Antigen test paper and application thereof

Abstract

The invention provides antigen detection test paper and application thereof, and the antigen detection test paper comprises a sample pad; the combination pad is in lap joint with the sample pad; the combination pad comprises an antibody A1 which is combined with a marker and corresponds to human secretory immunoglobulin A and an antibody B1 which is combined with a marker and corresponds to an antigen to be detected; the reaction pad is in lap joint with the combination pad; the reaction pad comprises a detection area and a quality control area, and an antibody B2 corresponding to the antigen to be detected is fixedly arranged in the detection area; an antibody A2 corresponding to the human secretory immunoglobulin A is fixedly arranged in the quality control area; and the water absorption pad is lapped with the reaction pad. According to the application, the antigen detection test paper can judge whether the to-be-detected sample is valid or not by judging whether a signal corresponding to the marker appears in the quality control area or not, so that false negative of a detection result caused by an invalid sample is reduced, and the risk of missing detection is further reduced.

Description

Technical Field

[0001] The present application relates to the field of rapid antigen detection, and in particular to an antigen detection test strip and its application. Background Art

[0002] Rapid antigen testing is a test performed on a test strip by specific binding of antigens and antibodies. By adding known antibodies and markers, known antigens are fixed and detected. This method is convenient and fast, and generally 15 to 20 minutes are enough to get results. It is widely used in disease screening and diversion management of suspected people in communities, grassroots hospitals, airports, customs and homes.

[0003] Common rapid antigen detection methods currently on the market include colloidal gold method, fluorescent immunochromatography and latex method, and the main difference lies in the different markers. Regardless of the labeling method, the internal reference uses a secondary antibody that can react specifically with the marker on the test paper, and whether the antigen test paper is in a valid state is determined based on whether a specific reaction occurs. However, the problem is that when the collected sample is invalid, the above-mentioned antigen test paper will produce a false negative result, and there is a risk of missed detection.

[0004] Therefore, it is necessary to provide an antigen detection test paper that can reduce the risk of missed detection. Summary of the invention

[0005] The present application provides an antigen detection test strip and its application. The antigen detection test strip adds human secretory immunoglobulin A (SIgA) as an internal reference, which can be used to monitor invalidly collected samples, thereby reducing false negatives in test results and further reducing the risk of missed detection.

[0006] In a first aspect, the present application provides an antigen detection test paper, comprising: a sample pad for receiving a liquid sample to be tested; a binding pad overlapped with the sample pad, for receiving the sample to be tested that flows from the sample pad to the binding pad through chromatography; the binding pad comprises: an antibody A1 corresponding to human secretory immunoglobulin A bound to a marker and an antibody B1 corresponding to the antigen to be tested bound to a marker, wherein the antibody A1 and the antibody B1 are used to dissolve in the sample to be tested and bind to the corresponding antigen, so that the corresponding antigen in the sample to be tested binds to the marker; the binding pad overlapped with the binding pad The reaction pad is used to receive the sample to be tested that flows from the binding pad to it through chromatography; the reaction pad includes a detection area and a quality control area, the detection area is fixedly provided with an antibody B2 corresponding to the antigen to be tested, and is used to fix the antigen to be tested that is combined with a marker to the detection area; the quality control area is fixedly provided with an antibody A2 corresponding to human secretory immunoglobulin A, and is used to fix human secretory immunoglobulin A that is combined with a marker to the quality control area; the absorbent pad overlapped with the reaction pad is used to make the sample to be tested flow along the sample pad to the absorbent pad under chromatography, and pass through the binding pad and the reaction pad in sequence.

[0007] According to the present application, the conjugate pad of the antigen test paper contains antibody A1 corresponding to human secretory immunoglobulin A combined with a marker. Antibody A1 can specifically bind to SIgA in the sample to be tested, and then specifically bind to antibody A2 fixed in the quality control area on the reaction pad. Therefore, whether the sample to be tested is valid can be judged by whether a signal corresponding to the marker appears in the quality control area, thereby reducing false negative test results caused by invalid samples and further reducing the risk of missed detection.

[0008] In some embodiments, the concentration ratio of the antibody A1 to the antibody B1 is 1:0.7-4; preferably, the concentration ratio is 1:0.8-1.2.

[0009] In some embodiments, the antigen to be tested is a disease antigen corresponding to one of influenza A virus, influenza B virus, novel coronavirus, adenovirus and parainfluenza virus; preferably, the antigen to be tested is a disease antigen corresponding to influenza A virus or novel coronavirus COVID-9.

[0010] In some embodiments, the marker includes at least one of colloidal gold, fluorescent microspheres, latex particles and nanosilver; preferably, the marker is colloidal gold with a particle size of 20 to 40 nm.

[0011] In some embodiments, the sample pad and the conjugate pad are made of glass fiber membrane.

[0012] In some embodiments, the material of the reaction pad is nitrocellulose membrane and / or cellulose acetate membrane.

[0013] In some embodiments, a bottom plate is further included for supporting the sample pad, conjugate pad, reaction pad and absorbent pad; optionally, the bottom plate is one of a polyvinyl chloride plate, a polyethylene plate, a polypropylene plate and a polyurethane plate.

[0014] In a second aspect, the present application provides a kit for antigen detection, comprising: an antigen detection test paper according to any embodiment of the first aspect; a shell for accommodating the antigen detection test paper, the shell comprising: a sample addition hole arranged corresponding to the position of the sample pad; and an observation window arranged corresponding to the position of the detection area and the quality control area.

[0015] In some embodiments, the kit includes: a sample processing solution, wherein the sample processing solution includes a surfactant with a mass percentage of 0.1% to 0.5%; optionally, the surfactant includes at least one of Tween 20 and polyethylene glycol octylphenyl ether.

[0016] In some embodiments, the kit includes: an instruction manual, which includes a method for using the kit, including the following steps: mixing a nasopharyngeal swab with a sample processing solution to obtain a sample to be tested; adding the sample to be tested to a sample pad through a sample addition hole, and waiting for a certain period of time; judging whether the sample to be tested contains the antigen to be tested based on whether a signal corresponding to the marker appears in the detection area and the quality control area in the observation window, and the judgment method is: if no signal appears in the quality control area, it is impossible to judge whether the sample to be tested contains the antigen to be tested; if a signal appears in the quality control area and no signal appears in the detection area, it is judged that the sample to be tested does not contain the antigen to be tested; if a signal appears in the quality control area and a signal appears in the detection area, it is judged that the sample to be tested contains the antigen to be tested.

[0017] The antigen test strip provided by the present application uses SIgA as an internal reference, and simultaneously detects disease antigens and internal reference SIgA through the principles of immune capture and double antibody sandwich method. This antigen test strip realizes sample quality control of nasopharyngeal swab-derived samples through internal references, and can realize full-process monitoring of sample sampling, extraction, and detection. It can serve as a powerful supplement to quality control products, reduce the generation of false negative test results, and reduce the risk of missed detection.

[0018] The antigen detection test strip provided in this application can be used for antigen detection of samples derived from nasopharyngeal swabs, and can be applied to antigen detection of various diseases, such as influenza, new coronavirus COVID-19, etc.

[0019] The antigen detection test paper provided in the present application has the advantages of strong specificity, simple use, low price, intuitive and rapid reading results, etc. It does not require special equipment and professional personnel to operate, and provides sample quality control supplements for antigen detection of various diseases, widely reducing the occurrence of false negatives in antigen detection, and has important application backgrounds in clinical and home testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0021] Figure 1 Schematic diagram of an antigen detection test strip according to an embodiment of the present application.

[0022] Figure 2 For packaging such as Figure 1 Schematic diagram of the kit for the antigen detection test strip shown.

[0023] Figure 3 The color development results of the C line when the antigen detection test paper in Example 2 detects different samples.

[0024] Figure 4 The middle left picture shows the color development results of the C line when the commercially available new coronavirus antigen test strips are used to detect different samples.

[0025] Figure 4 The middle right picture shows the color development results of the C line when the antigen detection test paper in Example 3 detects different samples.

[0026] Figure 5 This is the color development result of the C line when the antigen detection test paper in Example 2 and Example 3 detects the standard sampling.

[0027] Figure 6 This is the color development result of the C line when the antigen test strips in Example 2 and Example 4 detect samples with a small sampling amount.

[0028] Explanation of the accompanying reference numerals: 1. card shell, 2. sample addition hole, 3. observation window, 4. sample pad, 5. binding pad, 6. reaction pad, 7. water absorption pad, 8. detection line, 9. quality control line, 10. bottom plate.

[0029] The above drawings have shown clear embodiments of the present application, which will be described in more detail later. These drawings and text descriptions are not intended to limit the scope of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0030] The various embodiments or implementation schemes in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments.

[0031] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the implementation or examples are included in at least one implementation or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same implementation or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more implementations or examples in a suitable manner.

[0032] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0033] As described in the background technology above, the current antigen detection test strips are prone to false negatives when used for antigen detection. Analysis shows that the main reason for the false negatives is that the current antigen detection test strips cannot effectively screen out invalid samples, resulting in missed detections.

[0034] In response to the above problems, antigen detection can be performed simultaneously with the target disease antigen using the characteristic components in the test sample, so as to determine whether the test sample is effective based on whether specific antigen-antibody binding occurs with the characteristic components. Due to the convenience of antigen detection, samples from nasopharyngeal swabs are generally tested, and there are many characteristic components in nasopharyngeal swabs. In order to obtain antigen detection test strips that are common to nasal swabs and throat swabs, it is necessary to select characteristic components common to nasal swabs and throat swabs. On the other hand, since the antigen test strip detection of disease antigens adopts the double antibody sandwich method, it is necessary to select characteristic components with a larger molecular weight. The characteristic protein components in nasopharyngeal swabs are more suitable as internal references for antigen detection.

[0035] Since the characteristic protein component is used as an internal reference, it is equivalent to detecting two antigens in the sample at the same time. Although the antigen-antibody reaction has strong specificity, there will also be certain interference. In addition, the content of the characteristic protein component in the nasopharyngeal swab will also affect whether it can accurately judge whether the test sample is valid. Therefore, it is necessary to select a suitable characteristic protein component derived from the nasopharyngeal swab.

[0036] Based on this, the present application provides an antigen test strip, which uses SIgA as an internal reference, and can effectively screen out invalid samples, thereby reducing the false negatives of the test results and further reducing the risk of missed detection. The implementation methods of the present application are described in detail below.

[0037] In a first aspect, the present application provides an antigen detection test paper, comprising: a sample pad for receiving a liquid sample to be tested; a binding pad overlapped with the sample pad, for receiving the sample to be tested that flows from the sample pad to the binding pad through chromatography; the binding pad comprises: an antibody A1 corresponding to human secretory immunoglobulin A bound to a marker and an antibody B1 corresponding to the antigen to be tested bound to a marker, wherein the antibody A1 and the antibody B1 are used to be dissolved in the sample to be tested and bind to the corresponding antigen, so that the corresponding antigen in the sample to be tested binds to the marker; a reaction pad overlapped with the binding pad The pad is used to receive the sample to be tested that flows from the binding pad to it through chromatography; the reaction pad includes a detection area and a quality control area, the detection area is fixedly provided with an antibody B2 corresponding to the antigen to be tested, which is used to fix the antigen to be tested combined with a marker to the detection area; the quality control area is fixedly provided with an antibody A2 corresponding to human secretory immunoglobulin A, which is used to fix human secretory immunoglobulin A combined with a marker to the quality control area; the absorbent pad overlapped with the reaction pad is used to make the sample to be tested flow along the sample pad to the absorbent pad under chromatography, and pass through the binding pad and the reaction pad in turn.

[0038] According to the present application, the antigen detection test paper includes a sample pad, a conjugation pad, a reaction pad and an absorbent pad. When performing antigen detection, the sample pad is used to receive the liquid sample to be tested. Under the action of chromatography, the sample to be tested flows through the conjugation pad, the reaction pad and the absorbent pad in sequence, wherein the conjugation pad contains an antibody A1 conjugated with a marker and an antibody B2 conjugated with a marker. When the sample to be tested flows through the conjugation pad, the antibody A1 conjugated with a marker and the antibody B2 conjugated with a marker will both dissolve in the sample to be tested and bind to the corresponding antigen in the sample to be tested. SIgA can specifically bind to the antibody A1 conjugated with a marker, which is equivalent to containing SIgA conjugated with a marker in the sample to be tested. Then the sample to be tested flows through the reaction pad, and the quality control area in the reaction pad is fixedly provided with antibody A2, which can specifically bind to SIgA conjugated with a marker, thereby fixing the marker to the quality control area. Therefore, by monitoring whether the corresponding signal of the marker appears in the quality control area, it can be determined whether there is a suitable concentration of SIgA in the sample to be tested, and then whether the sample to be tested is valid. It should be noted that the strength of the corresponding signal of the marker in the quality control area is linearly correlated with the concentration of SIgA in the sample within a certain range, and the concentration of SIgA in the sample is related to whether the sampling is standard. Therefore, judging whether the sample to be tested is valid is not limited to whether the corresponding signal of the marker appears. The strength of the corresponding signal of the marker can also be used as a basis for judging whether the sample to be tested is valid. Those skilled in the art can determine the standard for judging whether the sample to be tested is valid based on the corresponding signal of the marker according to actual needs. The above-mentioned judging whether the sample is valid based on whether the corresponding signal of the marker appears is only exemplary.

[0039] Correspondingly, the antigen to be tested can specifically bind to the antibody B1 and antibody B2 combined with the marker. By monitoring whether the corresponding signal of the marker appears in the detection area, it can be determined whether the antigen to be tested exists in the sample to be tested. Combined with the above results of whether the sample to be tested is valid, if the sample is invalid, it is necessary to resample for antigen detection; if the sample is valid and the antigen to be tested exists, the sample to be tested is positive; if the sample is valid and the antigen to be tested does not exist, the sample to be tested is negative. Therefore, judging whether the sample is valid through the quality control area can effectively reduce the problem of false negatives caused by non-standard sampling, thereby reducing the risk of missed detection.

[0040] In addition, since SIgA is a characteristic protein component common in nasal swabs and throat swabs, the antigen test paper can be used to detect samples derived from nasopharyngeal swabs.

[0041] In some embodiments, the concentration ratio of antibody A1 to antibody B1 is 1:0.7-4; preferably, the concentration ratio is 1:0.8-1.2.

[0042] In the above embodiment, the concentration ratio of antibody A1 and antibody B1 is further limited. This is because the concentration of the antibody bound to the marker in the conjugate pad is limited by the loading amount of the conjugate pad. Therefore, the concentration ratio of the two has a certain influence on the accuracy of antigen detection. If there is too much antibody A1, the detection limit of SIgA will be too low, making it difficult to distinguish samples with non-standard sampling and unable to obtain semi-quantitative results. If there is too little antibody A1, the detection limit of SIgA will be too high, the sampling requirements will be too high, the proportion of invalid results will increase, and the cost of antigen detection will increase. It is also worth mentioning that the content of antibody B1 against the antigen to be tested in the conjugate pad will also affect the detection limit of the antigen to be tested. Compared with other characteristic protein components in nasopharyngeal swabs (such as lysozyme, lactoferrin, etc.), SIgA has a higher content in nasopharyngeal swabs and a wider linear range of detection. Therefore, the concentration ratio of antibody A1 and antibody B1 can be controlled at 1:0.7-4. At this time, the antigen detection paper has a more suitable detection limit for SIgA and better sensitivity to the antigen to be tested, thereby having better accuracy and a lower risk of missed detection. For example, the concentration ratio of antibody A1 to antibody B1 can be 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, or any range thereof. More preferably, the concentration ratio can be 1:0.8 to 1.2.

[0043] In some embodiments, the antigen to be tested is a corresponding disease antigen of influenza A virus, influenza B virus, new coronavirus, adenovirus and parainfluenza virus.

[0044] In the above embodiment, several antigens to be tested are specifically listed. It is understood that the antigens to be tested include but are not limited to the above. Since the source of the test sample of the antigen test paper is a nasopharyngeal swab, it can be used to detect diseases in which the corresponding antigens can be detected in the nasopharyngeal swab. Those skilled in the art can choose according to actual needs, such as respiratory infections caused by viruses for disease antigens. Preferably, the antigen to be tested is the disease antigen corresponding to influenza A virus or the new coronavirus COVID-9, and the antigen test paper has a lower risk of missed detection.

[0045] In some embodiments, the marker includes at least one of colloidal gold, fluorescent microspheres, latex particles and nanosilver; preferably, the marker is colloidal gold with a particle size of 20 to 40 nm.

[0046] In the above embodiment, several markers are specifically listed. It is understandable that the antigens to be tested include but are not limited to the above. As long as the marker binding antibody has little effect on the antigen-antibody specific binding and can display the corresponding signal under certain conditions, it can be used as a marker, and those skilled in the art can choose according to actual needs. Preferably, the marker is colloidal gold with a particle size of 20 to 40 nm. Colloidal gold is simple to prepare, and its negative surface charge can be firmly combined with the electrostatic interaction between the positively charged groups of the protein and the hydrophobic effect of the protein, and has no obvious effect on the biological activity of the protein. At the same time, colloidal gold aggregates in the quality control area or the detection area to appear obvious red, and no further processing is required to determine whether a signal is displayed.

[0047] As an example, colloidal gold can be prepared by reducing gold chlorate with sodium citrate, which specifically includes the following steps: heating the sodium citrate solution to boiling, adding the gold chlorate solution, boiling for 15 minutes and then cooling to obtain colloidal gold with a particle size of 20 to 40 nm and a colloidal gold concentration of 0.02wt% to 0.04wt%.

[0048] In some embodiments, the material of the sample pad and the conjugate pad is a glass fiber membrane. The glass fiber membrane has a good water absorption effect and low non-specific adsorption, and can well load the antibody and the sample to be detected that are bound to the marker without being adsorbed. It is understandable that the material is not limited to the glass fiber membrane, and those skilled in the art can select it according to actual needs.

[0049] In some embodiments, the sample pad is prepared by the following method: soaking a sample pad raw material in a pad treatment buffer and drying to obtain a sample pad.

[0050] In some embodiments, the conjugate pad is prepared by the following method: soaking the conjugate pad raw material in a pad treatment buffer and drying to obtain a pretreated conjugate pad; spraying a protein preservation solution containing antibody A1 conjugated with a marker and antibody B1 conjugated with a marker onto the pretreated conjugate pad and drying to obtain a conjugate pad.

[0051] In some embodiments, the pad treatment buffer is a 0.01 mol / L PBS buffer at pH=8 containing 0.1 wt% to 0.5 wt% Tween-20 and 1 wt% to 5 wt% bovine serum albumin (BSA).

[0052] In some embodiments, the protein preservation solution is an aqueous solution containing 0.1 wt% to 0.5 wt% Tween-20, 1 wt% to 5 wt% bovine serum albumin (BSA), and 1 wt% to 5 wt% sucrose.

[0053] In some embodiments, the material of the reaction pad is nitrocellulose membrane and / or cellulose acetate membrane. Nitrocellulose membrane and cellulose acetate membrane can fix antibody A2 and antibody B2 on the reaction pad through electrostatic interaction and hydrophobic interaction, so that the marker can show more obvious signals in the quality control area and detection area of ​​the reaction pad.

[0054] In some embodiments, the quality control area and the detection area on the reaction pad are separated by 0.3 to 1.5 cm along the sample chromatography direction to prevent interference between the two.

[0055] In some embodiments, the quality control area is a quality control line (C line) perpendicular to the sample chromatography direction, and the detection area is a detection line (T line) perpendicular to the sample chromatography direction, and the detection line is arranged near one side of the sample pad.

[0056] In some embodiments, the reaction pad is prepared by the following method: spraying an antibody A2 aqueous solution with a concentration of 1 to 3 mg / mL at one place of the reaction pad raw material, and drying to obtain a quality control area; spraying an antibody B2 aqueous solution with a concentration of 1 to 3 mg / mL at another place of the reaction pad raw material, and drying to obtain a detection area to obtain a reaction pad.

[0057] In some embodiments, the material of the absorbent pad is nitrocellulose membrane and / or cellulose acetate membrane, which has good water absorption and good binding ability with proteins, and can collect and fix the samples after the test to reduce contamination.

[0058] In some embodiments, a bottom plate is also included for carrying a sample pad, a conjugation pad, a reaction pad and a water-absorbing pad; optionally, the bottom plate is one of a polyvinyl chloride plate, a polyethylene plate, a polypropylene plate and a polyurethane plate. The bottom plate is used to carry the sample pad, the conjugation pad, the reaction pad and the water-absorbing pad. The bottom plate is a thin sheet that does not absorb water and has a supporting function, so that the sample to be tested can be smoothly chromatographed from the sample pad to the water-absorbing pad. Therefore, the bottom plate can be any of the above materials, and it can be understood that the bottom plate is not limited to the above materials.

[0059] As an example, Figure 1 A schematic diagram of an antigen detection test paper according to an embodiment of the present application is shown, in which a sample pad 4, a conjugation pad 5, a reaction pad 6 and a water absorbent pad 7 are overlapped in sequence on a section of a bottom plate 10, and the overlapping length between adjacent pads is 1 to 2 mm. The sample pad 4 is overlapped on the conjugation pad 5, and the conjugation pad 5 and the water absorbent pad 7 are overlapped at both ends of the reaction pad 6. A detection line 8 is provided on the side of the reaction pad 6 close to the conjugation pad 5, and a quality control line 9 is provided on the side away from the conjugation pad 5.

[0060] In a second aspect, the present application provides a kit for antigen detection, comprising: an antigen detection test paper according to any embodiment of the first aspect; a shell for accommodating the antigen detection test paper, the shell comprising: a sample addition hole arranged corresponding to the position of the sample pad; and an observation window arranged corresponding to the position of the detection area and the quality control area.

[0061] According to the present application, since the kit includes the antigen detection test paper of any embodiment of the first aspect, it has the beneficial effects of the first aspect. In addition, the antigen detection test paper is packaged to reduce the risk of contamination, which is convenient for antigen detection.

[0062] The shapes of the sample adding hole and the observation window are not limited and can be selected according to actual needs. As an example, Figure 2 The package is shown as Figure 1 The schematic diagram of the reagent kit of the antigen detection test paper is shown, and the shell 1 includes a sample addition hole 2 and an observation window 3. The sample addition hole 2 is arranged corresponding to the position of the sample pad, and the observation window 3 is arranged corresponding to the position of the detection line 8 and the quality control line 9.

[0063] In some embodiments, the kit includes: a sample processing solution, the sample processing solution includes a surfactant with a mass percentage of 0.1% to 0.5%; optionally, the surfactant includes at least one of Tween 20 and polyethylene glycol octylphenyl ether.

[0064] In the above embodiment, adding a certain surfactant to the sample processing liquid can effectively reduce the occurrence of nonspecific binding, thereby improving the accuracy of detection. In addition, the surfactant can be Tween 20 or polyethylene glycol octylphenyl ether, both of which are mild non-ionic surfactants, have little effect on the structure of proteins, and can improve the specificity of antigen-antibody binding.

[0065] In some embodiments, the sample treatment solution is a 0.01 mol / L PBS buffer with a pH of 8 and containing 0.1 wt% to 0.5 wt% Tween 20.

[0066] In some embodiments, the kit includes: an instruction manual, which includes a method for using the kit, including the following steps: mixing a nasopharyngeal swab with a sample processing solution to obtain a test sample; adding the test sample to the sample pad through the sample addition hole, and waiting for a certain period of time; judging whether the test sample contains the test antigen based on whether a signal corresponding to the marker appears in the detection area and the quality control area in the observation window, and the judgment method is: if no signal appears in the quality control area, it is impossible to judge whether the test sample contains the test antigen; if a signal appears in the quality control area and no signal appears in the detection area, it is judged that the test sample does not contain the test antigen; if a signal appears in the quality control area and a signal appears in the detection area, it is judged that the test sample contains the test antigen.

[0067] In the above embodiment, the instructions describe the method for using the kit, which is easy to operate and can screen out invalid samples, reduce the problem of false negatives, and thus reduce the risk of missed detection.

[0068] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0069] It should be noted that the antibody codes in the following embodiments are only used to distinguish antibodies corresponding to different antigens, and have no corresponding relationship with the antibody codes in the above specific embodiments.

[0070] The sample pad, conjugate pad, reaction pad, water absorbent pad and cartridge used in the following examples were purchased from Shanghai Jie Ning Biotechnology Co., Ltd. The reaction pad was an NC membrane of model Sartorius CN 140.

[0071] The gold-labeled protein preservation solution in the following examples is prepared as follows: Tween-20, BSA and sucrose are dissolved in triple-distilled water so that the mass percentage of Tween-20 is 0.25%, the mass percentage of BSA is 1%, and the mass percentage of sucrose is 5% to obtain the gold-labeled protein preservation solution.

[0072] The pad treatment buffer in the following examples was prepared as follows: Tween-20 and BSA were dissolved in 0.01 mol / L PBS buffer at pH 8.0 to make the mass percentage of Tween-20 0.25% and the mass percentage of BSA 1%, to obtain a membrane treatment buffer.

[0073] The sample treatment solution in the above-mentioned embodiment is prepared as follows: Tween-20 is dissolved in 0.01 mol / L PBS buffer solution with a pH of 8.0 so that the mass percentage of Tween-20 is 0.25%, thereby obtaining the sample treatment solution.

[0074] Example 1

[0075] Preparation of antigen test strips for detecting influenza A virus:

[0076] (I) Preparation of colloidal gold

[0077] Preparation of colloidal gold by sodium citrate reduction chloroauric acid method: Soak the glassware used overnight in advance and rinse with deionized water. Add triple distilled water to the beaker, then heat the sodium citrate solution to boiling, then add the chloroauric acid solution, boil for 15 minutes, cool, and store at 4°C. The particle size of the colloidal gold particles is 20-40nm, and the concentration of colloidal gold is 0.02%.

[0078] (II) Preparation of colloidal gold labeled SIgA antibody A1 and influenza A virus antibody B1

[0079] Take 10mL of the above colloidal gold solution and place it in a test tube. Add 15-40μL 0.01mol / L K2CO3 to adjust the pH of the solution. Stir well and add antibody A1 or B1. Stir for 30 minutes and then add 20μL 10% BSA solution. Stir for another 30 minutes and then centrifuge at 12000rpm for 30 minutes. After centrifugation, discard the supernatant and add 1mL of the above gold-labeled protein preservation solution. The synthesized gold-labeled antibody A1 and gold-labeled antibody B1 are stored at 4°C.

[0080] (III) Coating of gold-labeled antibody A1 and gold-labeled antibody B1 at the binding pad

[0081] The conjugate pad was soaked in the pad treatment buffer for half an hour at a soaking temperature of 37°C. The conjugate pad was dried under the same temperature for 4 hours to obtain a pretreated conjugate pad. The gold-labeled antibody A1 and the gold-labeled antibody B1 synthesized above were mixed in a ratio of 1:1 to obtain a gold-labeled antibody mixture, and the gold-labeled antibody mixture was sprayed onto the pretreated conjugate pad using the XYZ3050 spray film system of BioDot, and dried under the same temperature to obtain a conjugate pad coated with the gold-labeled antibody A1 and the gold-labeled antibody B1.

[0082] (IV) Coating of T-line and C-line antibodies at the reaction pad

[0083] Antibody B2 corresponding to influenza A virus antigen and antibody A2 corresponding to human SIgA antibody were used to prepare the T-line and C-line antibody coatings at the reaction pad, respectively. Antibody B2 and antibody A2 were prepared into 1 mg / mL solutions with triple distilled water, and the XYZ3050 spray film system of BioDot was used to spray antibody B2 to the T-line position of the reaction pad (nitrocellulose membrane), and antibody A2 to the C-line position of the reaction pad. Drying was performed for 4 hours under the same temperature conditions as above to obtain a reaction pad with a test line and a quality control line coating.

[0084] (V) Overall assembly of test strips

[0085] Soak the sample pad in the pad treatment buffer for half an hour at 37°C. Dry it for 4 hours at the same temperature to obtain the treated sample pad. In a 100,000-class clean room with a relative humidity of less than 10%, add the treated sample pad, binding pad and reaction pad, add a water-absorbing pad and a backing (polyvinyl chloride rubber sheet) according to the schematic diagram. Figure 1 Assemble as shown: stick the sample pad, conjugate pad, reaction pad and absorbent pad on one end of the backing in sequence, with the former and the latter overlapping by 1-2mm, and the covering relationship is that the sample pad covers the conjugate pad, and the conjugate pad and absorbent pad cover both ends of the reaction pad. Then use BioDot's CM4000 cutting system to cut the whole material into test strips with a width of 3mm, put them into the card shell and seal them with an aluminum foil bag containing desiccant for use.

[0086] Evaluation of the above antigen test strips for detecting influenza A virus:

[0087] Detection steps: Use a precise pipette to take 60 μL of the sample to be tested and slowly add it vertically to the sample pad of the antigen test paper. After 15 minutes, observe the appearance of red bands on the C line and T line with the naked eye.

[0088] (I) C-line detection sensitivity

[0089] Human SIgA and normal human throat swabs were used as test samples to determine the sensitivity of the C line in the antigen detection test paper of Example 1.

[0090] Human SIgA was mixed with the above sample treatment solution to prepare samples of a series of concentrations, and tested using the antigen test paper of Example 1. The results showed that the sensitivity of the antigen test paper C line to human SIgA was 12-120 ng / mL.

[0091] The normal human throat swab was mixed with the above sample treatment solution and diluted to a series of concentrations, and tested using the antigen test paper of Example 1. The results showed that the detection limit of the antigen test paper C line for throat swabs was 1:100.

[0092] (II) T-line detection sensitivity

[0093] The sensitivity of the T line in the antigen detection test strip of Example 1 was determined using the recombinant NP protein of influenza A virus as a test sample.

[0094] The recombinant NP protein of influenza A virus was detected using the antigen test paper of Example 1. The results showed that the sensitivity of the antigen test paper T line to the recombinant NP protein of influenza A virus was 80-800 ng / mL, and the detection of human SIgA by the C line did not interfere with the detection of influenza A virus antigen.

[0095] (III) Comparison of normal and abnormal sampling results

[0096] The antigen detection test paper of Example 1 is used to detect standard sampling, irregular sampling of sampling position, less sampling of sampling amount, empty sampling and insufficient mixing of samples. Wherein, the standard pharyngeal swab sampling adopted is: the pharyngeal swab is extended to the throat over the tongue, and the left and right tonsils are wiped three times respectively, and then the pharyngeal wall in the middle is wiped up and down three times and then taken out, and the pharyngeal swab is immersed in the sample treatment solution for more than 30s. The sampling position adopted is irregular sampling, and the pharyngeal swab stays on the tongue without extending to the throat, and is wiped three times on the tongue, and then the left and right upper jaws at the parallel position are wiped three times and then taken out, and the pharyngeal swab is immersed in the sample treatment solution for more than 30s. The sampling amount adopted is less sampling: the pharyngeal swab is extended to the throat over the tongue, and the left and right tonsils are wiped once respectively, and then the pharyngeal wall in the middle is wiped up and down once and then taken out (normal sampling concentration 1 / 3), and the pharyngeal swab is immersed in the sample treatment solution for more than 30s. The empty sampling method is as follows: insert the pharyngeal swab into the oral cavity, scratch three times in the oral cavity and throat wall before taking it out, and immerse the pharyngeal swab in the sample processing solution for more than 30 seconds. The incompletely mixed sample is as follows: extend the pharyngeal swab over the tongue to the throat, wipe the left and right tonsils three times each, and then wipe the middle posterior pharyngeal wall up and down three times before taking it out, and immerse the pharyngeal swab in the sample processing solution for 15 seconds. The test results are as follows Figure 3, from left to right are the test results of standard sampling, sampling at irregular sampling positions, sampling with insufficient sampling volume, empty sampling and insufficient mixing. In addition to the standard sampling, the test results of abnormal sampling samples show that the color of the quality control line C becomes lighter, which proves that the present invention can monitor the sample quality.

[0097] Example 2

[0098] Antigen test strips for detecting the novel coronavirus (COVID-19)

[0099] Similar to Example 1, the only difference is that the influenza A virus antibodies B1 and B2 are replaced by the new coronavirus antibodies C1 and C2.

[0100] Evaluation of Antigen Test Strips for Detection of Novel Coronavirus:

[0101] Detection steps: Use a precise pipette to take 60 μL of the sample to be tested and slowly add it vertically to the sample pad of the antigen detection test paper of Example 2. After 15 minutes, observe the appearance of red bands on the C line and T line with the naked eye.

[0102] (I) C-line detection sensitivity

[0103] Human SIgA and normal human throat swabs were used as test samples to determine the sensitivity of the C line in the antigen detection test paper of Example 2.

[0104] Human SIgA was mixed with the above sample treatment solution to prepare samples of a series of concentrations, and tested using the antigen test paper of Example 2. The results showed that the sensitivity of the antigen test paper C line to human SIgA was 12-120 ng / mL.

[0105] The normal human throat swab was mixed with the above sample treatment solution and gradiently diluted into a series of concentration samples, and tested using the antigen detection test paper of Example 2. The results showed that the detection limit of the antigen detection test paper for throat swabs was 1:100.

[0106] (II) T-line detection sensitivity

[0107] The recombinant NP protein of the new coronavirus was used as the test sample to determine the sensitivity of the T line in the antigen detection test paper of Example 2.

[0108] The recombinant NP protein of the new coronavirus was detected using the antigen test paper of Example 2. The results showed that the sensitivity of the T line of the antigen test paper to the recombinant NP protein of the new coronavirus was 15-150 ng / mL. And the detection of human SIgA by the C line did not interfere with the detection of the new coronavirus antigen.

[0109] (III) Comparison of normal and abnormal sampling results

[0110] The antigen test strips of Example 2 were used to test standard samples, samples with irregular sampling locations, samples with less sampling volume, empty samples, and samples with insufficient mixing. The sampling methods for each sample were the same as those of Example 1. The test results are shown in FIG. Figure 4 , from left to right are the test results of standard sampling, sampling at irregular sampling positions, sampling with insufficient sampling volume, empty sampling and insufficient mixing. In addition to the test results of standard sampling, the color of the quality control line C becomes lighter, which proves that the present invention can monitor the sample quality.

[0111] (IV) Comparison of test results with commercially available COVID-19 monitoring kits

[0112] Normal and abnormal throat swabs collected from normal subjects were used as test samples to perform internal reference line differentiation between Example 2 of the present invention and a commercially available COVID-19 monitoring kit.

[0113] like Figure 4 It can be seen that the commercially available COVID-19 monitoring kits are insensitive to abnormal sampling swabs, while Example 2 can clearly distinguish between abnormal samples and normal samples, thus playing a role in sample quality control.

[0114] Example 3

[0115] Antigen test strips for detecting the novel coronavirus (COVID-19)

[0116] Similar to Example 2, the only difference is that the gold-labeled antibody A1 and the gold-labeled antibody B2 are mixed in a ratio of 1:4 to obtain a gold-labeled antibody mixture.

[0117] Evaluation of Antigen Test Strips for Detection of Novel Coronavirus:

[0118] Detection steps: Use a precise pipette to take 60 μL of the sample to be tested and slowly add it vertically to the sample pad of the antigen detection test paper of Example 3. After 15 minutes, observe the appearance of the red band of line C with the naked eye.

[0119] The antigen test strips of Example 2 and Example 3 were used to test the standard samples, and the standard sampling method was the same as that of Example 1. The test results are shown in Figure 5 , Figure 5 The left side is the test result of the test paper in Example 2, and the right side is the test result of Example 3. The color of the C line in the antigen test paper obtained in Example 3 is significantly lighter, indicating that its sampling requirements are too high, which may cause the antigen test paper to be more inefficient than Example 2.

[0120] Example 4

[0121] Antigen test strips for detecting the novel coronavirus (COVID-19)

[0122] Similar to Example 2, the only difference is that the gold-labeled antibody A1 and the gold-labeled antibody B2 are mixed in a ratio of 1:0.7 to obtain a gold-labeled antibody mixture.

[0123] Evaluation of Antigen Test Strips for Detection of Novel Coronavirus:

[0124] Detection steps: Use a precise pipette to take 60 μL of the sample to be tested and slowly add it vertically to the sample pad of the antigen detection test paper of Example 3. After 15 minutes, observe the appearance of the red band of line C with the naked eye.

[0125] The antigen test strips of Example 2 and Example 4 were used to test the samples with less sampling volume. The sampling method for the samples with less sampling volume was the same as that of Example 1. The test results are as follows: Figure 6 , Figure 6 The left side is the test result of the test paper in Example 4, and the right side is the test result of Example 2. The color of the C line in the antigen test paper obtained in Example 4 is obviously darker than the color of the C line in Example 2, indicating that it cannot effectively screen out materials with a small sampling amount, and the accuracy of the antigen test paper is not as good as the antigen test paper obtained in Example 2.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An antigen detection test paper, characterized in that include: A sample pad, used for receiving a liquid sample to be tested; The binding pad overlapped with the sample pad is used to receive the sample to be tested that flows from the sample pad to it through chromatography; the binding pad comprises: an antibody A1 corresponding to human secretory immunoglobulin A combined with a marker and an antibody B1 corresponding to the antigen to be tested combined with a marker, wherein the antibody A1 and the antibody B1 are used to be dissolved in the sample to be tested and combined with the corresponding antigen, so that the corresponding antigen in the sample to be tested is combined with the marker; The reaction pad overlapped with the binding pad is used to receive the sample to be tested that flows from the binding pad to it through chromatography; the reaction pad includes a detection area and a quality control area, the detection area is fixedly provided with an antibody B2 corresponding to the antigen to be tested, and is used to fix the antigen to be tested combined with a marker to the detection area; the quality control area is fixedly provided with an antibody A2 corresponding to human secretory immunoglobulin A, and is used to fix human secretory immunoglobulin A combined with a marker to the quality control area; The water-absorbing pad overlapped with the reaction pad is used to make the sample to be tested flow from the sample pad to the water-absorbing pad under the action of chromatography, and pass through the binding pad and the reaction pad in sequence.

2. The antigen test paper according to claim 1, characterized in that: The concentration ratio of the antibody A1 to the antibody B1 is 1:0.7-4; preferably, the concentration ratio is 1:0.8-1.

2.

3. The antigen detection test paper according to claim 1 or 2, characterized in that: The antigen to be tested is a disease antigen corresponding to influenza A virus, influenza B virus, new coronavirus, adenovirus and parainfluenza virus; preferably, the antigen to be tested is a disease antigen corresponding to influenza A virus or new coronavirus COVID-9.

4. The antigen test paper according to claim 1, characterized in that: The marker includes at least one of colloidal gold, fluorescent microspheres, latex particles and nanosilver; preferably, the marker is colloidal gold with a particle size of 20 to 40 nm.

5. The antigen detection test paper according to claim 1, characterized in that: The sample pad and the conjugate pad are made of glass fiber membrane.

6. The antigen detection test paper according to claim 1, characterized in that: The material of the reaction pad is nitrocellulose membrane and / or cellulose acetate membrane.

7. The antigen detection test paper according to claim 1, characterized in that: It also includes a bottom plate for carrying the sample pad, the conjugate pad, the reaction pad and the absorbent pad; optionally, the bottom plate is one of a polyvinyl chloride plate, a polyethylene plate, a polypropylene plate and a polyurethane plate.

8. A kit for antigen detection, characterized in that: include: The antigen detection test paper according to any one of claims 1 to 7; A shell for accommodating the antigen detection test paper, the shell comprising: a sample addition hole arranged corresponding to the position of the sample pad; An observation window is arranged corresponding to the position of the detection area and the quality control area.

9. The kit according to claim 8, characterized in that include: The sample processing liquid comprises a surfactant with a mass percentage of 0.1% to 0.5%; optionally, the surfactant comprises at least one of Tween 20 and polyethylene glycol octylphenyl ether.

10. The kit according to claim 8, characterized in that include: Instructions, the instructions including the method for using the kit, comprising the following steps: Mixing the nasopharyngeal swab with the sample processing solution to obtain a sample to be tested; Add the sample to be tested to the sample pad through the sample adding hole and wait for a certain period of time; According to whether the signal corresponding to the marker appears in the detection area and the quality control area in the observation window, it is judged whether the sample to be tested contains the antigen to be tested, and the judgment method is: If no signal appears in the quality control area, it is impossible to determine whether the test sample contains the antigen to be tested. If a signal appears in the quality control area and no signal appears in the detection area, it is determined that the sample to be tested does not contain the antigen to be tested. If a signal appears in the quality control area and a signal appears in the detection area, it is determined that the sample to be tested contains the antigen to be tested.

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