 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, or any value in any interval above.

In one embodiment, the activation reaction time is between 16 and 24 hours, such as 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any of the above Any value in the spacing. It is worth noting that the activation reaction indicated in the commercially available microspheres generally takes between 15 minutes and 30 minutes. It is worth mentioning that by extending the activation reaction time of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the microspheres, the effect of improving the coupling rate of the first antibody of the microspheres is achieved. It is helpful to improve the sensitivity of the microspheres to detect the object to be tested, thereby achieving the effect that the test piece 3 can be colored at the same time to detect whether multiple types of test objects (for example, four types) are present in the sample.

In one embodiment, the step of mixing the first mixed solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide further comprises mixing 1-ethyl-3-(3-diimide). (Methylaminopropyl) carbodiimide, the first mixed solution and N-hydroxybutanediimide, or mixed 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide The amine, the first mixed solution, and the sulfonated N-hydroxysuccinimide can also be mixed and reacted with other additives that contribute to the activation reaction at the same time as required.

In one embodiment, the coupling solution is a buffer solution containing phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propanesulfonic acid, N,N-bis(2- Hydroxyethyl)glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N-morpholine ) Ethanesulfonic acid or a combination of the foregoing.

In one embodiment, in the step of mixing the first antibody and the second mixed solution, the weight ratio of the first antibody to the microspheres in the second mixed solution is about 1:40 to 1:120, such as 1:40, 1:50 , 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, or any ratio in any of the above intervals. In one embodiment, after the coupling action, it includes adding Ethanolamine to the solution containing the first antibody and the second mixed solution. In one embodiment, adding Ethanolamine to the solution containing the first antibody and the second mixed solution After that, a washing solution containing a stabilizer (such as casein) and a buffer solution is included to wash and suspend the microspheres.

In one embodiment, the bonding pad solution includes a stabilizer, a dispersant, an emulsifier, and a buffer solution.

In one embodiment, the stabilizer can be used to prevent the decomposition of protein and the non-specific identification of the antibody to the analyte. In some embodiments, the stabilizer of the binding pad solution includes serum protein (for example, bovine serum albumin), casein (for example, derived from cow milk), amino acid (for example, glycine), artificial polymer, or a combination of the foregoing . In some embodiments, the weight percentage of the bonding pad solution is 100%, and the weight percentage of the stabilizer is between about 0.05% and about 2%, such as 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2% or any value in any interval above. It is worth mentioning that when the weight percentage of the stabilizer is between 0.1 to 0.5% and/or casein is used as the stabilizer, the bonding pad solution may have better fluidity.

In one embodiment, the dispersant can prevent the solution from settling or agglomerating, so as to improve the storage stability and maintain the storage concentration. In some embodiments, the dispersant of the bonding pad solution includes polyvinylpyrrolidone (e.g., polyvinylpyrrolidone-10), polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropylcellulose, or a combination of the foregoing. In some embodiments, the weight percentage of the bonding pad solution is 100%, and the weight percentage of the dispersant is between about 0.05% and about 10%, such as 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any value in any interval above.

In one embodiment, the emulsifier is used to stabilize the microspheres present on the bonding pad 320, and the emulsifier can be a nonionic emulsifier, including polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, and polysorbate. 20, or a combination thereof. In some embodiments, the weight percentage of the bonding pad solution is 100%, and the weight percentage of the emulsifier is between about 0.1% and about 5%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5% or any value in any interval above.

It is worth noting that the components between the lysis solution 210, the sample pad solution, and the binding pad solution can optionally contain the same or similar components, which helps to improve the smoothness of the flow of the specimen on the test strip 3.

Please refer to Figures 2-5 at the same time. In some embodiments of the present disclosure, a method for detecting multiple test objects at the same time is further provided, including the following steps: providing a sample; providing a test kit 1, which includes a test Piece 3 and lysis solution 210. Test piece 3 includes a sample pad 310, a bonding pad 320, a fiber membrane 330, and an absorbent pad 340, which are sequentially overlapped on the support plate 350; add the sample to the lysis solution 210; add the test piece 3 Immerse the sample pad 310 into the lysis solution 210 containing the sample in the downward direction, and the binding pad 320 will not contact the lysis solution 210, so as to avoid affecting the sensitivity of the binding of the microspheres in the solution of the binding pad 320 to the test object; Next, determine whether the strips 331 on the test strip 3 are colored. When one or more of the strips 331 are colored, it means that the specimen contains the specific test object corresponding to the colored strips 331.

In one embodiment, in the step of adding the sample to the lysis solution 210, the action time of the sample in the lysis solution 210 is between about 0.5 minutes and 5 minutes, such as 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes. Minutes, 5 minutes, or any value in any interval above.

In one embodiment, the duration of the step of immersing the test piece 3 in the lysis solution 210 containing the sample with the sample pad 310 facing downward is between about 5 minutes and 15 minutes, such as 5 minutes, 6 minutes, 7 minutes. Minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes or any value in any interval above.

In order to further test the inspection kit 1 provided by the various embodiments of the present disclosure and the method of simultaneously detecting multiple analytes, the following implementations are carried out. It should be noted that the following embodiments are provided for exemplary purposes only, rather than limiting the present invention.

Example 1. Preparation and composition of test kit

First, in order to obtain a test kit that can simultaneously detect multiple test objects in the same specimen, in this embodiment, four respiratory viruses (adenovirus, influenza A virus, influenza B virus, and respiratory fusion Virus) is used as the test object to improve the lysis solution in the test kit and the solution in the test piece.

In the past, different lysing solutions required different formulas to lyse different viruses. Therefore, if four types of viruses were to be lysed, four lysing solutions with different ingredients or contents were required. The manpower and time required for preparation were all different. Cause a certain degree of loss. The lysis solution of this embodiment (see Table 1) can effectively lyse the virus at the same time by using one formula, so that the four viruses can release viral antigens. In this embodiment, the solvent of the lysis solution is water. In addition, the compatibility of the sample pad solution and the binding pad solution is also taken into consideration, and the smoothness of the flow of the lysis solution in the test piece is improved.

The sample pad in the test piece is soaked in the sample pad solution and then dried. The sample pad can be a glass fiber pad. The sample pad solution contains the composition shown in Table 2, and the solvent is water.

The bonding pad in the test piece may be a polyester fiber gasket, and the composition of the bonding pad solution contained therein is shown in Table 3, and the solvent of the bonding pad solution is water. The preparation process of the bonding pad is as follows. First, the bonding pad is soaked in the bonding pad solution and then air-dried, and then the latex solution (Latex Solution) is sprayed on the bonding pad, and it is obtained by drying. The amount of latex sprayed can be 2 to 4 microliters (μl) of latex sprayed on the bonding pad per square centimeter. The dispersant in the bonding pad solution can promote the separation of particles in the latex solution to avoid precipitation or agglomeration, so as to improve storage stability and increase the coloring effect of the test piece.

Table 1. Composition of lysis solution serial number Element Content (weight percentage) 1 Sodium Chloride 1.97% 2 Potassium Chloride 0.02% 3 Sodium Phosphate Dibasic Dihydrate 0.18% 4 Potassium Dihydrogen Phosphate 0.03% 5 Nonylphenol-40 (NP-40) 1% 6 Sodium Azide 0.08% 7 Bovine Serum Albumin (BSA) 1%

Table 2. Composition of sample pad solution serial number Composition Content (weight percentage) 1 Sodium Azide 0.07% 2 Tris(hydroxymethyl)aminomethane; Tris 0.5% 3 Casein From Bovine Milk 0.48% 4 Polyvinylpyrrolidone-10 (Polyvinylpyrrolidone -10; PVP-10) 0.95% 5 Nonylphenol-40 (NP-40) 0.9% 6 1N HCl Solution 3.5%

Table 3. Composition of bonding pad solution serial number Composition Content (weight percentage) 1 Sodium Phosphate Dibasic Dihydrate 0.88% 2 Casein From Bovine Milk 0.49% 3 Polyvinylpyrrolidone-10 (Polyvinylpyrrolidone -10; PVP-10) 0.49% 4 Sodium Azide 0.02% 5 Polyoxyethylene (35) castor oil (KOLLIPHOR ^® EL) 0.1% 6 1N HCl Solution 0.49%

The latex liquid contains four antibody-coupled microspheres that can identify four viruses respectively. The preparation of each antibody-coupled microsphere includes the following steps. First, mix colored latex microspheres (purchased from Thermo Fisher, commercial models DR1040HA, DR1040CA, DB1040HA, DB1040CA, and DBK1040CA) with a washing solution (20mM MES buffer, pH 6.5). The mixed solution is ultrasonically shaken and washed After washing the liquid, resuspend and quantify to obtain a first mixed liquid containing 8.5 mg/ml latex microspheres.

Next, add sulfonated N-hydroxysuccinimide to 1 ml of the first mixed solution, adjust the concentration of sulfonated N-hydroxysuccinimide to 0.4M, and shake to treat the sulfonated N-hydroxybutane. The first mixed solution of diimide. Then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the first mixed solution containing sulfonated N-hydroxysuccinimide to make 1-ethyl- The concentration of 3-(3-dimethylaminopropyl)carbodiimide in the first mixed solution is 0.2M. The first mixture containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfonated N-hydroxysuccinimide is shaken and rotated at room temperature, The first mixed solution is uniformly mixed to activate the functional groups on the latex microspheres for 16 to 24 hours.

Next, mix the activated latex microspheres with the coupling solution (50mM PB buffer, pH 7.5, Na ₂ HPO ₄ • 2H ₂ O: 7.223 g/L, NaH ₂ PO ₄ •H ₂ O: 1.29807 g/L) After washing the latex microspheres with the coupling solution several times, resuspend the latex microspheres in the coupling solution so that the concentration of the latex microspheres is between 7.5 mg/ml and 9 mg/ml to obtain a second mixed solution.

Next, add the first antibody to the second mixed solution so that the concentration (w/v) of the first antibody in the second mixed solution is respectively adenovirus antibody: 150 μg/ml, influenza A virus antibody: 150 μg/ Ml, influenza B virus antibody: 100 μg/ml, respiratory tract fusion virus antibody: 100 μg/ml, rotate for 2 hours at room temperature, add ethanolamine (action concentration is 1 M), and act at room temperature for 30 minutes. To stop the binding of antibodies and latex microspheres.

Finally, after washing the antibody-coupled microspheres with a washing solution containing a stabilizer and a buffer solution, the microspheres are resuspended with 400 microliters of washing solution to obtain the latex solution.

Example 2: Use the test kit to detect four respiratory viruses at the same time

In order to further test the detection effect of the test kit of Example 1, therefore, in the same respiratory tract specimen, the detection limit of the test kit for four respiratory viruses was determined and compared with four commercially available kits (one kit To compare the detection limit of a respiratory virus individually), the detection limit of the virus is expressed as the number of diseased viruses in 50% of the cultured cells (Tissue Culture Infection Dose 50; TCID ₅₀ ). The results are shown in Table 4.

Table 4. Comparison of the detection limit of the test kit in Example 1 and the commercially available kit Example 1 inspection kit Commercially available sets Adenovirus CerTest Adenovirus One Step card test 2X10 ³ TCID ₅₀ /ml 2X10 ³ TCID ₅₀ /ml Flu A Quidel ^® QuickVue Influenza A/B Test 2.64X10 ⁵ TCID ₅₀ /ml 2.64X10 ⁵ TCID ₅₀ /ml Flu B Quidel ^® QuickVue Influenza A/B Test 3.71X10 ⁵ TCID ₅₀ /ml 3.71X10 ⁵ TCID ₅₀ /ml RSV QuickVue RSV Test 1.12X10 ³ TCID ₅₀ /ml 1.12X10 ³ TCID ₅₀ /ml

The results show that the test kits provided by the embodiments of the present disclosure only need to test the specimen once, and the results that had to be tested separately by four commercially available kits in the past can be obtained, and the detection limit of each virus is the same as that of the market. The sale sets are equivalent.

The present disclosure achieves good applicability for lysing a variety of test substances by preparing a lysis solution of appropriate components; in addition, it improves the formulation of the solution contained in the test piece, especially the preparation method of the microspheres, and improves the performance of the microsphere-coupled antibody. Efficiency is achieved in the limited antibody holding capacity on the binding pad, which can detect more than four test objects at the same time.

The inspection kit of the present disclosure can detect a variety of objects to be tested, and the benefits achieved are as follows: 1. Reduce operation steps and time: If you use a commercially available kit, the collection and detection steps need to be repeated four times, and the sampling tube and test piece need to be marked separately. If the number of samples is large, the sampling tube and the test card will be upgraded The risk of error increases the waste of manpower and material resources. However, if the test reagent of the present disclosure is used, the tester only needs to perform the steps once, and the time of labeling and operation can be saved. 2. Reduce the probability of error: Due to the use of a variety of commercially available kits, it is necessary to repeat multiple steps to increase the probability of operational errors or sample contamination. However, the inspection kit of this disclosure only performs one set of steps to get the result, which can reduce Mistakes, improve accuracy.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be subject to the definition of the attached patent application scope.

1: Inspection kit 2: Lysis reagent group 210: Lysis solution 220: Collection tube 230: sampling rod 3: test piece 310: sample pad 320: Combination pad 330: fiber membrane 331: Strip 340: Absorbent pad 350: support plate

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows: Figure 1 illustrates a schematic diagram of a test kit in some embodiments of the present disclosure. Figure 2 illustrates a schematic diagram of a lysis reagent set in some embodiments of the present disclosure. Figure 3 illustrates a schematic diagram of a test strip in some embodiments of the present disclosure. Figure 4 illustrates a schematic diagram of the inspection results in some embodiments of the present disclosure. Figure 5 illustrates a flow chart of simultaneous detection of multiple analytes in some embodiments of the present disclosure.

1: Inspection kit

2: Lysis reagent group

3: test piece

Claims (22)

A test kit for simultaneously detecting multiple test objects, including a lysis solution and a test piece, wherein the test piece includes a sample pad, a bonding pad, a fiber membrane, and an absorbent pad sequentially overlapped with a support plate superior, The binding pad includes a plurality of antibody-coupled microspheres, and the antibody-coupled microspheres identify a variety of test objects. The test kit according to claim 1, wherein the preparation method of the antibody-coupled microspheres includes the following steps: Provide a microsphere; Provide a flushing fluid; Mixing the microspheres and the rinsing liquid to obtain a first mixed liquid; Mix the first mixed solution with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbon An activation reaction is performed between the diimide and the microspheres in the first mixed solution, and the activation reaction time is between 16 to 24 hours; Mixing the microspheres after the activation reaction with a coupling liquid to obtain a second mixed liquid; and A first antibody and the second mixed solution are mixed, so that the first antibody and the microspheres in the second mixed solution perform a coupling action at room temperature to obtain the antibody-coupled microspheres. The inspection kit described in claim 1, One of the antibody-coupled microspheres identifies one of the test objects, any one of the antibody-coupled microspheres has a first antibody on the surface, and any one of the antibody-coupled microspheres penetrates The first antibody recognizes the specific one of the test objects, Wherein when the first antibodies of one group of the antibody-coupled microspheres recognize the same object to be tested, the antibody-coupled microspheres have the same color, and when the antibodies of the different groups of the antibody-coupled microspheres When the first antibody recognizes different test objects, the antibody-coupled microspheres have different colors; The fibrous membrane includes a plurality of strips, any one of the strips includes a second antibody, and the second antibody recognizes the analyte, and the second antibody on different strips recognizes different analytes, The second antibody of each of the bands and one of the first antibodies respectively identify the same analyte. The test kit according to claim 1, wherein the test objects are respiratory viruses, including adenovirus, influenza A virus, influenza B virus, influenza C virus, respiratory fusion virus, rhinovirus, coronavirus, Or a combination. The inspection kit according to claim 1 or 3, wherein the lysis solution is used for pre-inspection processing, the weight percentage of the lysis solution is 100%, and the lysis solution includes: A salt, the weight percentage is between about 0.5% to 5%; A surfactant, the weight percentage is between about 0.5% to about 5%; A stabilizer, the weight percentage is between about 0.5% to about 5%; and A buffer solution. The test kit according to claim 5, wherein the salt contains sodium chloride, potassium chloride or a combination of the foregoing. The test kit according to claim 5, wherein the surfactant comprises sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series or a combination of the foregoing. The test kit according to claim 5, wherein the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing. The test kit according to claim 5, wherein the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propane sulfonic acid, N,N-bis (2-Hydroxyethyl)glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N -Morpholine) ethanesulfonic acid or a combination of the foregoing. The inspection kit according to claim 5, wherein the lysis solution further contains sodium azide. The inspection kit according to claim 1 or 3, wherein the sample pad contains a sample pad solution, the weight percentage of the sample pad solution is calculated as 100%, and the sample pad solution includes: A surfactant, the weight percentage is between about 0.5% to 5%; A stabilizer, the weight percentage is between about 0.05% to about 10%; A dispersant, the weight percentage is between about 0.5% to about 5%; and A buffer solution. The test kit according to claim 11, wherein the surfactant comprises sodium lauryl sulfate, polysorbate series, triton series, nonylphenol series or a combination of the foregoing. The test kit according to claim 11, wherein the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing. The test kit according to claim 11, wherein the dispersant comprises polyvinylpyrrolidone, polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropyl cellulose, or a combination of the foregoing. The test kit according to claim 11, wherein the buffer solution contains phosphoric acid, carbonic acid, citric acid, acetic acid, boric acid, tris, trimethylol methylamino propane sulfonic acid, N,N-bis (2-Hydroxyethyl)glycine, N-tris-(hydroxymethyl)methylaminoacetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt, 3-(N -Morpholine) ethanesulfonic acid or a combination of the foregoing. The inspection kit according to claim 11, wherein the sample pad solution further contains sodium azide. The test kit according to claim 1 or 3, wherein the bonding pad further includes a bonding pad solution, and the bonding pad solution includes a stabilizer, a dispersant, an emulsifier, and a buffer solution. The test kit according to claim 17, wherein the weight percentage of the bond pad solution is calculated as 100%, The weight percentage of the stabilizer is between about 0.05% to 2%, The weight percentage of the dispersant is between about 0.05% and 10%, and the weight percentage of the emulsifier is between about 0.1% and about 5%. The test kit according to claim 17, wherein the stabilizer comprises serum protein, casein, artificial polymer, or a combination of the foregoing. The test kit according to claim 17, wherein the dispersant comprises polyvinylpyrrolidone, polyethylene glycol, oleic acid, polyacrylic acid, hydroxypropyl cellulose or a combination of the foregoing. The inspection kit according to claim 17, wherein the emulsifier includes a non-ionic emulsifier, including polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, polysorbate 20, or a combination thereof. The test kit according to claim 3, wherein the total concentration of the first antibody is not higher than 600 μg/ml, and the total concentration of the second antibody is not higher than 7.2 mg/ml.

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