A test strip for combined detection of cTnT and cTnl

Abstract

The application relates to the technical field of myocardial injury index detection, and specifically discloses a test strip for combined detection of cTnT and cTnI. A nitrocellulose membrane of the test strip is provided with a detection line and a quality control line; a preparation method of the detection line comprises the step of diluting detection antibodies with a detection line diluent and then drawing lines on the nitrocellulose membrane; and components of the detection line diluent comprise: 10-50 mM PB buffer, 5-10 wt% sodium chloride, 0.1-1 wt% sucrose, 0.1-1 wt% trehalose, 0.01%-0.1 wt% BSA, 0.05-0.15 wt% preservative, 0.05-0.15 wt% epigallocatechin gallate and 0.05-0.15 wt% 1,4-butanediamine. The test strip of the application can significantly improve tailing of the detection line during detection, and the detection is more efficient and accurate.

Description

Technical Field

[0001] This application relates to the technical field of myocardial injury index detection, and more specifically, it relates to a test strip for the combined detection of cTnT and cTnI. Background Technology

[0002] Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are both indicators for myocardial detection. Studies have shown that there is no significant difference between cTnI and cTnT in the diagnosis of acute myocardial infarction (AMI), and both can identify myocardial damage that CK-MB cannot detect.

[0003] Relevant detection methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and radioimmunoassay. Among them, chemiluminescent immunoassay is widely used for the detection of cTnT and cTnI due to its advantages such as high sensitivity, anti-interference ability, and simple operation.

[0004] Currently, both cTnT and cTnI have their advantages in clinical testing: compared to cTnT, cTnI testing has higher specificity, but it still has drawbacks such as the inability to standardize test results and gender differences in results; compared to cTnI, cTnT testing can achieve standardized test results and is gender-neutral, and has the advantages of a longer detection window and better stability, but it still suffers from low specificity. In clinical testing, for example, cTnT increases more frequently than cTnI in patients with unstable angina; and in predicting 30-day mortality after AMI, cTnT testing has a higher predictive accuracy than cTnI.

[0005] Therefore, detecting only one indicator is insufficient. Current detection methods for these two indicators focus on detecting one indicator alone, resulting in low detection efficiency. Therefore, it is necessary to provide a method for jointly detecting both indicators.

[0006] Currently, the widely used immunochromatographic assay typically involves preparing test strips. These strips include a sample conjugation pad, a glass fiber pad, a nitrocellulose membrane (containing a test line and a control line), and absorbent paper. The detection principle utilizes the specific binding of antigens and antibodies to detect the target analyte. Specifically, the sample conjugation pad contains a solid-phase coated and fluorescently labeled antibody, for example, using nanospheres as a solid-phase carrier to load fluorescently labeled antibodies. The test line contains the detection antibody. After the antigen binds to the antibodies at both locations, it emits light under certain conditions. Quantitative detection is achieved through the linear relationship between the luminescence intensity and the antigen concentration.

[0007] In the above methods, the detection line contains antibodies. When the binding between the antibodies in the detection line and the nitrocellulose membrane is unstable, the detection line becomes dispersed and severely tailed, resulting in unacceptable test results and requiring retesting. Summary of the Invention

[0008] To improve the problem of detection line trailing, this application provides a test strip for the combined detection of cTnT and cTnI.

[0009] The test strip for the combined detection of cTnT and cTnI provided in this application adopts the following technical solution:

[0010] A test strip for the combined detection of cTnT and cTnI, the test strip comprising a housing, the housing comprising a sample binding region and at least two sample detection regions connected to the sample binding region;

[0011] The sample binding area of ​​the cartridge is provided with a sample binding pad, and each sample detection area is provided with a glass fiber pad, a nitrocellulose membrane and absorbent paper that are sequentially overlapped. The glass fiber pad and the sample binding pad are connected, and the nitrocellulose membrane is provided with a detection line and a quality control line.

[0012] The method for preparing the detection line includes the step of diluting the detection antibody with a detection line diluent and then streaking it onto the nitrocellulose membrane. The components of the detection line diluent include:

[0013] 10-50 mM PB buffer, 5-10 wt% sodium chloride, 0.1-1 wt% sucrose, 0.1-1 wt% trehalose, 0.01%-0.1 wt% BSA, 0.05-0.15 wt% preservative, 0.05-0.15 wt% epigallocatechin gallate, 0.05-0.15 wt% 1,4-butanediamine.

[0014] Nitrocellulose membranes possess a high concentration of hydroxyl and nitro groups. Epigallocatechin gallate is a polyhydroxy water-soluble substance, while 1,4-butanediamine is a polyamino substance. By loading epigallocatechin gallate and 1,4-butanediamine onto the nitrocellulose membrane, the amount of hydroxyl and amino groups on the membrane is significantly altered, resulting in a richer functional group composition. The antigens being detected are all peptides, containing a high number of amino and carboxyl groups. Using the above technical approach, the hydroxyl and amino groups on the nitrocellulose membrane can interact with the amino and carboxyl groups on the antigen, further enhancing the membrane's ability to capture peptides and proteins such as antigens and antibodies, as well as the binding stability between antigens, antibodies, and the nitrocellulose membrane. This ensures that during detection, once the corresponding antigen is present, the detection line is clear and distinct without tailing, thus achieving efficient and effective antigen detection.

[0015] In this scheme, the polyhydroxy compounds (such as epigallocatechin gallate) and polyamino compounds (1,4-butanediamine) need to be water-soluble. After these two types of substances are dissolved in an aqueous solution, they are then soaked in a nitrocellulose membrane so that they can be stably loaded onto the nitrocellulose membrane, thereby exerting their function of stably and firmly capturing the target antigen or antibody.

[0016] Optionally, the detection antibody is diluted with the detection line diluent to achieve a concentration of 0.5-1.5 mg / mL.

[0017] Optionally, the method for preparing the control line includes the step of diluting the control material with a control line diluent and then streaking it onto the nitrocellulose membrane. The components of the control line diluent include: 10-50 mM PB buffer, 5-10 wt% sodium chloride, 0.1-1 wt% sucrose, 0.1-1 wt% trehalose, 0.01%-0.1 wt% BSA, 0.05-0.15 wt% preservative, 0.05-0.15 wt% epigallocatechin gallate, and 0.05-0.15 wt% 1,4-butanediamine.

[0018] By adopting the above technical solution, the distribution, type and number of groups on the nitrocellulose membrane are significantly changed by loading gallic catechin gallate and 1,4-butanediamine, thereby significantly improving its ability to capture antigens and antibodies, and thus significantly improving the tailing of the quality control line.

[0019] Optionally, the quality control material is diluted with the quality control line diluent to achieve a concentration of 0.5-1.5 mg / mL.

[0020] Optionally, the quality control material can be goat anti-chicken IgY or DNP antigen.

[0021] Optionally, the preparation method of the sample binding pad includes the steps of soaking a blank glass fiber pad in a sample diluent and then drying it. The sample diluent comprises: 10-200 mM PB buffer, 10-200 mM Tris buffer, 0.01-1 wt% surfactant, 0.05-0.15 wt% preservative, 0.2-0.6 mg / mL blocking agent, 0.5-2 wt% sodium casein, 0.5-2 wt% polyvinyl alcohol, 0.05-0.5 mg / mL fluorescent nanosphere-labeled cTnI monoclonal antibody, 0.05-0.5 mg / mL fluorescent nanosphere-labeled cTnT monoclonal antibody, and 0.05-0.5 mg / mL fluorescent nanosphere-labeled quality control antibody.

[0022] However, the presence of substances like IgG in the sample can affect the detection results and cause false positives. Therefore, an inhibitor is added to the sample dilution buffer. This inhibitor can bind to substances like HAMA and rheumatoid factor RF, preventing these substances from binding to antibodies, reducing their impact on the test results, and improving the accuracy of the results.

[0023] Optionally, the nanofluorescent microsphere-labeled quality control antibody is a nanofluorescent microsphere-labeled chicken IgY monoclonal antibody or a nanofluorescent microsphere-labeled anti-DNP monoclonal antibody.

[0024] In this protocol, the fluorescent microsphere-labeled control antibody in the sample diluent corresponds to the quality control material on the control line. For example, when the fluorescent microsphere-labeled control antibody is specifically selected as a fluorescent microsphere-labeled chicken IgY monoclonal antibody, the quality control material is goat anti-chicken IgY; when the fluorescent microsphere-labeled control antibody is specifically selected as a fluorescent microsphere-labeled anti-DNP monoclonal antibody, the quality control material is DNP antigen.

[0025] Optionally, the surfactant is selected from any one or more of S9, TWEEN80, TWEEN20, and S17.

[0026] Optionally, the method for preparing the glass fiber mat includes the step of soaking a blank glass fiber mat in a glass fiber mat solution and then drying it, wherein the glass fiber mat comprises: 10-50 mM Tris-HCl and 0.5-5 wt% disaccharide.

[0027] By adopting the above technical solution, the release of cTnT monoclonal antibodies and cTnI monoclonal antibodies labeled with fluorescent microspheres can be delayed, and the residue of cTnT monoclonal antibodies and cTnI monoclonal antibodies labeled with fluorescent microspheres on the glass fiber pad can be reduced, thereby improving the accuracy of the detection results.

[0028] Optionally, the disaccharide is composed of sucrose and trehalose in a mass ratio of 1:(0.5-1.5).

[0029] Optionally, when detecting an antigen, the detection antibody is a mixture of one or more monoclonal antibodies corresponding to the antigen.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. This application modifies the surface of nitrocellulose membrane by simultaneously adding epigallocatechin gallate and 1,4-butanediamine to the detection line diluent, thereby increasing the variety and quantity of its surface active groups (hydroxyl and amino groups) to achieve stable capture of target proteins and significantly improve the tailing of the detection line.

[0032] 2. In this application, epigallocatechin gallate and 1,4-butanediamine are also added to the quality control line diluent to solve the quality control line tailing problem. Attached Figure Description

[0033] Figure 1 This is a structural diagram of the test strip cartridge case of this application;

[0034] Figure 2 This is a schematic diagram of the overall structure of the test strip in this application;

[0035] Figure 3 The standard curve of cTnT detection in Example 2;

[0036] Figure 4 This is the standard curve of cTnI detection in Example 2;

[0037] Figure descriptions: 1. Clamping shell; 11. Sample binding area; 111. Sample binding pad; 12. First sample detection area; 121. First glass fiber pad; 122. First nitrocellulose membrane; 123. First absorbent paper; 13. Second sample detection area; 131. Second glass fiber pad; 132. Second nitrocellulose membrane; 133. Second absorbent paper. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: unless otherwise specified in the following embodiments, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0039] In this application, BSA refers to bovine serum albumin.

[0040] Example

[0041] A test strip for the combined detection of cTnT and cTnI has the following structure: Figure 1 As shown, the test strip includes a casing 1, on which a sample binding area 11 and two sample detection areas connected to the sample binding area are respectively named the first sample detection area 12 and the second sample detection area 13.

[0042] A sample binding pad 111 is installed on the sample binding area 11 of the casing. The two sample detection areas are similarly configured, as follows: The first sample detection area 12 has a first glass fiber pad 121, a first nitrocellulose membrane 122, and a first absorbent paper 123 sequentially overlapped; the second sample detection area 13 has a second glass fiber pad 131, a second nitrocellulose membrane 132, and a second absorbent paper 133 sequentially overlapped. Both the first glass fiber pad 121 and the second glass fiber pad 131 overlap with the sample binding pad 111. Detection lines and control lines are provided on both the first nitrocellulose membrane 122 and the second nitrocellulose membrane 132. The components in the first sample detection area 12 are used to detect one substance, and the components in the second sample detection area 13 are used to detect another substance.

[0043] Example 1

[0044] A test strip for the combined detection of cTnT and cTnI has the structure described above.

[0045] The sample binding pad is prepared by soaking a blank glass fiber pad in sample diluent and then drying it at 45°C for 4 days. The sample diluent consisted of: 10 mM PB buffer (pH 7.4), 200 mM Tris buffer (pH 8.0), 0.01 wt% TWEEN 80, 0.15 wt% Proclin 300 preservative, 0.2 mg / mL blocking agent (purchased from Feipeng Biotechnology Co., Ltd., product model HIER-R-001), 2 wt% sodium casein, 0.5 wt% polyvinyl alcohol, 0.05 mg / mL fluorescent nanosphere-labeled cTnI monoclonal antibody (purchased from Feipeng Biotechnology Co., Ltd., product model CTNI-REAB-30#), 0.05 mg / mL fluorescent nanosphere-labeled cTnT monoclonal antibody (purchased from Qingdao Handerson Biotechnology, product model HDS-A3026), and 0.05 mg / mL fluorescent nanosphere-labeled chicken IgY monoclonal antibody.

[0046] The first glass fiber mat is prepared by immersing a blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The glass fiber mat solution comprises: 10 mM Tris-HCl (pH 8.0), and 5 wt% disaccharide, which is a mixture of sucrose and trehalose in a mass ratio of 1:0.5.

[0047] The second glass fiber mat is prepared by immersing a second blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The composition of the glass fiber mat solution is as described above.

[0048] The detection line on the first nitrocellulose membrane was obtained by diluting the cTnT antibody with the first detection line solution, streaking it onto the first nitrocellulose membrane, and then drying it. The components of the first detection line solution were: 10 mM PB buffer (pH 7.4), 5 wt% sodium chloride, 0.1 wt% sucrose, 0.1 wt% trehalose, 0.01 wt% BSA, 0.05 wt% preservative Proclin 300, 0.05 wt% epigallocatechin gallate, and 0.05 wt% 1,4-butanediamine. When the cTnT monoclonal antibody labeled with fluorescent nanospheres (purchased from Qingdao Henderson Biotechnology, product model HDS-A3026) was diluted with the first detection line diluent, the concentration of the cTnT monoclonal antibody labeled with fluorescent nanospheres in the first detection line diluent was 0.5 mg / mL.

[0049] The detection line on the second nitrocellulose membrane is obtained by diluting the cTnI antibody with the second detection line solution, streaking it onto the second nitrocellulose membrane, and then drying it. The components of the second detection line solution are: 10 mM PB buffer (pH 7.4), 5 wt% sodium chloride, 0.1 wt% sucrose, 0.1 wt% trehalose, 0.01 wt% BSA, 0.05 wt% preservative Proclin 300, 0.05 wt% epigallocatechin gallate, and 0.05 wt% 1,4-butanediamine. The cTnI antibody is specifically prepared by mixing cTnI monoclonal antibody labeled with fluorescent nanospheres (purchased from Feipeng Biotechnology Co., Ltd., product model CTNI-REAB-30#) and cTnI monoclonal antibody labeled with fluorescent nanospheres (purchased from Feipeng Biotechnology Co., Ltd., product model CTNI-MCAB-29#) at a 1:1 mass ratio. When diluting the cTnI antibody with the second detection line diluent, the concentration of cTnI antibody in the second detection line diluent is 0.5 mg / mL.

[0050] The control lines on the first nitrocellulose membrane were obtained by diluting the control sample with the first control line diluent, streaking it onto the membrane, and then drying it. The components of the first control line diluent were: 10 mM PB buffer (pH 7.4), 5 wt% sodium chloride, 0.1 wt% sucrose, 0.1 wt% trehalose, 0.01 wt% BSA, 0.05 wt% preservative Proclin 300, 0.05 wt% epigallocatechin gallate, and 0.05 wt% 1,4-butanediamine. The control sample was specifically goat anti-chicken IgY (purchased from Shenzhen Heavy Chain Biotechnology). The control sample was diluted with the first control line diluent to a concentration of 0.5 mg / mL.

[0051] The control lines on the second nitrocellulose membrane were obtained by streaking the control material onto the membrane after dilution with the second control line diluent, followed by drying. The second control line diluent consisted of: 10 mM PB buffer (pH 7.4), 5 wt% sodium chloride, 0.1 wt% sucrose, 0.1 wt% trehalose, 0.01 wt% BSA, 0.05 wt% preservative Proclin 300, 0.05 wt% epigallocatechin gallate, and 0.05 wt% 1,4-butanediamine. The control material was specifically goat anti-chicken IgY (purchased from Shenzhen Heavy Chain Biotechnology). The control material was diluted with the second control line diluent to a concentration of 0.5 mg / mL.

[0052] Example 2

[0053] A test strip for the combined detection of cTnT and cTnI has the structure described above.

[0054] The sample binding pad was prepared by immersing a blank glass fiber pad in sample diluent and drying it at 45°C for 4 days. The sample diluent consisted of: 150 mM PB buffer (pH 7.4), 100 mM Tris buffer (pH 8.0), 0.5 wt% TWEEN20, 0.1 wt% preservative Proclin 300, 0.4 mg / mL blocking agent, 1.5 wt% sodium casein, 1.5 wt% polyvinyl alcohol, 0.25 mg / mL fluorescent nanosphere-labeled cTnI monoclonal antibody, 0.25 mg / mL fluorescent nanosphere-labeled cTnT monoclonal antibody, and 0.25 mg / mL fluorescent nanosphere-labeled anti-DNP monoclonal antibody.

[0055] The first glass fiber mat is prepared by immersing a blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The glass fiber mat solution comprises: 30 mM Tris-HCl (pH 8.0), and 2.5 wt% disaccharide, which is a mixture of sucrose and trehalose in a 1:1 mass ratio.

[0056] The second glass fiber mat is prepared by immersing a second blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The composition of the glass fiber mat solution is as described above.

[0057] The detection line on the first nitrocellulose membrane was obtained by diluting the cTnT antibody with the first detection line solution, streaking it onto the membrane, and then drying it. The components of the first detection line solution were: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, 0.1 wt% epigallocatechin gallate, and 0.1 wt% 1,4-butanediamine. The cTnT antibody was the same as in Example 1. The cTnT antibody was diluted with the first detection line diluent to a concentration of 1.0 mg / mL.

[0058] The detection line on the second nitrocellulose membrane was obtained by diluting the cTnI antibody with the second detection line solution, streaking it onto the membrane, and then drying it. The components of the second detection line solution were: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, 0.1 wt% epigallocatechin gallate, and 0.1 wt% 1,4-butanediamine. The cTnI antibody was the same as in Example 1. The cTnI antibody was diluted with the second detection line diluent to a concentration of 1.0 mg / mL.

[0059] The control lines on the first nitrocellulose membrane were obtained by diluting the control material with the first control line diluent, streaking it onto the membrane, and then drying it. The components of the first control line diluent were: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, 0.1 wt% epigallocatechin gallate, and 0.1 wt% 1,4-butanediamine. The control material was DNP antigen (purchased from Shenzhen Heavy Chain Biotechnology). After diluting the control material with the first control line diluent, the concentration of the control material in the first control line diluent was 1.0 mg / mL.

[0060] The control lines on the second nitrocellulose membrane were obtained by diluting the control material with the second control line diluent, streaking it onto the membrane, and then drying it. The components of the second control line diluent were: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, 0.1 wt% epigallocatechin gallate, and 0.1 wt% 1,4-butanediamine. The control material was DNP antigen (purchased from Shenzhen Heavy Chain Biotechnology). The control material was diluted with the second control line diluent to a concentration of 1.0 mg / mL.

[0061] Example 3

[0062] A test strip for the combined detection of cTnT and cTnI has the structure described above.

[0063] The sample binding pad was prepared by soaking a blank glass fiber pad in the sample diluent and then drying it at 45°C for 4 days. The sample diluent consisted of: 200 mM PB buffer (pH 7.4), 10 mM Tris buffer (pH 8.0), 1 wt% S9, 0.05 wt% preservative Proclin 300, 0.6 mg / mL blocking agent, 0.5 wt% sodium casein, 2 wt% polyvinyl alcohol, 0.5 mg / mL fluorescent nanosphere-labeled cTnI monoclonal antibody, 0.5 mg / mL fluorescent nanosphere-labeled cTnT monoclonal antibody, and 0.5 mg / mL fluorescent nanosphere-labeled chicken IgY monoclonal antibody.

[0064] The first glass fiber mat is prepared by immersing a blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The glass fiber mat solution comprises: 50 mM Tris-HCl (pH 8.0), and 0.5 wt% disaccharide, which is a mixture of sucrose and trehalose in a mass ratio of 1:1.5.

[0065] The second glass fiber mat is prepared by immersing a second blank glass fiber mat in a glass fiber mat solution, followed by drying at 45°C for 4 days. The composition of the glass fiber mat solution is as described above.

[0066] The detection line on the first nitrocellulose membrane was obtained by diluting the cTnT antibody with the first detection line solution, streaking it onto the membrane, and then drying it. The components of the first detection line solution were: 50 mM PB buffer (pH 7.4), 10 wt% sodium chloride, 1 wt% sucrose, 1 wt% trehalose, 0.1 wt% BSA, 0.15 wt% preservative Proclin 300, 0.15 wt% epigallocatechin gallate, and 0.15 wt% 1,4-butanediamine. The cTnT antibody was the same as in Example 1. The cTnT antibody was diluted with the first detection line diluent to a concentration of 1.5 mg / mL.

[0067] The detection line on the second nitrocellulose membrane was obtained by diluting the cTnI antibody with the second detection line solution, streaking it onto the membrane, and then drying it. The components of the second detection line solution were: 50 mM PB buffer (pH 7.4), 10 wt% sodium chloride, 1 wt% sucrose, 1 wt% trehalose, 0.1 wt% BSA, 0.15 wt% preservative Proclin 300, 0.15 wt% epigallocatechin gallate, and 0.15 wt% 1,4-butanediamine. The cTnI antibody was the same as in Example 1. The cTnI antibody was diluted with the second detection line diluent to a concentration of 1.5 mg / mL.

[0068] The control lines on the first nitrocellulose membrane were obtained by diluting the control material with the first control line diluent, streaking it onto the first nitrocellulose membrane, and then drying it. The components of the first control line diluent were: 50 mM PB buffer (pH 7.4), 10 wt% sodium chloride, 1 wt% sucrose, 1 wt% trehalose, 0.1 wt% BSA, 0.15 wt% preservative Proclin 300, 0.15 wt% epigallocatechin gallate, and 0.15 wt% 1,4-butanediamine. The control material was the same as in Example 1. After diluting the control material with the first control line diluent, the concentration of the control material in the first control line diluent was 1.5 mg / mL.

[0069] The control lines on the second nitrocellulose membrane were obtained by diluting the control antibody with the second control line diluent, streaking it onto the second nitrocellulose membrane, and then drying it. The components of the second control line diluent were: 50 mM PB buffer (pH 7.4), 10 wt% sodium chloride, 1 wt% sucrose, 1 wt% trehalose, 0.1 wt% BSA, 0.15 wt% preservative Proclin 300, 0.15 wt% epigallocatechin gallate, and 0.15 wt% 1,4-butanediamine. The specific quality control material was the same as in Example 1. The quality control material was diluted with the second control line diluent to a concentration of 1.5 mg / mL in the second control line diluent.

[0070] Example 4

[0071] The difference between this embodiment and Embodiment 2 is that neither of them contains epigallocatechin gallate in the quality control line diluent, as detailed below:

[0072] The components of the first quality control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0073] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0074] Example 5

[0075] The difference between this embodiment and Example 2 is that the quality control line diluent does not contain 1,4-butanediamine, as detailed below:

[0076] The components of the first control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0077] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0078] Example 6

[0079] The difference between this embodiment and Example 2 is that the quality control line diluent does not contain 1,4-butanediamine and epigallocatechin gallate, as detailed below:

[0080] The components of the first control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0081] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0082] Comparative Example

[0083] Comparative Example 1

[0084] The difference between this embodiment and Embodiment 2 is that the components of the first detection line solution do not contain epigallocatechin gallate, and the content of 1,4-butanediamine is 0.2 wt%; the components of the first control line diluent do not contain epigallocatechin gallate, and the content of 1,4-butanediamine is 0.2 wt%; the components of the second detection line solution do not contain epigallocatechin gallate, and the content of 1,4-butanediamine is 0.2 wt%; the components of the second control line diluent do not contain epigallocatechin gallate, and the content of 1,4-butanediamine is 0.2 wt%.

[0085] Specifically as follows:

[0086] The components of the first detection line solution are: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0087] The second detection line solution consists of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0088] The components of the first quality control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0089] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% 1,4-butanediamine.

[0090] Comparative Example 2

[0091] The difference between this embodiment and Embodiment 2 is that the components of the first detection line solution do not contain 1,4-butanediamine, and the content of epigallocatechin gallate is 0.2 wt%; the components of the first quality control line diluent do not contain 1,4-butanediamine, and the content of epigallocatechin gallate is 0.2 wt%; the components of the second detection line solution do not contain 1,4-butanediamine, and the content of epigallocatechin gallate is 0.2 wt%; the components of the second quality control line diluent do not contain 1,4-butanediamine, and the content of epigallocatechin gallate is 0.2 wt%.

[0092] Specifically as follows:

[0093] The first detection line solution consists of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0094] The second detection line solution consists of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0095] The components of the first control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0096] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, 0.1 wt% preservative Proclin 300, and 0.2 wt% epigallocatechin gallate.

[0097] Comparative Example 3

[0098] The difference between this embodiment and Embodiment 2 is that the components of the first detection line solution do not contain 1,4-butanediamine and epigallocatechin gallate; the components of the first quality control line diluent do not contain 1,4-butanediamine and epigallocatechin gallate; the components of the second detection line solution do not contain 1,4-butanediamine and epigallocatechin gallate; and the components of the second quality control line diluent do not contain 1,4-butanediamine and epigallocatechin gallate.

[0099] Specifically as follows:

[0100] The first detection line solution consists of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0101] The second detection line solution consists of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0102] The components of the first control line diluent are: 20 mM PB buffer (PB buffer pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0103] The second control line diluent consisted of: 20 mM PB buffer (pH 7.4), 8.5 wt% sodium chloride, 0.5 wt% sucrose, 0.5 wt% trehalose, 0.05 wt% BSA, and 0.1 wt% preservative Proclin 300.

[0104] Performance testing

[0105] 1. Standard Curve

[0106] 1.1 Preparation of the standard curve for cTnT detection

[0107] The test strips of Example 2 were used to perform combined detection of cTnT and cTnI.

[0108] The steps for preparing the standard curve for cTnT detection are as follows:

[0109] Standard curves were plotted using the Roche assignment method with lyophilized samples. Specifically, lyophilized samples were used to prepare solutions with concentrations of 5.01, 10.05, 20.07, 58.8, 125.4, 286.3, 521.02, 1354.66, 5013.21, and 9869 ng / L. The samples were then tested using the method described in Example 2. Each sample was placed on a WIZ A101 portable immunoassay analyzer from Weizheng Biotechnology for detection, and graphs were plotted using Log concentration and Log(T / C), where T is the detection line and C is the control line. The linearity obtained from Log concentration, log(T / C) (i.e., the luminescence intensity of the detection line and control line), and cTnT concentration was 0.03-32 ng / L. Figure 2 As shown, the linear regression equation is obtained: y = 0.7951x - 2.4618; the linear range of cTnT is 5-10000ng / L.

[0110] 1.2 Preparation of the standard curve for cTnI detection

[0111] The test strips of Example 2 were used to perform combined detection of cTnT and cTnI.

[0112] The steps for preparing the standard curve for cTnI detection are as follows:

[0113] A standard curve was prepared using the Siemens assignment method from the lyophilized samples.

[0114] Solutions with concentrations of 5.01, 10.05, 20.07, 58.8, 125.4, 286.3, 521.02, 1354.66, 5013.21, and 9869 ng / mL were prepared from the lyophilized samples. The samples were tested using the method described in Example 2. Each sample was tested on the same equipment as described above, and a standard curve was plotted based on the luminescence intensity and the data described above. Figure 3 As shown, the linear regression equation is obtained: y = 0.7865x - 0.8373, R0 2 =0.9979; the linear range of cTnI is 0.03-40 ng / mL.

[0115] 2. Specificity:

[0116] Different samples were tested using the Roche high-sensitivity troponin T assay kit to obtain the cTnT values ​​for each sample. Different samples were also tested using the Siemens high-sensitivity cardiac troponin I (cTnI) assay kit (direct chemiluminescence immunoassay) to obtain the cTnI values ​​for each sample. Finally, the method described in Example 2 of this application was used to obtain the cTnT and cTnI values ​​for each sample. The detection values ​​of the corresponding indicators from the two methods were compared to obtain the detection result deviation, and the specific results are shown in Table 1. The results in Table 1 show that when using the assay kit of this application for combined cTnT and cTnI detection, the data deviation of the detection results is within the range of 0-15%, indicating that the assay kit of this application has good specificity.

[0117] Table 1 Comparison of detection data from different detection methods

[0118]

[0119] 2. Statistics on the trailing effect of the testing line

[0120] 180 positive samples of cTnT and cTnI were tested using test strips from Examples 1-3 and Comparative Examples 1-3, respectively. The tailing of the test line for each test strip was statistically analyzed, and the specific results are shown in Table 2.

[0121] Table 2. Test line trailing on the test strips

[0122]

[0123] The data in Table 2 show that adding 1,4-butanediamine and epigallocatechin gallate to the test line diluent simultaneously significantly reduces test line tailing, resulting in a higher success rate and more accurate results. When using the test strips from Examples 1-3 for combined detection of cTnT and cTnI, the tailing rate of both cTnT and cTnI test lines was less than 1%, specifically controlled within the range of 0-0.56%.

[0124] Comparing the results of Example 2 and Comparative Examples 1-3, it was found that, for example, in Example 1 and Comparative Example 2, simply adding epigallocatechin gallate to the detection line diluent could alleviate the detection line tailing issue to some extent, but more than 4% of the samples still exhibited detection line tailing. However, the methods of simply adding epigallocatechin gallate to the detection line diluent and simply adding 1,4-butanediamine to the detection line diluent were more effective in resolving detection line tailing. For example, in Comparative Example 1, more than 5% of the samples exhibited detection line tailing.

[0125] When neither 1,4-butanediamine nor epigallocatechin gallate was added to the test line diluent, the tailing of the test line was more severe, with the tailing rate reaching 11.11%.

[0126] 3. Quality control line tailing statistics

[0127] The test strips from Examples 2 and 4-5 were used to test 180 positive samples of cTnT and cTnI, respectively. The tailing of the test line for each test strip was statistically analyzed. The specific results are shown in Table 3.

[0128] Table 3. Test line tailing on the test strips

[0129]

[0130]

[0131] The data in Table 3 show that by simultaneously adding 1,4-butanediamine and epigallocatechin gallate to the control line diluent, the tailing of the control line can be significantly reduced, resulting in a higher success rate and more accurate test results.

[0132] Comparing the results of Examples 2 and 4-6, it was found that epigallocatechin gallate and 1,4-butanediamine need to be added simultaneously to the quality control line diluent in order to effectively solve the problem of quality control line tailing.

[0133] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A test strip for the combined detection of cTnT and cTnI, characterized in that, The test strip includes a housing, the housing including a sample binding area and at least two sample detection areas connected to the sample binding area; The sample binding area of ​​the cartridge is provided with a sample binding pad, and each sample detection area is provided with a glass fiber pad, a nitrocellulose membrane and absorbent paper that are sequentially overlapped. The glass fiber pad and the sample binding pad are connected, and the nitrocellulose membrane is provided with a detection line and a quality control line. The method for preparing the detection line includes the step of diluting the detection antibody with a detection line diluent and then streaking it onto the nitrocellulose membrane. The components of the detection line diluent include: 10-50 mM PB buffer, 5-10 wt% sodium chloride, 0.1-1 wt% sucrose, 0.1-1 wt% trehalose, 0.01%-0.1 wt% BSA, 0.05-0.15 wt% preservative, 0.05-0.15 wt% epigallocatechin gallate, and 0.05-0.15 wt% 1,4-butanediamine. The method for preparing the control line includes the step of diluting the control material with a control line diluent and then streaking it onto the nitrocellulose membrane. The components of the control line diluent include: 10-50 mM PB buffer, 5-10 wt% sodium chloride, 0.1-1 wt% sucrose, 0.1-1 wt% trehalose, 0.01%-0.1 wt% BSA, 0.05-0.15 wt% preservative, 0.05-0.15 wt% epigallocatechin gallate, and 0.05-0.15 wt% 1,4-butanediamine.

2. The test strip for combined detection of cTnT and cTnI according to claim 1, characterized in that, The detection antibody is diluted with the detection line diluent to achieve a concentration of 0.5-1.5 mg / mL.

3. The test strip for combined detection of cTnT and cTnI according to claim 1, characterized in that, The quality control material is diluted with the quality control line diluent to achieve a concentration of 0.5-1.5 mg / mL.

4. The test strip for combined detection of cTnT and cTnI according to claim 1, characterized in that, The method for preparing the sample binding pad includes the steps of soaking a blank glass fiber pad in a sample diluent and then drying it. The sample diluent comprises: 10-200 mM PB buffer, 10-200 mM Tris buffer, 0.01-1 wt% surfactant, 0.05-0.15 wt% preservative, 0.2-0.6 mg / mL blocking agent, 0.5-2 wt% sodium casein, 0.5-2 wt% polyvinyl alcohol, 0.05-0.5 mg / mL fluorescent nanosphere-labeled cTnI monoclonal antibody, 0.05-0.5 mg / mL fluorescent nanosphere-labeled cTnT monoclonal antibody, and 0.05-0.5 mg / mL fluorescent nanosphere-labeled quality control antibody.

5. The test strip for combined detection of cTnT and cTnI according to claim 4, characterized in that, The quality control antibody is either a chicken IgY monoclonal antibody labeled with fluorescent nanospheres or an anti-DNP monoclonal antibody labeled with fluorescent nanospheres.

6. The test strip for combined detection of cTnT and cTnI according to claim 4, characterized in that, The surfactant is selected from any one or more of S9, TWEEN80, TWEEN20, and S17.

7. The test strip for combined detection of cTnT and cTnI according to claim 1, characterized in that, The method for preparing the glass fiber mat includes the steps of soaking a blank glass fiber mat in a glass fiber mat solution and then drying it. The glass fiber mat solution comprises: 10-50 mM Tris-HCl and 0.5-5 wt% disaccharide.

8. The test strip for combined detection of cTnT and cTnI according to claim 7, characterized in that, The disaccharide is composed of sucrose and trehalose in a mass ratio of 1:(0.5-1.5).

9. The test strip for combined detection of cTnT and cTnI according to claim 1, characterized in that, When detecting an antigen, the detection antibody is a mixture of one or more monoclonal antibodies corresponding to the antigen.

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