Hypertension multi-item combined determination kit

Abstract

The invention provides a multi-item combined quantitative detection method for five hypertension markers, namely aldosterone (ALD), cortisol, adrenocorticotropic hormone (ACTH), renin (Renin) and angiotensin II (Angiotensin II, A II), and the detection method is composed of a test card, an IC (Integrated Circuit) card, a sample diluent and an operating instruction. The invention further provides a method for the multi-item combined quantitative detection of the five hypertension markers, namely the aldosterone (ALD), the cortisol, the adrenocorticotropic hormone (ACTH), the renin (Renin) and the angiotensin II (AII). The sample diluent comprises a Tris-HCl buffer solution, bovine serum albumin and a dissociation reagent; the test card consists of a test strip and a plastic card shell, wherein the main components on the test strip are as follows: a) monoclonal antibodies of five items of hypertension (Cortisol, ACTH and Renin) and micromolecular antigens (ALD and A II) coupled with BSA (bovine serum albumin) are sequentially fixed on a nitrocellulose membrane respectively; b) a rabbit anti-chicken IgY polyclonal antibody fixed on the nitrocellulose membrane; and c) adding a fluorescence-labeled monoclonal antibody of five hypertension markers and a fluorescence-labeled chicken IgY antibody on the pretreated glass fiber.

Description

Technical Field

[0001] The invention belongs to the fields of biology and medicine, and in particular relates to a hypertension multiple combined determination kit. Background Art

[0002] Hypertension is the most common cardiovascular disease in humans. It is an important risk factor for coronary heart disease, myocardial infarction, stroke, renal insufficiency and even death, with high disability and mortality rates. Domestic and foreign practices have proved that hypertension is a disease that can be prevented and controlled. Lowering the blood pressure level of hypertensive patients can significantly reduce stroke and heart disease events, significantly improve the quality of life of patients, and effectively reduce the burden of disease. Hypertension is divided into two categories: primary and secondary. In the vast majority of patients, the cause of hypertension is unclear, and the main manifestation is non-specific continuous increase in blood pressure, which is called primary hypertension, accounting for more than 90% of all hypertensive patients; in less than 10% of patients, increased blood pressure is a clinical manifestation of certain diseases, which has a clear and independent cause, called secondary hypertension.

[0003] In clinical practice, in addition to asking patients about their medical history in detail, a comprehensive physical examination will be conducted for patients with hypertension, especially some auxiliary examinations, the purpose of which is to identify the cause of abnormally high blood pressure and to distinguish between primary and secondary hypertension. The European Society of Hypertension and the European Society of Cardiology pointed out that all hypertensive people should undergo a simple secondary hypertension screening. Plasma aldosterone (ALD), cortisol, adrenocorticotrophic hormone (ACTH), renin, and angiotensin Ⅱ (AⅡ) are of great significance for the identification of secondary hypertension and the classification diagnosis and treatment of primary hypertension. They can also be used to perform joint detection and analysis of the renin-angiotensin-aldosterone system (RAAS) and the hypothalamus-pituitary gland-adrenal cortex (HPA) axis to differentially diagnose various diseases caused by abnormal RAAS system and HPA axis. Given the importance of the five hypertension tests in clinical applications, the reliability of their test results has received increasing attention in clinics and laboratories.

[0004] Serum cortisol is the most important glucocorticoid synthesized and secreted by the zona fasciculata of the adrenal cortex, and its secretion is regulated by the hypothalamus-pituitary-adrenal (HPA) axis. Cortisol is the main information factor of the HPA axis function, and its level changes rhythmically in the human body. When the HPA axis regulatory function is disordered, the cortisol level will change and reversely affect the regulatory function of the HPA axis. There is a certain connection between increased cortisol and blood pressure, and high levels of serum cortisol can lead to hypertension. ACTH is secreted by the anterior pituitary gland. Studies have shown that increased ACTH levels can cause ectopic ACTH syndrome and cause symptoms such as hypertension. When the body is stressed, corticotropin-releasing hormone (CRH) is released through the hypothalamus-pituitary-adrenal (HPA) axis, which causes the pituitary to release adrenocorticotropic hormone (ACTH). ACTH acts on the adrenal cortex to release glucocorticoids. Glucocorticoids feedback inhibit the release of peptide hormones from the hypothalamus and pituitary to achieve a self-stabilizing effect. Disorders of the HPA axis can lead to adrenal diseases such as hypercortisolism, pheochromocytoma, primary aldosteronism, and congenital adrenal hyperplasia, causing secondary hypertension. Therefore, detection of the HPA axis is of great significance for the diagnosis of secondary hypertension.

[0005] The renin-angiotensin-aldosterone system (RAAS) is a group of hormones and receptors that interact and regulate each other secreted by the kidneys and liver. It plays a key role in the regulation of blood pressure and is of great significance to the occurrence and development of hypertension. Renin acts on angiotensinogen to produce angiotensin I, and angiotensin I is hydrolyzed by angiotensin converting enzyme to produce angiotensin II. Angiotensin II causes hypertension by promoting vasoconstriction and the secretion of aldosterone by the adrenal cortex.

[0006] Renin, angiotensin, and aldosterone are a connected system that regulates blood pressure, water, and electrolyte balance by controlling blood volume and peripheral resistance to maintain a constant internal environment. When blood pressure drops, the kidneys begin to secrete renin, which can convert angiotensinogen into angiotensin I and generate angiotensin II under the action of angiotensin converting enzyme. Angiotensin II causes blood vessels to constrict, leading to increased blood pressure. It can also act as an effector molecule to stimulate the release of aldosterone, which has the function of retaining sodium and excreting potassium, and can cause increased blood pressure, leading to hypertension. Therefore, the detection of RAAS indicators is of great significance for the diagnosis, classification, and guidance of medication of hypertension. Summary of the invention

[0007] The purpose of the present invention is to provide a kit for the combined determination of five hypertension items, namely, plasma aldosterone (ALD), cortisol (Cortisol), adrenocorticotrophic hormone (ACTH), renin (renin), and angiotensin Ⅱ (AⅡ).

[0008] In order to achieve the above object, the present invention provides the following technical solutions: A multiple combined determination kit for hypertension, the kit consisting of a test card, an IC card, and a sample diluent; Sample diluents include Tris-HCl buffer, bovine serum albumin, and dissociation reagents; The test card consists of a test strip and a plastic card shell. The main components on the test strip are: a) Monoclonal antibodies and small molecule antigens for five hypertension items, fixed on nitrocellulose membrane; b) rabbit anti-chicken IgY polyclonal antibody, immobilized on nitrocellulose membrane; c) Five fluorescently labeled monoclonal antibodies against hypertension, fixed on glass fibers; d) Fluorescently labeled chicken IgY antibody, immobilized on glass fiber.

[0009] Furthermore, the monoclonal antibodies and small molecule antigens fixed on the nitrocellulose membrane are Cor mouse monoclonal antibody, ACTH mouse monoclonal antibody, Rein mouse monoclonal antibody, and small molecule antigens of ALD and AⅡ coupled with bovine serum albumin (BSA) (BSA-ALD and BSA-AⅡ). The Cor mouse monoclonal antibody, ACTH mouse monoclonal antibody, and Rein mouse monoclonal antibody can specifically capture Cor, ACTH, and Rein antigens in the sample to be tested, respectively. The small molecule antigens of ALD and AⅡ coupled with bovine serum albumin (BSA) can specifically compete with ALD and AⅡ in the sample to be tested to bind to the fluorescently labeled ALD and AⅡ monoclonal antibodies. The order of them on the nitrocellulose membrane is Cor mouse monoclonal antibody, BSA-AⅡ, ACTH mouse monoclonal antibody, BSA-ALD, and Rein mouse monoclonal antibody, which are 3-5 mm apart from each other. Further, the rabbit anti-chicken IgY polyclonal antibody is fixed on a nitrocellulose membrane, and can specifically capture fluorescently labeled chicken IgY at 3-5 mm behind the position of the Rein mouse monoclonal antibody as a common reference quality control line. Further, the preparation method of the fluorescent labeled antibody is: NHS activated biotin is dissolved in anhydrous DMF organic solvent, reacted with the amino group on the antibody in a PBS buffer system to generate a biotin-coupled labeled monoclonal antibody, and then stirred and mixed with a fluorescent protein with streptavidin fusion expression in a PBS solution containing BSA at room temperature for 10 minutes to obtain antibody protein fluorescent particles. Biotin is added to terminate the reaction, and the excess biotin is removed by dialysis 3 times. The fluorescent substance labeled with the antibody can also be fluorescent microspheres, quantum dots, time-resolved fluorescent microspheres and other fluorescent substances. The pH of the PBS reaction system is 6.5-8.0, and the BSA concentration is 0.5%-5.0%. Furthermore, the molar ratio of NHS-activated biotin to monoclonal antibody is 4-40:1, and the reaction ratio of biotin-labeled antibody to streptavidin fluorescent protein is 1:2-10. Furthermore, the five fluorescent-labeled antibodies ALD, Cor, ACTH, Renin, and AⅡ are mixed with fluorescent-labeled chicken IgY antibodies at a concentration of 0.05 mg / mL-0.5 mg / mL and sprayed on a pretreated glass fiber mat. Furthermore, the sample diluent is mainly composed of Tris-HCl buffer, bovine serum albumin, etc. as stabilizers and blocking agents. The immune reaction pH environment is adjusted by Tris-HCl. The pH is 5.5-8.4. The reactants are evenly dispersed by a surfactant to promote the reaction, and the surfactant is Tween-20 with a concentration of 0.05%-0.3% (v / v).Proteins and sugars are used as blocking agents and stabilizers to block non-specific binding reactions. The proteins may be bovine serum albumin, casein, etc., with a concentration of 0.5%-5.0% (m / v). Sugars may be sucrose, trehalose, etc., with a concentration of 0.3%-4.0% (m / v). The dissociation reagent is a structural analogue with a stronger affinity for glucocorticoid binding protein than Cortisol, and may include one or more of progesterone, ANS salt, etc., for competitively dissociating Cortisol from glucocorticoid binding protein in the sample, and by adjusting the binding force of its analogues with glucocorticoid binding protein to the optimal conditions, the cortisol in the sample is completely released, and the cross-reaction of the five markers on the same test strip is minimized.

[0010] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: the kit of the present invention utilizes lateral flow chromatography technology and immunofluorescence labeling technology, and adopts the double antibody sandwich method and competition method principle for detection. Under the action of the excitation light source, the fluorescent substance emits a fluorescent signal of a specific wavelength, and the fluorescent immunoassay analyzer captures the fluorescent signal, which is automatically converted into a quantitative value through signal conversion and a set calibration curve, and the concentrations of five hypertension markers in the sample, namely, plasma aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II, are calculated. The accuracy and sensitivity of the kit can be improved by adding a dissociation reagent to the sample diluent. The detection method is unique, portable, fast, and can produce results in only 10-15 minutes, with high specificity. The combined detection of plasma aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II has important clinical value for identifying the causes of abnormally elevated blood pressure, distinguishing whether hypertension is primary or secondary, and guiding medication, and has great market prospects.

[0011] However, the current relevant detection methods are mainly based on chemiluminescence immunoassay and liquid chromatography-tandem mass spectrometry, which have problems such as complex operation, high cost, slow speed, or require an indicator for analysis during detection and analysis.

[0012] For example, the method and kit for simultaneously detecting aldosterone and renin activity in blood samples invented by Feng Zhen et al. CN13049697 A can simultaneously detect aldosterone and renin activity. The method for simultaneously detecting multiple typical biomarkers related to elevated blood pressure invented by Tan Xiaojie CN115541741 A successfully achieved the purpose of simultaneously detecting 10 biomarkers related to elevated blood pressure, including aldosterone and cortisol, which improved the efficiency of analyzing the causes of elevated blood pressure, solved the problems of throughput and efficiency, and greatly simplified the operation process. The screening kit and diagnosis and typing diagnosis system for primary aldosteronism invented by Liu Pengyun et al. CN 114895043 A use magnetic beads with surface-bonded hydrophilic-lipophilic balanced polymers for sample pretreatment, optimize the process conditions, and then accurately detect the contents of five markers in the sample, including aldosterone, angiotensin I, angiotensin II, cortisol, and 18-hydroxycorticosterone, at one time by liquid chromatography-tandem mass spectrometry. These tests all use liquid chromatography-tandem mass spectrometry to optimize the detection, but the overall operation requirements are high, and there are problems such as complex operation, higher requirements for clinical use and long detection time. The adrenocorticotropic hormone chemiluminescence detection kit CN108548930 B invented by Huang Yanling et al. uses a double antibody method of anti-adrenocorticotropic hormone monoclonal antibodies coated with magnetic particles and labeled with acridinium ester for detection. And Tan Zishen disclosed a chemiluminescent immunoassay kit CN 117031011 A, which is based on an improved double antibody sandwich method for the detection of antigens of small molecule compounds, coupling the first antibody to magnetic beads and the second antibody to the chemiluminescent agent acridinium ester. The chemiluminescence detection method has the characteristics of high sensitivity and good stability, but it also has the problems of expensive instruments and equipment, complex operation, long time and intelligent detection of one indicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the structure of five markers of hypertension.

[0014] Figure 2 The scatter plots and conversion calibration curves are for the five marker concentrations and T / C.

[0015] Figure 3 The correlation between Example 1 and the commercial chemiluminescent kit and chemiluminescence. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Example 1 This embodiment provides a multiple combined determination kit for five hypertension markers, namely, aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II, the kit comprising a test card, an IC card, and a sample diluent; The main components of the sample diluent are: 0.1MTris-HCl buffer, 0.5% bovine serum albumin, 100μg / mL progesterone, 0.2% ANS salt, 0.05% proclin 300, and the buffer pH is 7.4.

[0018] Antibody coating: Cor mouse monoclonal antibody at a concentration of 1.0 mg / mL, BSA-AⅡ at a concentration of 0.5 mg / mL, ACTH mouse monoclonal antibody at a concentration of 1.0 mg / mL, BSA-ALD at a concentration of 0.2 mg / mL, and Rein mouse monoclonal antibody at a concentration of 1.0 mg / mL were fixed on nitrocellulose by a spray-stripping instrument, with an interval of 3-5 mm (e.g. Figure 1 As shown), the coating solution was 0.05 M CBS, pH was 9.6, the streaking liquid speed was 1 μL / cm, and it was dried in an oven at 50°C for 5 hours.

[0019] Preparation of antibody fluorescent protein labeling: The molar ratio of NHS-activated biotin to monoclonal antibody is 20:1, the reaction buffer is 0.05M PBS buffer at pH 7.0, the reaction is carried out at 30°C for 30 minutes, and the reaction is terminated by adding 1mM NH4Cl for 10 minutes. The reaction ratio of biotin-labeled antibody to streptavidin fluorescent protein is 1:2 for Cor, 1:2 for AⅡ, 1:4 for ACTH, 1:2 for ALD, and 1:4 for Renin. Fluorescently labeled Cor monoclonal antibody was diluted to a final concentration of 0.3 mg / mL with 0.1 M Tris-HCl buffer and 0.5% bovine serum albumin to a final concentration of 0.3 mg / mL, AⅡ monoclonal antibody to a final concentration of 0.3 mg / mL, ACTH monoclonal antibody to a final concentration of 0.4 mg / mL, ALD monoclonal antibody to a final concentration of 0.3 mg / mL, Renin monoclonal antibody to a final concentration of 0.4 mg / mL, and chicken IgY concentration of 0.2 mg / mL, and then sprayed onto a 7 mm wide conjugate pad at a speed of 3 μL / cm using a spray film streak instrument and dried in an oven at 37°C for 16 hours.

[0020] Preparation of test strips: Place the nitrocellulose membrane coated with antibodies on the conjugate pad and sample pad absorbent paper. Figure 1 The test paper is bonded to a low-viscosity backing plate in the order shown, cut into 4 mm wide test strips by a strip cutter, and assembled in a plastic card shell to obtain a test card.

[0021] Example 2 This embodiment provides a multiple combined determination kit for five hypertension markers, namely, aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II, the kit comprising a test card, an IC card, and a sample diluent; The main components of the sample diluent are: 0.1M Tris-HCl buffer, 0.5% bovine serum albumin, 100μg / mL progesterone, 0.2% ANS salt, 0.05% proclin 300, and the buffer pH is 7.4.

[0022] Antibody coating: Cor mouse monoclonal antibody at a concentration of 0.5 mg / mL, BSA-AⅡ at a concentration of 0.2 mg / mL, ACTH mouse monoclonal antibody at a concentration of 1.0 mg / mL, BSA-ALD at a concentration of 0.2 mg / mL, and Rein mouse monoclonal antibody at a concentration of 1.0 mg / mL were fixed on nitrocellulose by a spray-stripping instrument, with a spacing of about 4 mm (e.g. Figure 1 As shown), the coating solution was 0.05 M CBS, pH was 9.6, the streaking liquid speed was 1 μL / cm, and it was dried in an oven at 50°C for 5 hours.

[0023] Preparation of antibody fluorescent protein labeling: The molar ratio of NHS-activated biotin to monoclonal antibody is 4:1, the reaction buffer is 0.05M PBS buffer at pH 7.0, the reaction is carried out at 30°C for 30 minutes, and the reaction is terminated by adding 1mM NH4Cl for 10 minutes. The reaction ratio of biotin-labeled antibody to streptavidin fluorescent protein is 1:2 for Cor, 1:2 for AⅡ, 1:4 for ACTH, 1:2 for ALD, and 1:4 for Renin. Fluorescently labeled Cor monoclonal antibody was diluted to a final concentration of 0.3 mg / mL with 0.1 M Tris-HCl buffer and 0.5% bovine serum albumin to a final concentration of 0.3 mg / mL, AⅡ monoclonal antibody to a final concentration of 0.3 mg / mL, ACTH monoclonal antibody to a final concentration of 0.4 mg / mL, ALD monoclonal antibody to a final concentration of 0.3 mg / mL, Renin monoclonal antibody to a final concentration of 0.4 mg / mL, and chicken IgY concentration of 0.2 mg / mL, and then sprayed onto a 7 mm wide conjugate pad at a speed of 3 μL / cm using a spray film streak instrument and dried in an oven at 37°C for 16 hours.

[0024] Preparation of test strips: Place the nitrocellulose membrane coated with antibodies on the conjugate pad and sample pad absorbent paper. Figure 1 The test paper is bonded to a low-viscosity backing plate in the order shown, cut into 4 mm wide test strips by a strip cutter, and assembled in a plastic card shell to obtain a test card.

[0025] Example 3 This embodiment provides a multiple combined determination kit for five hypertension markers, namely, aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II, the kit comprising a test card, an IC card, and a sample diluent; The main components of the sample diluent are: 0.1M Tris-HCl buffer, 0.5wt% bovine serum albumin, 100μg / mL progesterone, 0.2% ANS salt, 0.05% proclin 300, and the buffer pH is 7.4.

[0026] Antibody coating: Cor mouse monoclonal antibody at a concentration of 1.5 mg / mL, BSA-AⅡ at a concentration of 0.05 mg / mL, ACTH mouse monoclonal antibody at a concentration of 1.0 mg / mL, BSA-ALD at a concentration of 0.1 mg / mL, and Rein mouse monoclonal antibody at a concentration of 1.0 mg / mL were fixed on nitrocellulose by a spray-stripping instrument, with an interval of about 4 mm (e.g. Figure 1 As shown), the coating solution was 0.05 M CBS, pH was 9.6, the streaking liquid speed was 1 μL / cm, and it was dried in an oven at 50°C for 5 hours.

[0027] Preparation of antibody fluorescent protein labeling: The molar ratio of NHS-activated biotin to monoclonal antibody is 10:1, the reaction buffer is 0.05M PBS buffer at pH 7.0, the reaction is carried out at 30°C for 30 minutes, and the reaction is terminated by adding 1mM NH4Cl for 10 minutes. The reaction ratio of biotin-labeled antibody to streptavidin fluorescent protein is 1:4 for Cor, 1:4 for AⅡ, 1:6 for ACTH, 1:4 for ALD, and 1:6 for Renin. Fluorescently labeled Cor monoclonal antibody was diluted to a final concentration of 0.3 mg / mL with 0.1 M Tris-HCl buffer and 0.5% bovine serum albumin to a final concentration of 0.3 mg / mL, AⅡ monoclonal antibody to a final concentration of 0.3 mg / mL, ACTH monoclonal antibody to a final concentration of 0.4 mg / mL, ALD monoclonal antibody to a final concentration of 0.3 mg / mL, Renin monoclonal antibody to a final concentration of 0.4 mg / mL, and chicken IgY concentration of 0.2 mg / mL. The mixture was sprayed onto a 7 mm wide conjugate pad at a speed of 3 μL / cm using a spray film streak instrument and dried in an oven at 37°C for 16 hours.

[0028] Preparation of test strips: Place the nitrocellulose membrane coated with antibodies on the conjugate pad and sample pad absorbent paper. Figure 1 The test paper is bonded to a low-viscosity backing plate in the order shown, cut into 4 mm wide test strips by a strip cutter, and assembled in a plastic card shell to obtain a test card.

[0029] Example 4 This embodiment provides a process scheme for preparing a kit for the combined determination of five hypertension markers, namely, aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II. The kit consists of a test card, an IC card, and a sample diluent. The main components of the sample diluent are: 0.1M Tris-HCl buffer, 0.5% bovine serum albumin, 50μg / mL progesterone, 0.1% ANS salt, 0.05% proclin 300, and the buffer pH is 7.4.

[0030] Antibody coating: Cor mouse monoclonal antibody at a concentration of 0.5 mg / mL, BSA-AⅡ at a concentration of 0.05 mg / mL, ACTH mouse monoclonal antibody at a concentration of 1.0 mg / mL, BSA-ALD at a concentration of 0.1 mg / mL, and Rein mouse monoclonal antibody at a concentration of 1.0 mg / mL were fixed on nitrocellulose by a spray-stripping instrument, with an interval of about 4 mm (e.g. Figure 1 As shown), the coating solution was 0.05 M CBS, pH was 9.6, the streaking liquid speed was 1 μL / cm, and it was dried in an oven at 50°C for 5 hours.

[0031] Preparation of antibody fluorescent protein labeling: The molar ratio of NHS-activated biotin to monoclonal antibody is 20:1, the reaction buffer is 0.05M PBS buffer at pH 7.0, the reaction is carried out at 30°C for 30 min, and the reaction is terminated by adding 1mM NH4Cl for 10 min. The reaction ratio of biotin-labeled antibody to streptavidin fluorescent protein is 1:8 for Cor, 1:8 for AⅡ, 1:10 for ACTH, 1:8 for ALD, and 1:10 for Renin. Fluorescently labeled Cor monoclonal antibody was diluted to a final concentration of 0.2 mg / mL with 0.1 M Tris-HCl buffer and 0.5% bovine serum albumin to a final concentration of 0.2 mg / mL, AⅡ monoclonal antibody to a final concentration of 0.2 mg / mL, ACTH monoclonal antibody to a final concentration of 0.3 mg / mL, ALD monoclonal antibody to a final concentration of 0.2 mg / mL, Renin monoclonal antibody to a final concentration of 0.3 mg / mL, and chicken IgY concentration of 0.2 mg / mL. The mixture was sprayed onto a 7 mm wide conjugate pad at a speed of 3 μL / cm using a spray film streak instrument and dried in an oven at 37°C for 16 hours.

[0032] Preparation of reinforcement pad: Based on dense glass fiber pad, with 0.01M PBS buffer pH 7.4, add 0.50% BSA, 0.05% Tween 20, 2% D-trehalose, 0.1% dextran 20,000, 0.25% PVP 40K, 0.3% HPMC for immersion treatment for 5 minutes, dry at 37 degrees overnight and cut into 7mm The 300mm pad is assembled on the bottom plate and overlaps the nitrocellulose membrane by 2mm, which increases the reaction uniformity and enhances the fluorescence intensity during the reaction.

[0033] Test card preparation: Place the antibody-coated nitrocellulose membrane on the conjugate pad and sample pad absorbent paper. Figure 1 The test paper is bonded to a low-viscosity backing plate in the order shown, cut into 4 mm wide test strips by a strip cutter, and assembled in a plastic card shell to obtain a test card.

[0034] Example 5 This implementation case provides five marker single detection schemes as control tests for the five joint tests, with implementation case 1 as the parameters. For control case 1, Cor is a competitive method, coated with BSA-Cor antigen, the concentration is 0.1 mg / mL, the fluorescent labeled antibody is consistent with that in implementation case 1, and the concentration of the fixed quality control line on the nitrocellulose membrane is consistent with that in implementation case 1. The parameters of control case 2 AII, control case 3 ACTH, control case 4 ALD, and control case 5 Rein are consistent with those of implementation case 1, except that the detection line is only drawn in the middle of the nitrocellulose membrane for one case marker antibody or antigen, that is, the structure is similar. Figure 1 As shown, all control case NC membranes used a 2.5 cm width.

[0035] Example 6 This implementation case calibrates the multiple combined determination kit for five hypertension markers, including aldosterone, cortisol, adrenocorticotropic hormone, renin, and angiotensin II, used in Implementation Case 1, and performs performance tests such as sensitivity, precision, and clinical relevance.

[0036] Using the fluorescent immunoassay analyzer FIA-T-01 of Guangzhou Tianbao Songyuan Biotechnology Development Co., Ltd. as the detection instrument, the calibrator and sample of the kit of implementation case 1 were tested. The test steps are as follows: Take 100μL of sample (or calibration quality control) and add it to 100μL of diluent, mix it by pipetting 10 times, and take 100μL of the mixture and add it to the sample well of the test card. Use FIA-T-01 as the detector to read the fluorescence signal value, calculate the T / C value of each of the five items, and indicate the concentration of the analyte (aldosterone, cortisol, adrenocorticotropic hormone, renin, angiotensin II) in the sample. Cortisol, adrenocorticotropic hormone, and renin are fitted with a quadratic curve, and aldosterone and angiotensin II are fitted with a quadratic curve through reciprocal data conversion; the standard curve is written into the IC card, and the standard curve information in the IC card is read by the FIA-T-01 detector.

[0037] Analytical sensitivity testing: The multiple combined tests of the present invention can realize the simultaneous detection of 5 indicators at one time, and the analytical sensitivity is tested in Example 1 and control cases 1-5 respectively. The calibration parameters are written into the IC card by standard curve fitting calibration. After the IC card information is read in the instrument, the blank calibration product is used as a sample for detection. The detection results can be obtained according to the regression fitting equation. The control case and the implementation case are repeated 20 times. The average value (AVE) and standard deviation (SD) are calculated to obtain the analytical sensitivity (blank limit), that is, AVE+2SD. The specific results are shown in Table 1.

[0038] Table 1 Analytical sensitivity results

[0039] Precision (CV) test: The multiple combined tests of the present invention can detect five indicators at a time. The analytical sensitivity is tested in Example 1 and control cases 1-5 respectively. The calibration parameters are written into the IC card by standard curve fitting calibration. After the IC card information is read in the instrument, the low, medium and high concentration samples of five markers Cor, AII, ACTH, ALD and Rein are used for detection. The test results can be obtained according to the regression fitting equation. The control case and the implementation case are repeated 10 times. The average value (AVE) and standard deviation (SD) are calculated, and CV) = SD / AVE The precision is calculated by 100%, and the specific results are shown in Table 2-1, Table 2-2 and Table 2-3.

[0040] Table 2-1 Low concentration precision results

[0041] Table 2-2 Medium concentration precision results

[0042] Table 2-3 High concentration precision results

[0043] Clinical Methodology Comparison Test: A comparative analysis was conducted on Example 1 and a commercial chemiluminescent reagent. 30, 31, 29, 45 and 44 clinical samples of Cor, AII, ACTH, ALD and Rein were taken and tested simultaneously by the two methods. The test results were as follows: Figure 3 A and Figure 3 B.

Claims

1. A multiple combined determination kit for hypertension, characterized in that: The kit consists of a test card, an IC card, and sample dilution; Sample diluents included Tris-HCl buffer, bovine serum albumin, and dissociation reagent; The test card consists of a test strip and a plastic card shell. The main components on the test strip are: a) Monoclonal antibodies and small molecule antigens for five hypertension items, fixed on nitrocellulose membrane; b) rabbit anti-chicken IgY polyclonal antibody, immobilized on nitrocellulose membrane; c) Five fluorescently labeled monoclonal antibodies against hypertension, immobilized on pretreated glass fibers; d) Fluorescently labeled chicken IgY antibody, immobilized on pre-treated glass fiber.

2. The hypertension multiple combined determination kit according to claim 1, characterized in that: The dissociation reagent is a structural analogue with stronger affinity to glucocorticoid binding protein than Cortisol, and may include one or more of progesterone, ANS salt, etc., for competitively dissociating Cortisol from glucocorticoid binding protein in the sample. Tris-HCl buffer, bovine serum albumin, etc. are used as stabilizers and blocking agents, and the binding force between the analogue and glucocorticoid binding protein is adjusted to the optimal condition, so that the cross-interference is reduced when the five markers are detected simultaneously on a test strip.

3. The hypertension multiple combined determination kit according to claim 1, characterized in that: The nitrocellulose membrane on the test strip is coated and fixed with Cortisol, ACTH, Renin monoclonal antibodies and ALD and AⅡ small molecule antigens coupled with BSA in a forward and backward order. ACTH and Renin monoclonal antibodies are used to capture the Cortisol, ACTH, Renin molecules to be tested in the sample and form a sandwich reaction with the Cortisol, ACTH, Renin fluorescently labeled monoclonal antibodies released and labeled on the glass fiber, while the ALD and AⅡ small molecule antigens coupled with BSA can compete with the ALD and AⅡ small molecule test substances in the sample to bind to the fluorescently labeled antibodies on the glass fiber mat. The Cortisol fluorescently labeled antibodies on the glass fiber have no cross reaction with the analogs in the dissociation agent.

4. The hypertension multiple combined determination kit according to claim 1, characterized in that: The preparation method of the fluorescent substance is as follows: NHS activated biotin is dissolved in anhydrous DMF organic solvent, combined with the amino group on the antibody in a PBS buffer system, and the fluorescent protein expressed by streptavidin fusion is stirred and mixed in a PBS solution containing BSA at room temperature for 10 minutes to obtain antibody protein fluorescent particles. The fluorescent substance labeled with the antibody can also be fluorescent microspheres, quantum dots, time-resolved fluorescent microspheres, etc.

5. The hypertension multiple combined determination kit according to claim 4, characterized in that: The molar ratio of biotin-labeled antibody to fluorescent protein fused with streptavidin is 1:2-10. At the end of the reaction, 0.1-10 mM excess biotin is added for pre-blocking and termination of the reaction.

6. The hypertension multiple combined determination kit according to claim 3, characterized in that: The detection method combines the Cortisol, ACTH, Renin double antibody sandwich method and the ALD, AⅡ competition method on the same test strip, calibrates different reaction modes through the IC card standard curve, and fixes the rabbit anti-chicken IgY polyclonal antibody on the nitrocellulose membrane as an independent internal reference quality control line. Compared with the traditional sheep anti-mouse reference quality control line, it can better eliminate the changes in the reference quality control value caused by the changes in the concentration of different markers in multiple joint tests. The competition method can also use the small molecule double antibody sandwich method. The rabbit anti-chicken IgY polyclonal antibody quality control line can also use the same type of DNP antibody as an independent quality control to control the difference in the influence of the concentration difference of different items on the reference line.

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