Joint detection test paper, kit and method for quantitatively detecting five items of hypertension

Abstract

The embodiment of the invention belongs to the technical field of biological detection, and relates to joint detection test paper, a kit and a method for quantitatively detecting five items of hypertension, the joint detection test paper comprises a PVC bottom plate, and a sample pad, a combination pad, a coating film and a water absorption pad which are sequentially connected on the PVC bottom plate; wherein the combination pad is coupled with a fluorescent microsphere labeled mouse anti-human ACTH antibody, a fluorescent microsphere labeled COR antibody, a fluorescent microsphere labeled ALD antibody, a fluorescent microsphere labeled REN antibody, a fluorescent microsphere labeled AngII antibody and a fluorescent microsphere labeled first antibody; a first detection line coated with an ACTH monoclonal antibody, a second detection line coated with a COR coupled BSA antigen, a third detection line coated with an ALD coupled BSA antigen, a fourth detection line coated with an REN monoclonal antibody, a fifth detection line coated with an AngII monoclonal antibody and a quality control line coated with a second antibody are arranged on the coating film. The detection accuracy is effectively improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a combined test strip, a kit and a method for quantitatively detecting five items of hypertension. Background Art

[0002] Hypertension is a common cardiovascular disease, which refers to a clinical syndrome characterized by increased systemic arterial blood pressure (systolic and / or diastolic blood pressure) (systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg), which may be accompanied by functional or organic damage to organs such as the heart, brain, and kidneys. Hypertension can be divided into two categories: primary hypertension and secondary hypertension. Primary hypertension is an independent disease with increased blood pressure as the main manifestation and an unknown cause, accounting for more than 90% of all hypertensive patients. Patients often have a family history of the disease, and its pathogenesis is related to multiple factors, including diabetes, obesity, hyperlipidemia, and atherosclerosis. Secondary hypertension is also called symptomatic hypertension. In this type of disease, the cause is clear, and hypertension is only one of the manifestations of the disease. Blood pressure may increase temporarily or permanently.

[0003] At present, five main indicators are tested to determine primary hypertension and secondary hypertension, namely adrenocorticotropic hormone (ACTH), cortisol (COR), aldosterone (ALD), renin (REN), and angiotensin II (AngII). When the body is stressed, the HPA axis (hypothalamus-pituitary-adrenal cortex axis) releases corticotropin-releasing hormone (CRH), which causes the pituitary gland to release adrenocorticotropic hormone (ACTH). ACTH acts on the adrenal cortex to secrete and release cortisol. Cortisol in turn feedback inhibits the release of peptide hormones from the hypothalamus and pituitary gland to achieve a self-stabilizing effect. Disorders in the HPA axis can lead to adrenal diseases such as hypercortisolism, pheochromocytoma, primary aldosteronism, and congenital adrenal hyperplasia, causing secondary hypertension. Therefore, detecting the HPA axis is of great significance for the determination of secondary hypertension.

[0004] Renin, angiotensin, aldosterone three are a connected action system, called renin-angiotensin-aldosterone system (RAAS), by the control of blood volume and peripheral yang force, regulate human blood pressure, water and electrolyte balance, maintain body internal environment constant.When blood pressure decreases, kidney begins to secrete renin. Renin can convert angiotensinogen into angiotensin I, and generates angiotensin II under the effect of angiotensin converting enzyme. Angiotensin II contracts blood vessels, causing blood pressure to rise, and can also be used as effector molecule to stimulate the release of aldosterone, and aldosterone has the effect of sodium retention and potassium excretion, can cause blood pressure to rise, and causes hypertension.And studies have found that hypertensive patients' adrenocorticotropic hormone, renin, angiotensin II, aldosterone, cortisol levels are more obviously raised, and combined detection of blood adrenocorticotropic hormone, renin, angiotensin, aldosterone, cortisol levels is good for the determination and prognosis evaluation value of hypertensive patients.

[0005] In summary, the combined detection of blood adrenocorticotropic hormone, renin, angiotensin II, aldosterone, and cortisol is an important indicator for the determination and research of primary and secondary hypertension. At present, the existing detection technology mainly uses single card or two-card detection kits using ELISA, chemiluminescence and other methods, which cannot achieve quantitative detection of the five hypertension items in one test. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a combined test strip, a test kit and a method for quantitatively detecting five items of hypertension, thereby effectively improving the detection accuracy.

[0007] In order to solve the above technical problems, the present application embodiment provides a combined test strip for quantitatively detecting five items of hypertension, which adopts the following technical solution:

[0008] A combined test strip for quantitatively detecting five items of hypertension, the combined test strip comprising a PVC base plate and a sample pad, a binding pad, a coating film and a water-absorbing pad sequentially connected on the PVC base plate;

[0009] Wherein, the conjugate pad is coupled with fluorescent microsphere-labeled mouse anti-human ACTH antibody, fluorescent microsphere-labeled COR antibody, fluorescent microsphere-labeled ALD antibody, fluorescent microsphere-labeled REN antibody, fluorescent microsphere-labeled AngII antibody and fluorescent microsphere-labeled first antibody;

[0010] The coating membrane is provided with a first detection line coated with ACTH monoclonal antibody, a second detection line coated with COR coupled with BSA antigen, a third detection line coated with ALD coupled with BSA antigen, a fourth detection line coated with REN monoclonal antibody, a fifth detection line coated with AngII monoclonal antibody and a quality control line coated with the second antibody.

[0011] Furthermore, the first antibody is a goat anti-chicken IgY antibody; and / or

[0012] The second antibody is chicken IgY antibody.

[0013] Furthermore, the coating concentrations of the ACTH monoclonal antibody of the first test line, the COR-coupled BSA antigen of the second test line, the ALD-coupled BSA antigen of the third test line, the REN monoclonal antibody of the fourth test line, the AngII monoclonal antibody of the fifth test line and the second antibody of the quality control line are all 1-1.5 mg / mL.

[0014] Furthermore, the fluorescent microspheres include europium time-resolved fluorescent microspheres.

[0015] Further, the sample pad is prepared by the following process:

[0016] The glass cellulose membrane is soaked in the sample pad treatment solution and dried at 37-50° C. to obtain the sample pad.

[0017] Furthermore, the sample pad treatment solution includes, by mass concentration, 0.02 M phosphate buffer at pH 7.4, 0.5% to 1% bovine serum albumin, 0.1% Tween 80 and 3% to 5% sucrose.

[0018] Furthermore, the conjugate pad is prepared by the following method:

[0019] respectively preparing fluorescent microsphere-labeled anti-ACTH, fluorescent microsphere-labeled COR, fluorescent microsphere-labeled ALD, fluorescent microsphere-labeled REN, fluorescent microsphere-labeled AngII antibody, and fluorescent microsphere-labeled primary antibody;

[0020] The fluorescent microsphere-labeled anti-ACTH, fluorescent microsphere-labeled COR, fluorescent microsphere-labeled ALD, fluorescent microsphere-labeled REN, fluorescent microsphere-labeled AngII antibody, and fluorescent microsphere-labeled first antibody are mixed in a volume ratio of 2:2:2:2:2:1 to obtain a mixed antibody solution;

[0021] The mixed antibody solution is sprayed on the initial conjugate pad at a spray rate of 3.0-4.0 μL / cm, and after drying, the conjugate pad is obtained.

[0022] In order to solve the above technical problems, the present application also provides a kit for quantitatively detecting five items of hypertension, which adopts the following technical solution:

[0023] A kit for quantitatively detecting five items of hypertension, the kit comprising a detection card, the detection card comprising a fluorescent card shell and the above-mentioned joint detection test paper, the joint detection test paper being arranged in the fluorescent card shell.

[0024] Furthermore, the kit also includes a diluent and a chip;

[0025] The diluent includes 0.5% Tween-20 and 0.01M pH 7.4 PBS buffer solution in terms of mass concentration.

[0026] In order to solve the above technical problems, the embodiment of the present application also provides a method for quantitatively detecting five items of hypertension, which adopts the following technical solution:

[0027] A method for quantitatively detecting five items of hypertension using the above-mentioned kit comprises the following steps:

[0028] S1: Mix the sample to be tested and the diluent evenly, add them to the sample well of the test card, and let them stand for treatment;

[0029] S2: Put the test card into the test instrument to obtain the output test result.

[0030] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0031] This application uses time-resolved fluorescent microsphere immunochromatography technology to measure five indicators of hypertension in the blood (ACTH / COR / ALD / REN / AngII). This application has the following two advantages: through one sample addition, five indicators can be detected simultaneously, so as to timely diagnose the type of hypertensive patients; this joint test strip uses a combination of time-resolved fluorescence and immunochromatography technology, and uses europium time-resolved fluorescent microspheres to label antibodies, which is conducive to reducing the background fluorescence intensity, improving the resolution and detection sensitivity, and has high accuracy. In addition, the sample is simple, the instrument is easy to operate, the price is low, and the total time for sample addition and result output is within 20 minutes, which is convenient and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 It is a schematic diagram of the structure of the combined test strip for quantitatively detecting five items of hypertension of the present application;

[0034] Figure 2 It is a schematic diagram of the structure of the test card of the kit for quantitatively detecting five items of hypertension of the present application;

[0035] Figure 3is a flow chart of an embodiment of the method for quantitatively detecting five items of hypertension of the present application;

[0036] Figure 4 It is a clinical correlation result diagram of the kit for quantitative detection of five hypertension items of the present application;

[0037] Figure 5 is another clinical relevance result diagram of the kit for quantitatively detecting five items of hypertension of the present application;

[0038] Figure 6 is another clinical relevance result diagram of the kit for quantitatively detecting five items of hypertension of the present application;

[0039] Figure 7 is another clinical relevance result diagram of the kit for quantitatively detecting five items of hypertension of the present application;

[0040] Figure 8 This is another clinical relevance result diagram of the kit for quantitatively detecting five items of hypertension of the present application.

[0041] Figure numerals: 1, sample pad; 2, binding pad; 3, coating membrane; 4, absorbent pad; 5, PVC bottom plate; 6, detection line; 7, quality control line; 8, sample addition hole; 9, reading area; 10, hand-held area. DETAILED DESCRIPTION

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0043] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0044] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0045] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0046] The present application provides a combined test strip for quantitatively detecting five items of hypertension, such as Figure 1 As shown, Figure 1 It is a schematic diagram of the structure of the combined test strip for quantitatively detecting five hypertension items of the present application. The five hypertension items of the present application include ACTH (corticosteroid), COR (cortisol), ALD (aldosterone), REN (renin) and AngII (angiotensin II).

[0047] The joint test strip comprises a PVC base plate 5 and a sample pad 1, a conjugation pad 2, a coating membrane 3 and a water-absorbing pad 4 connected in sequence on the PVC base plate 5, wherein the conjugation pad 2 is coupled with mouse anti-human ACTH antibody labeled with fluorescent microspheres, COR antibody labeled with fluorescent microspheres, ALD antibody labeled with fluorescent microspheres, REN antibody labeled with fluorescent microspheres, AngII antibody labeled with fluorescent microspheres and a first antibody (sheep anti-chicken IgY antibody) labeled with fluorescent microspheres;

[0048] The coating membrane 3 is provided with a detection line 6 and a quality control line 7, wherein the detection line 6 comprises: a first detection line coated with ACTH monoclonal antibody, a second detection line coated with COR coupled with BSA antigen, a third detection line coated with ALD coupled with BSA antigen, a fourth detection line coated with REN monoclonal antibody and a fifth detection line coated with AngII monoclonal antibody; the quality control line 7 is coated with a second antibody (chicken IgY antibody).

[0049] In this embodiment, the joint test paper includes a PVC base plate and a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad connected in sequence on the PVC base plate. The right side of the sample pad is covered with a conjugation pad of 1 to 2 mm, and the conjugation pad and the water-absorbing pad are respectively at both ends of the nitrocellulose coating membrane, wherein the conjugation pad covers 1 to 2 mm on the nitrocellulose membrane, and the water-absorbing pad covers 2 to 3 mm on the nitrocellulose coating membrane. The first test line, the second test line, the third test line, the fourth test line, the fifth test line and the quality control line are arranged in parallel. Five index values ​​are detected by one sample addition. The distances between the test line and the quality control line of the above-mentioned coating membrane are (2 ± 0.5) mm, respectively, the distance between the quality control line and the side of the coating membrane close to the water-absorbing pad is (6 ± 0.5) mm, and the distance between the first test line and the side of the coating membrane close to the conjugation pad is (6 ± 0.5) mm.

[0050] As an option in the present application, the coating concentrations of the ACTH monoclonal antibody of the first detection line, the COR-coupled BSA antigen of the second detection line, the ALD-coupled BSA antigen of the third detection line, the REN monoclonal antibody of the fourth detection line, the AngII monoclonal antibody of the fifth detection line and the second antibody of the quality control line are all 1-1.5 mg / mL.

[0051] In this embodiment, the optional concentrations include 1-1.2 mg / mL, 1.2-1.5 mg / mL, for example, 1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.5 mg / mL.

[0052] Furthermore, the fluorescent microspheres include europium time-resolved fluorescent microspheres.

[0053] In this embodiment, the fluorescent microspheres use the rare earth element europium as a marker. The rare earth element europium has a large Stokes shift, and the difference between its excitation wavelength and emission wavelength is about 245 nm, which can effectively eliminate the interference of various non-specific fluorescence.

[0054] As an option of the present application, the sample pad is prepared by the following method: soaking the glass cellulose membrane in a sample pad treatment solution and drying at 37-50° C. to obtain the sample pad. In terms of mass concentration (i.e., mass volume ratio, W / V), the sample pad treatment solution includes 0.02M phosphate buffer at pH 7.4, 0.5% to 1% bovine serum albumin, 0.1% Tween 80, and 3% to 5% sucrose.

[0055] In this embodiment, the raw material of the sample pad is glass cellulose membrane. Before use, the glass cellulose membrane is soaked with sample pad treatment solution, dried overnight in a forced air drying oven at a temperature of 37-50° C., and sealed for storage.

[0056] As an option in this application, the bonding pad is prepared by the following method:

[0057] 1) Fluorescent microspheres are used to label anti-ACTH antibodies (fluorescent microspheres are used to label anti-COR, ALD, REN, AngII antibodies, and goat anti-chicken IgY in the same steps): 100 μL of fluorescent microspheres with a solid content of 1% are mixed in 2 mL of 0.05M PH 6.0MES buffer, and 10 μL of 50 mg / mL N-hydroxysuccinimide (NHS) and 10 μL of 50 mg / mL N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) are added, and the mixture is mixed for 30 min. 40-60 μg, preferably 50 μg of anti-ACTH antibody is added, and after rotating for 90 min, 500 μL of labeling blocking solution (10% BSA) is added, and the mixture is rotated for 60 min. After centrifugation to remove the supernatant, labeling preservation solution (1% casein sodium salt, 0.1% Tween 20, 0.05M Tris-HCl buffer at pH 8.0) is added for storage.

[0058] 2) Spray pad: Mix the fluorescent microsphere-labeled anti-ACTH, fluorescent microsphere-labeled COR, fluorescent microsphere-labeled ALD, fluorescent microsphere-labeled REN, fluorescent microsphere-labeled AngII antibody, and fluorescent microsphere-labeled first antibody (sheep anti-chicken IgY) obtained in the above steps in a ratio of 2:2:2:2:2:1, and spray them on the initial binding pad at a spray volume of 3.0-4 μL / cm, preferably 3.0 μL / cm. Place the sprayed initial binding pad in an environment of 37-50°C and humidity ≤30% and dry overnight (16-24) hours to obtain a binding pad. Add an appropriate amount of desiccant, seal it in a ziplock bag, and label it for later use.

[0059] As an option of the present application, the coating membrane is prepared by the following process:

[0060] ACTH monoclonal, COR-coupled BSA antigen (COR recombinant antigen), ALD-coupled BSA antigen (ALD recombinant antigen), REN monoclonal antibody, AngII monoclonal antibody, chicken IgY antibody were respectively prepared into a coating solution with a concentration of 1 mg / ml using a coating buffer (0.01M phosphate buffer at pH 7.4, 1% sucrose, 1% trehalose). Coat the nitrocellulose membrane at a concentration of 1-1.5 μL / cm, preferably 1 μL / cm. Place the sprayed coating membrane in an environment of 37-50°C and humidity ≤30% to dry overnight (16-24) hours. Add an appropriate amount of desiccant, seal in a ziplock bag, and label it for later use.

[0061] The present application also provides a kit for quantitatively detecting five items of hypertension, the kit comprising a test card, the test card comprising a fluorescent card shell and the above-mentioned joint test paper, the joint test paper being arranged in the fluorescent card shell. Figure 2 As shown, Figure 2It is a schematic diagram of the structure of the test card of the kit for quantitative detection of five items of hypertension of the present application. The test card comprises a sample addition hole 8, a reading area 9 and a hand-held area 10, wherein the sample addition hole corresponds to the area of ​​the sample pad of the joint test paper, the reading area corresponds to the area of ​​the coating membrane of the joint test paper, and the hand-held area corresponds to the water absorption pad of the joint test paper.

[0062] As an option in the present application, the kit further includes a diluent, a chip and instructions.

[0063] As an option for this application, the assembly process of the test card is as follows: (the production environment requires a temperature of (18-30)°C and a humidity of ≤30%), cut the absorbent paper into a size of (23±1) mm×(300±10) mm, set aside, and pay attention to drying. Pasting the marking pad: Gently peel off the protective film at the junction of the nitrocellulose membrane marking pad, paste the binding pad on it, and cover the coating membrane 1-2 mm. Pasting the sample pad: Cover and paste the sample pad about 1-2 mm below the marking pad. Cutting the joint test paper: Cut the prepared large board into joint test papers with a width of (3.85±0.05) mm; load each joint test paper into the first shell of the fluorescent card shell, cover the second shell of the fluorescent card shell after installation, place it on the card press, adjust the height of the shell press to 3.5 mm, so that the first shell and the second shell of the fluorescent card shell are tightly combined to obtain the test card. Put each test card into an aluminum foil bag, add 1 bag of 1g desiccant to each bag, and seal it.

[0064] As an option of the present application, the diluent is a phosphate buffered saline with Tween-20 added as a surfactant. In terms of mass concentration (ie, mass-to-volume ratio, W / V), the diluent includes 0.5% Tween-20, 0.01M pH 7.4 PBS buffer.

[0065] As an option for this application, the chip is prepared by the following steps: take a qualified test kit, ACTH, COR, ALD, REN, AngII calibrators, each calibrator has at least 5 concentrations, and each concentration is tested at least 3 times. According to the reagent instructions, operate on a dry fluorescent immunoassay analyzer, and use the average value of the T / C value obtained by the prepared concentration and each concentration test to establish a standard curve, obtain a linear equation, import the numerical value of the equation into the software, connect the blank original chip to the computer, write the standard curve information into the chip, complete the chip data configuration, and affix the chip label.

[0066] In this embodiment, the chip records the curve information of the relationship between the detection signal value and the detection concentration, and the detection data can be directly read out by inserting the ACTH / COR / ALD / REN / AngII detection card.

[0067] This application uses time-resolved fluorescent microsphere immunochromatography technology to measure five indicators of hypertension in the blood (ACTH / COR / ALD / REN / AngII). This application has the following two advantages: ① Through one-time sample addition, five indicators can be detected at the same time, so as to timely diagnose the type of hypertensive patients; ② This joint test strip uses a combination of time-resolved fluorescence and immunochromatography technology, and uses europium time-resolved fluorescent microspheres to label antibodies, which is beneficial to reduce the background fluorescence intensity, improve resolution and detection sensitivity, and has high accuracy. In addition, the sample is simple, the instrument is easy to operate, the price is low, and the total time for sample addition and result output is within 20 minutes, which is convenient and fast.

[0068] The present application also provides a method for quantitatively detecting five items of hypertension using the above kit, and further reference is made to Figure 3 , shows a flow chart of an embodiment of a method for quantitatively detecting five items of hypertension according to the present application. The method for quantitatively detecting five items of hypertension comprises the following steps:

[0069] S1: Mix the sample to be tested and the diluent evenly, add them to the sample well of the test card, and let them stand for treatment;

[0070] S2: Put the test card into the test instrument to obtain the output test result.

[0071] The detection method of the above-mentioned hypertension test kit is as follows:

[0072] 1) Turn on the fluorescent immunoassay analyzer and read the chip.

[0073] 2) Pipette 100 μl of serum / plasma sample and add it to 500 μl of diluent.

[0074] 3) Take 100 μl of the mixed liquid and add it to the sample well of the test card. After standing for 15 minutes, insert the test card into the fluorescent immunoassay analyzer and read the test results according to the labeled curve information in the chip.

[0075] The testing principle of this application: After the sample to be tested is dripped onto the sample pad, the liquid moves from left to right due to the chromatography effect. The human ACTH, COR, ALD, REN, and AngII in the sample to be tested are respectively combined with the mouse anti-human ACTH antibody labeled with fluorescent microspheres, the COR antibody labeled with optical microspheres, the ALD antibody labeled with fluorescent microspheres, the REN antibody labeled with fluorescent microspheres, and the AngII antibody labeled with fluorescent microspheres in the binding pad to form a fluorescently labeled antibody-antigen complex, which continues to move to the right. Among them, the ACTH, REN, and AngII fluorescently labeled antibody-antigen complex are respectively combined with the ACTH, REN, and AngII antibodies on the coating membrane to form a double-antibody sandwich complex that is fixed on the coating membrane, and the content of ACTH, REN, and AngII is reflected by the intensity of fluorescence. The COR and ALD fluorescently labeled antibody-antigen complexes will be directly chromatographed to the absorbent paper, while the fluorescently labeled antibodies that are not bound to COR and ALD are captured by the COR-coupled BSA antigen and ALD-coupled BSA antigen on the coating membrane, respectively. The stronger the fluorescence on the coating membrane, the less the corresponding COR and ALD content, and vice versa. On the other hand, the sheep anti-chicken IgY labeled with fluorescent microspheres on the binding pad also moves to the right with the solution and binds to the chicken IgY coated on the coating membrane. The signal value is used as the quality control line of this application.

[0076] The existing methods for detecting hypertension indicators only include single card or double card detection, but generally, the classification and prognosis of hypertensive patients require detection of the HPA axis and RAAS system, that is, the detection of the above five indicators. Therefore, this detection method has the advantages of being convenient and fast, and has a high detection range, sensitivity and accuracy. It can produce results faster and more accurately, and has the advantages of low cost and broad application prospects.

[0077] The performance data of the kit of this application are shown in the following table.

[0078] Table 1 Performance of the kit of this application

[0079]

[0080] Table 2 Performance test of the kit of this application

[0081]

[0082]

[0083]

[0084] Each item was tested on 50 clinical samples, and the test results were compared with the labeled values. The clinical relevance R 2 ≥0.975, meets the requirements. Clinical correlation of ACTH is as follows Figure 4 As shown, Figure 4This is a clinical correlation result diagram of the kit for quantitative detection of five hypertension items in this application; COR clinical correlation is as follows Figure 5 As shown, Figure 5 This is another clinical correlation result diagram of the kit for quantitative detection of five hypertension items of the present application; ALD clinical correlation is as follows Figure 6 As shown, Figure 6 This is another clinical correlation result diagram of the kit for quantitative detection of five items of hypertension in this application; REN clinical correlation is as follows Figure 7 As shown, Figure 7 This is another clinical correlation result diagram of the kit for quantitative detection of five hypertension items of the present application; AngII clinical correlation is as follows Figure 8 As shown, Figure 8 This is another clinical relevance result diagram of the kit for quantitatively detecting five items of hypertension of the present application.

[0085] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0086] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A combined test strip for quantitatively detecting five items of hypertension, characterized in that: The joint test paper comprises a PVC base plate and a sample pad, a binding pad, a coating film and a water-absorbing pad which are sequentially connected on the PVC base plate; Wherein, the conjugate pad is coupled with fluorescent microsphere-labeled mouse anti-human ACTH antibody, fluorescent microsphere-labeled COR antibody, fluorescent microsphere-labeled ALD antibody, fluorescent microsphere-labeled REN antibody, fluorescent microsphere-labeled AngII antibody and fluorescent microsphere-labeled first antibody; The coating membrane is provided with a first detection line coated with ACTH monoclonal antibody, a second detection line coated with COR coupled with BSA antigen, a third detection line coated with ALD coupled with BSA antigen, a fourth detection line coated with REN monoclonal antibody, a fifth detection line coated with AngII monoclonal antibody and a quality control line coated with the second antibody.

2. The combined test paper for quantitatively detecting five items of hypertension according to claim 1, characterized in that: The first antibody is a goat anti-chicken IgY antibody; and / or The second antibody is chicken IgY antibody.

3. The combined test paper for quantitatively detecting five items of hypertension according to claim 1, characterized in that: The coating concentrations of the ACTH monoclonal antibody of the first detection line, the COR-coupled BSA antigen of the second detection line, the ALD-coupled BSA antigen of the third detection line, the REN monoclonal antibody of the fourth detection line, the AngII monoclonal antibody of the fifth detection line and the second antibody of the quality control line are all 1-1.5 mg / mL.

4. The combined test paper for quantitatively detecting five items of hypertension according to claim 1, characterized in that: The fluorescent microspheres include europium time-resolved fluorescent microspheres.

5. The combined test paper for quantitatively detecting five items of hypertension according to claim 1, characterized in that: The sample pad is prepared by the following process: The glass cellulose membrane is soaked in the sample pad treatment solution and dried at 37-50° C. to obtain the sample pad.

6. The combined test strip for quantitatively detecting five items of hypertension according to claim 5, characterized in that: Measured by mass concentration, the sample pad treatment solution includes 0.02M phosphate buffer with a pH of 7.4, 0.5% to 1% bovine serum albumin, 0.1% Tween 80 and 3% to 5% sucrose.

7. The combined test strip for quantitatively detecting five items of hypertension according to claim 1, characterized in that: The conjugate pad is prepared by the following method: Prepare fluorescent microsphere-labeled anti-ACTH, fluorescent microsphere-labeled COR, fluorescent microsphere-labeled ALD, fluorescent microsphere-labeled REN, fluorescent microsphere-labeled AngII antibody, and fluorescent microsphere-labeled primary antibody respectively; The fluorescent microsphere-labeled anti-ACTH, fluorescent microsphere-labeled COR, fluorescent microsphere-labeled ALD, fluorescent microsphere-labeled REN, fluorescent microsphere-labeled AngII antibody, and fluorescent microsphere-labeled first antibody are mixed in a volume ratio of 2:2:2:2:2:1 to obtain a mixed antibody solution; The mixed antibody solution is sprayed on the initial conjugate pad at a spray rate of 3.0-4.0 μL / cm, and after drying, the conjugate pad is obtained.

8. A kit for quantitatively detecting five items of hypertension, characterized in that: The test kit comprises a detection card, wherein the detection card comprises a fluorescent card shell and a combined test strip according to any one of claims 1 to 7, wherein the combined test strip is arranged in the fluorescent card shell.

9. The kit for quantitatively detecting five items of hypertension according to claim 8, characterized in that: The kit also includes a diluent and a chip; The diluent includes 0.5% Tween-20 and 0.01M pH 7.4 PBS buffer solution in terms of mass concentration.

10. A method for quantitatively detecting five items of hypertension using the kit according to any one of claims 8 to 9, characterized in that: The steps include: S1: Mix the sample to be tested and the diluent evenly, add them to the sample well of the test card, and let them stand for treatment; S2: Put the test card into the test instrument to obtain the output test result.

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