A combined diagnostic model for acute allergic respiratory diseases and a detection product

Abstract

The application discloses a combined detection model and product of acute allergic respiratory diseases, and belongs to the fields of biological medicine and medicine. The application relates to application of substances for detecting trypsin-like enzyme concentration and substances for detecting eosinophil cationic protein concentration in preparation of products for diagnosing or assisting in diagnosing acute allergic respiratory diseases. The application detects trypsin-like enzyme concentration and eosinophil cationic protein concentration, and whether it is or is a candidate of "acute allergic respiratory diseases" is determined through calculation of a combined detection diagnosis prediction model. Compared with a single item diagnosis model, the diagnosis efficiency is significantly improved. Whether it is or is a candidate of "acute allergic respiratory diseases" is determined through diagnosis of a concentration change diagnosis model. Compared with a single time point diagnosis model, the diagnosis efficiency is significantly improved.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and medicine, specifically relating to a combined diagnostic model and detection product for acute allergic respiratory diseases. Background Technology

[0002] Allergic diseases are common worldwide, including allergic rhinitis, asthma, atopic dermatitis, and food allergies, which seriously affect patients' quality of life. Acute allergic diseases, in particular, can be life-threatening. Currently, the diagnosis of allergic diseases mainly involves three approaches: taking a medical history, in vivo testing (including skin prick tests, intradermal tests, and provocation tests), and in vitro testing (including total IgE and allergen-specific IgE).

[0003] In vivo testing carries certain risks. For patients with severe allergies, it may trigger systemic allergic reactions, such as anaphylactic shock, which can be life-threatening. It is also unsuitable for patients in the acute phase of an allergic reaction or with serious underlying diseases. Furthermore, test results are easily affected by various factors. For example, recent use of antihistamines or corticosteroids can suppress the immune response, leading to false negatives. Inflammation or skin damage can also affect the accuracy of the test.

[0004] The mainstream methods for in vitro diagnostics are the detection of total IgE and allergen-specific IgE. Total IgE is an immunoglobulin, and elevated levels of total IgE antibodies are common in patients with allergic diseases. Early studies considered total IgE levels the simplest way to identify such patients, but subsequent research found that only about 60% of patients had total IgE antibody levels above the normal range, and there was significant crossover between serum total IgE antibody levels in healthy individuals and patients with allergic diseases. Currently, total IgE testing is mostly used to preliminarily determine whether a patient has an allergic constitution. Allergen-specific IgE, on the other hand, can clearly identify a patient's sensitization to a specific allergen. Although in vitro testing is unaffected by medications, has no requirements on skin conditions, and is suitable for all types of patients, including those who cannot undergo in vivo testing, both total IgE and allergen-specific IgE tests have significant limitations. They can only assess whether a patient is sensitized to a certain allergen, but cannot definitively determine whether the patient is currently experiencing an allergic reaction. For example, a patient may have been sensitized for many years but has not recently been exposed to the allergen. In this case, tests for total IgE and allergen-specific IgE may still be positive, but this does not necessarily indicate an immediate allergic reaction. Furthermore, because IgE-mediated allergic reactions are only one part of allergic diseases, many allergic reactions are not mediated by IgE but through other mechanisms, such as the activation of mast cells and eosinophils via non-IgE receptors. Therefore, current in vitro detection methods cannot fully cover these non-IgE-mediated allergic reactions, leading to many allergy patients not receiving an accurate diagnosis.

[0005] Traditional in vivo and in vitro diagnostic methods have significant limitations and unmet needs in diagnosing allergic reactions. For example, the golden window for treating severe allergic reactions (such as anaphylactic shock) is from minutes to hours after onset, but traditional in vivo and in vitro diagnostic methods cannot provide rapid diagnostic evidence in emergency situations. Furthermore, patients with chronic allergic diseases (such as asthma and eczema) may experience both long-term sensitization and occasional acute attacks; traditional in vivo and in vitro diagnostic methods cannot distinguish between acute reactions and chronic inflammation to guide treatment. In clinical practice, there is an urgent need for an in vitro diagnostic method that can rapidly and accurately diagnose the state of an allergic reaction (especially acute allergies) to guide timely and effective treatment. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a combined diagnostic model and detection product for acute allergic respiratory diseases.

[0007] Tryptase is a serine protease primarily stored in the secretory granules of mast cells. During an allergic reaction, when an allergen binds to specific antibodies on the surface of mast cells, it activates the mast cells, causing them to degranulate and release large amounts of tryptase into the bloodstream. Tryptase has a short half-life in the body; its concentration typically rises rapidly within minutes to hours after an allergic reaction, peaking within 2-4 hours and returning to baseline within 24 hours. Therefore, detecting tryptase concentration in the blood can reflect the activation status of mast cells, thus determining whether a recent allergic reaction has occurred. Furthermore, tryptase exhibits high specificity; its elevated levels are closely related to mast cell-mediated allergic reactions, while elevations in tryptase are usually not significant in inflammatory responses caused by other non-allergic factors.

[0008] Eosinophil cationic protein (ECP) is a cationic protein synthesized and released by eosinophils, possessing strong cytotoxic and inflammatory regulatory functions. In allergic reactions, eosinophils are attracted by chemokines, migrate to the allergic reaction site, and are activated. Activated eosinophils release ECP, which can damage mucosal epithelial cells in the respiratory and digestive tracts, leading to tissue damage and exacerbated inflammation. Changes in blood ECP concentration are closely related to the severity of allergic reactions. During the acute phase of allergic diseases such as allergic asthma, allergic rhinitis, and atopic dermatitis, blood ECP levels are positively correlated with the degree of eosinophil activation, beginning to rise 6-12 hours after symptom onset and lasting for 2-3 days, reflecting the persistent inflammatory state of the allergic reaction. Therefore, detecting ECP levels can reflect the activation and infiltration of eosinophils, serving as an important indicator for assessing whether an allergic reaction is occurring and its severity.

[0009] This design combines the detection of trypsin and eosinophil cationic protein, allowing physicians to assess a patient's allergic state from multiple dimensions, including cellular and temporal stages, thus improving diagnostic accuracy and reliability and overcoming the limitations of single-indicator testing. With single-indicator testing, trypsin may be affected by a few non-allergic factors (such as mast cell proliferative disorders and chronic kidney disease), and ECP may also be affected by a few non-allergic factors such as parasitic infections and eosinophilic syndrome, leading to false positives. The combined detection significantly improves specificity through a "dual-cell pathway verification" mechanism: at the cellular level, trypsin reflects immediate mast cell activation (early events of an allergic reaction), while ECP reflects sustained eosinophil activation (mid-to-late stages of an allergic reaction and tissue damage); temporally, both show a "temporally synergistic increase" in allergic diseases, meaning that in acute allergies, trypsin rises first, followed by a subsequent increase and maintenance of ECP; in chronic allergies, both are continuously co-expressed, showing a mild to moderate elevation. When both trypsin and ECP are elevated simultaneously, and this aligns with the clinical timeline of an allergic reaction, it strongly suggests an ongoing allergic reaction (including IgE-mediated and non-IgE-mediated reactions), with a specificity approaching 90%. If only trypsin is elevated 6-12 hours after symptom onset, and the patient presents with moderate to severe symptoms, it suggests a non-allergic condition. Similarly, if only ECP is elevated, and the symptoms are in an acute phase, a non-allergic condition should also be suspected. Combined testing can also quantify the severity of the condition: combined test results can aid in grading; mild allergies may only show elevated trypsin, while moderate to severe allergies are often accompanied by significant elevations in both. Furthermore, ECP levels are positively correlated with the degree of tissue damage, providing a basis for clinical treatment selection.

[0010] This "dual-target, multi-dimensional" detection mode allows doctors to more accurately differentiate allergic reactions from other diseases involving mast cells or eosinophils, avoiding missed or misdiagnosed cases due to a single indicator. It is particularly suitable for rapid diagnosis of emergency allergic reactions, long-term management of chronic allergies, and differentiation of complex cases (such as allergies complicated by parasitic infections), helping doctors to develop timely and appropriate treatment plans and improve patient prognosis. It also provides a potential detection method for diagnosing non-IgE-mediated allergic reactions, filling a gap in existing in vitro diagnostic methods. However, currently, a combined quantitative detection system for trypsin and eosinophil cationic proteins has not been established for manufacturing in vitro diagnostic products (especially for acute allergic respiratory diseases) to diagnose the state of allergic reactions in patients, making it difficult to achieve multi-dimensional and accurate assessment of allergic diseases.

[0011] The technical solution adopted by this invention to solve its technical problem is: This invention provides the application of a substance for detecting trypsin concentration and a substance for detecting eosinophil cationic protein concentration in the preparation of products for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases.

[0012] This invention provides the application of a substance for detecting trypsin-like protein concentration, a substance for detecting eosinophil cationic protein concentration, and a readable carrier containing a diagnostic method in the preparation of products for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases; the readable carrier contains the following diagnostic method: The concentrations of trypsin and eosinophil cationic protein in the serum S1 of the test subjects were measured. The measured values ​​were then substituted into a combined detection diagnostic prediction model to calculate the model's predicted value. If the model's predicted value was ≤0.244, the test subject was considered not to be, or a candidate not to be, a patient with acute allergic respiratory disease; if the model's predicted value was >0.244, the test subject was considered to be, or a candidate for, a patient with acute allergic respiratory disease. Serum S1 was collected from the test subject within 0.5-6 hours after the onset of a suspected respiratory allergic reaction. The joint detection and diagnostic prediction model is shown in the following formula: Predicted value of the diagnostic model = -5.112 + 0.291 × T1 + 0.179 × E1; In the formula, T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1.

[0013] Preferably, the readable carrier also describes the following diagnostic method: The concentrations of trypsin and eosinophil cationic protein in the serum S2 of the test subjects were measured. The measured values ​​were then substituted into a concentration change diagnostic model to calculate M1 and M2. When M1 ≤ 0.8 and M2 ≥ 1.2, the test subject was considered to be or a candidate for acute allergic respiratory disease. When M1 > 0.8 and / or M2 < 1.2, the test subject was considered not to be or a candidate for acute allergic respiratory disease. Serum S2 was collected from the test subject within 20-28 hours after the onset of suspected respiratory allergic reaction. Serum S1 and serum S2 were samples taken from the same test subject at different time periods. The concentration change diagnostic model is shown in the following formula: M1=T2 / T1, M2=E2 / E1; In the formula, T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1; T2 represents the concentration of trypsin in the subject's serum S2; and E2 represents the concentration of eosinophil cationic protein in the subject's serum S2.

[0014] The readable medium may be a USB flash drive, optical disc, or the like that contains the diagnostic method.

[0015] The present invention provides an application of the above-described readable carrier containing diagnostic methods in the preparation of products for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases.

[0016] Preferably, the substance used to detect trypsin concentration is a substance that can specifically bind to trypsin; More preferably, the substance used to detect the concentration of trypsin is a trypsin antibody.

[0017] Preferably, the substance used to detect the concentration of eosinophil cationic protein is a substance that can specifically bind to eosinophil cationic protein; More preferably, the substance used to detect the concentration of eosinophil cationic protein is an eosinophil cationic protein antibody.

[0018] Preferably, the diagnosis or auxiliary diagnosis of acute allergic respiratory disease is for the diagnosis or auxiliary diagnosis of acute allergic respiratory disease in humans.

[0019] This invention provides a kit for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases, containing substances for detecting trypsin concentration and substances for detecting eosinophil cationic protein concentration.

[0020] Preferably, the kit further comprises the above-described readable carrier containing the diagnostic method.

[0021] Preferably, the kit contains a test card for detecting trypsin concentration and a test card for detecting eosinophil cationic protein concentration, or a test card for detecting trypsin concentration and eosinophil cationic protein concentration (combined test card).

[0022] More preferably, the test card for detecting trypsin concentration and eosinophil cationic protein concentration consists of a card shell and a test strip. The test strip includes a PVC base plate, a blood filtration membrane assembled on the PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The conjugate pad is loaded with fluorescent microspheres-trypsin antibody, fluorescent microspheres-eosinophil cationic protein antibody, and fluorescent microspheres-quality control material. The nitrocellulose membrane has two detection lines and a quality control line. The two detection lines are coated with trypsin antibody and eosinophil cationic protein antibody, respectively, and the quality control line is coated with quality control antibody.

[0023] More preferably, the quality control material is chicken IgY antibody or rabbit IgG antibody, and the quality control antibody is goat anti-chicken IgY antibody or goat anti-rabbit IgG antibody.

[0024] More preferably, the fluorescent microsphere-trypsin antibody is a trypsin antibody labeled with fluorescent microspheres by chemical coupling; the fluorescent microsphere-eosinophil cationic protein antibody is an eosinophil cationic protein antibody labeled with fluorescent microspheres by chemical coupling; and the fluorescent microsphere-quality control is a quality control material labeled with fluorescent microspheres by chemical coupling.

[0025] This invention provides a method for preparing a detection card for detecting trypsin concentration and eosinophil cationic protein concentration, comprising the following steps: I. Preparation of Sample Pads Preparation of sample pad treatment solution: Mix 0.1-1.0% (w / v) bovine serum albumin (BSA), 0.01-0.1% (v / v) Tween-20, and 0.01-0.1 mol / L phosphate buffer (PBS, pH 7.2-7.6) thoroughly to obtain the sample pad treatment solution; Immersion and Drying: Immerse the glass fiber completely in the sample pad treatment solution for 5-20 minutes. After removal, dry at 40-60 ℃ for 6-12 hours to prepare the sample pad. II. Preparation of the binding pad (a) Microsphere labeling Microsphere washing and activation: Fluorescent microspheres (100-400 nm) with carboxyl groups on the surface and filled with europium chelates of lanthanides were washed by centrifugation in 0.01-0.1 mol / L MES buffer (pH 5.0-6.0) at 5000-10000 rpm for 10-30 minutes, repeating the washing process 2-3 times. After washing, the microspheres were dispersed in activation buffer (0.01-0.1 mol / L MES buffer, pH 5.0-6.0, containing 0.1-1.0% (w / v) NaCl), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to a final concentration of 0.1-1.0 mg / mL. The mixture was shaken at room temperature for 15-60 minutes to activate the fluorescent microspheres.

[0026] Antibody or quality control conjugation: The activated microspheres were washed by centrifugation using conjugation buffer (0.01-0.1 mol / L PBS buffer, pH 7.2-7.6, containing 0.0-1.0% (w / v) BSA) at 5000-10000 rpm for 10-30 minutes, repeating the washing process 2-3 times. After washing, trypsin-like protein secondary antibody, eosinophil cationic protein secondary antibody, chicken IgY antibody, or rabbit IgG antibody were added to the microsphere solution in each tube, ensuring a final concentration of 0.1-1.0 mg / mL for each antibody and quality control. The mixture was incubated at room temperature with shaking for 1-3 hours to achieve conjugation between the antibody / quality control and the fluorescent microspheres.

[0027] Washing and Storage: After the coupling reaction, the labeled fluorescent microspheres were washed by centrifugation in 0.01-0.1 mol / L phosphate-buffered saline (PBS, pH 7.2-7.6) containing 0.01-0.1% (v / v) Tween-20 at 5000-10000 rpm for 10-30 minutes. This washing was repeated 2-3 times. After washing, the microspheres were dispersed in a microsphere storage solution (0.1-1.0% (w / v) bovine serum albumin (BSA), 0.01-0.1% (v / v) sodium azide, and 0.01-0.1 mol / L phosphate-buffered saline (PBS, pH 7.2-7.6)). (ii) Immersion and drying of the conjugate pad in treatment solution Preparation of conjugate pad treatment solution: Mix 0.1-1.0% (w / v) trehalose, 0.01-0.1% (v / v) polysorbate 80, and 0.01-0.1 mol / L Tris-HCl buffer (pH 7.5-8.5) evenly to obtain the conjugate pad treatment solution. Immersion and Drying: Immerse the glass fiber in the bonding pad treatment solution for 5-20 minutes. After removal, dry at 40-60℃ for 6-12 hours. (III) Preparation of the binding pad The labeled fluorescent microspheres were diluted with a microsphere preservation solution, and the diluted fluorescent microsphere solution was sprayed onto dried glass fiber at a rate of 2-6 μL / cm. The fiber was then dried at 40-60 ℃ for 6-12 hours to form a conjugate pad. III. Preparation of the coating membrane Preparation of coating buffer T1: Dissolve 0.1–1.0 mg / mL of trypsin-like antibody in 0.0–0.1% (v / v) Tween-20, 10–30% (v / v) methanol, and 0.01–0.1 mol / L phosphate-buffered saline (PBS, pH 7.2–7.6) to prepare coating buffer T1. Draw the detection line T1 on a nitrocellulose membrane (NC membrane) using coating buffer T1 at a speed of 1–5 μL / cm. Preparation of coating buffer T2: Dissolve 0.1-1.0 mg / mL of eosinophil cationic protein primary antibody in phosphate buffer (PBS, pH 7.2-7.6) containing 0.0-0.1% (v / v) Tween-20, 10-30% (v / v) methanol, and 0.01-0.1 mol / L to prepare coating buffer T2. Draw the detection line T2 on the NC membrane using coating buffer T2 at a speed of 1-5 μL / cm. Preparation of the quality control antibody coating solution: Dissolve goat anti-chicken IgY antibody or goat anti-rabbit IgG antibody in phosphate buffer (PBS, pH 7.2-7.6) containing 0.0-0.1% (v / v) Tween-20, 10-30% (v / v) methanol, and 0.01-0.1 mol / L to obtain the quality control antibody coating solution. Draw control lines on an NC membrane using the quality control antibody coating solution at a streaking speed of 1-5 μL / cm. Dry the marked NC film at 37-45 ℃ for 6-12 hours. IV. Assembly of the test strips Following the order from the sample application end to the absorbent end, the blood filtration membrane, sample pad, conjugate pad, coating membrane (nitrocellulose membrane), and absorbent paper are sequentially pasted onto the PVC base plate, with each component overlapping by 2-3 mm. The assembled test strip is then cut into strips 3-5 mm wide using a cutting machine to obtain the test strip. V. Assembly of the test card Insert the cut test strip into the housing, ensuring that the sample application end of the test strip and the observation window for the test result correspond to the corresponding positions on the housing. Seal the housing to complete the assembly of the test card.

[0028] This invention provides a system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases, comprising: (1) Reagents and / or instruments for detecting trypsin concentration and reagents and / or instruments for detecting eosinophil cationic protein concentration, or reagents and / or instruments for detecting trypsin concentration and eosinophil cationic protein concentration. (2) Device 1, which includes a data input module 1, a data processing module 1, a data comparison module 1 and a conclusion output module 1; The data input module 1 is used to input the detection values ​​of trypsin concentration and eosinophil cationic protein concentration in the serum S1 of the test subject obtained by (1); the serum S1 is obtained by sampling the test subject within a time range of 0.5-6 hours after the suspected respiratory allergic reaction occurs; The data processing module 1 is used to substitute the detected value into the joint detection diagnostic prediction model to calculate the diagnostic model prediction value; the joint detection diagnostic prediction model is shown in the following formula: Diagnostic model prediction value = -5.112 + 0.291 × T1 + 0.179 × E1; where T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1; The data comparison module 1 is used to compare the predicted value of the diagnostic model with a threshold; the threshold is 0.244. The conclusion output module 1 is used to output a conclusion. When the diagnostic model prediction value is ≤0.244, the conclusion "the subject is not or is not a candidate for acute allergic respiratory disease" is output; when the model prediction value is >0.244, the conclusion "the subject is or is a candidate for acute allergic respiratory disease" is output.

[0029] Preferably, the system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases further includes: (3) Device 2, which includes a data input module 2, a data processing module 2, a data comparison module 2 and a conclusion output module 2; The data input module 2 is used to input (1) the detection values ​​of trypsin concentration and eosinophil cationic protein concentration in serum S1 and S2 of the test subject; serum S1 is obtained by sampling the test subject within a time range of 0.5-6 hours after the suspected respiratory allergic reaction occurs; serum S2 is obtained by sampling the test subject within a time range of 20-28 hours after the suspected respiratory allergic reaction occurs. The data processing module 2 is used to substitute the detected values ​​into the concentration change diagnostic model to calculate M1 and M2; the concentration change diagnostic model is shown in the following formula: M1=T2 / T1, M2=E2 / E1; where T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1; T2 represents the concentration of trypsin in the subject's serum S2; E2 represents the concentration of eosinophil cationic protein in the subject's serum S2; The data comparison module 2 is used to compare M1 and M2 with thresholds; the threshold for M1 is 0.8; the threshold for M2 is 1.2. The conclusion output module 2 is used to output a conclusion. When M1≤0.8 and M2≥1.2, the conclusion "the subject is or is a candidate for acute allergic respiratory disease" is output; when M1>0.8 and / or M2<1.2, the conclusion "the subject is or is not a candidate for acute allergic respiratory disease" is output.

[0030] The reagents and / or instruments used to detect trypsin concentration and the reagents and / or instruments used to detect eosinophil cationic protein concentration can be ordinary commercial test cards that can accurately detect trypsin concentration and eosinophil cationic protein concentration.

[0031] The reagents and / or instruments used for detecting trypsin concentration and eosinophil cationic protein concentration are preferably the detection cards described above for detecting trypsin concentration and eosinophil cationic protein concentration.

[0032] The beneficial effects of this invention are: This invention provides a product for detecting trypsin and eosinophil cationic protein concentrations in acute allergic respiratory diseases. It uses a "dual-cell pathway validation" mechanism and a combined detection and diagnostic prediction model to diagnose whether or not an individual has acute allergic respiratory disease, significantly improving diagnostic efficiency compared to single-item diagnostic models. Furthermore, it utilizes a "concentration change" mechanism and a concentration change diagnostic model to diagnose whether or not an individual has acute allergic respiratory disease, significantly improving diagnostic efficiency compared to single-time-point diagnostic models. Combined with immunofluorescence chromatography, it enables rapid diagnosis of the disease and covers more types of allergic reactions, providing a new technical solution for the in vitro diagnosis of related diseases. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of an immunofluorescence chromatography test strip for the combined quantitative detection of trypsin-like proteins and eosinophil cationic proteins, provided by the present invention.

[0034] Among them, 1: PVC base plate; 2: blood filtration membrane; 3: sample pad; 4: conjugate pad; 5: nitrocellulose membrane; 6: detection line T1; 7: detection line T2; 8: quality control line; 9: absorbent paper.

[0035] Figure 2 ROC curves were plotted for the diagnostic model of trypsin, eosinophil cationic protein, and the combined detection of the two in Example 1. Detailed Implementation

[0036] The present invention will be further described below with reference to embodiments.

[0037] The following will clearly and completely describe the concept, specific solutions, and technical effects of the present invention with reference to embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. The various technical features in the present invention can be combined interactively without contradicting each other.

[0038] Example 1 This invention provides an immunofluorescence chromatography kit for the combined quantitative detection of trypsin-like proteins and eosinophil cationic proteins. The kit contains a test card, which consists of a card shell and a test strip, as shown below. Figure 1 As shown, the test strip includes a PVC base plate 1, a blood filtration membrane 2 assembled on the PVC base plate, a sample pad 3, a conjugate pad 4, a nitrocellulose membrane 5, and absorbent paper 9. The conjugate pad is loaded with a trypsin antibody labeled with fluorescent microspheres by chemical coupling, an eosinophil cationic protein antibody labeled with fluorescent microspheres by chemical coupling, and a quality control material labeled with fluorescent microspheres by chemical coupling. The nitrocellulose membrane has two detection lines (detection line T1 6 and detection line T2 7) and a quality control line 8. The two detection lines are coated with trypsin antibody and eosinophil cationic protein antibody, respectively, and the quality control line is coated with a quality control antibody.

[0039] This invention utilizes a combined detection reagent based on the principle of immunofluorescence chromatography, employing a double-antibody sandwich method to simultaneously and quantitatively detect trypsin and eosinophil cationic proteins. Its advantages include: (1) Compatible with whole blood, convenient sampling In addition to being compatible with serum and plasma, it also supports whole blood (including capillary blood) testing, making it especially suitable for children, elderly patients, and those with difficulty obtaining venous blood. Whole blood samples are added directly, avoiding centrifugation, simplifying the operation process to the greatest extent and improving the convenience of clinical testing.

[0040] (2) Quick results A single test can simultaneously obtain two key indicators, trypsin and eosinophil cationic protein, in just 15-20 minutes, which is 3 times more efficient than the traditional method (ELISA). It can be used for immediate auxiliary diagnosis in emergency / outpatient settings (such as anaphylactic shock and acute asthma attacks).

[0041] (3) Point-of-care testing (POCT) adapts to diverse scenarios Extremely simple to operate: Only a portable reader is needed, and primary healthcare workers can quickly get started. It is suitable for outpatient clinics, emergency rooms, community health service centers, and bedside rapid testing.

[0042] (4) It can be stored at room temperature and has strong stability. The reagents can be stored at room temperature (2-30℃) for 18 months. They are individually packaged and ready to use, making them suitable for grassroots and remote areas.

[0043] This invention provides a method for preparing the above-mentioned immunofluorescence chromatography kit for the combined quantitative detection of trypsin-like proteins and eosinophil cationic proteins, comprising the following steps: I. Preparation of Sample Pads Preparation of sample pad treatment solution: Mix 1.0% (w / v) bovine serum albumin (BSA), 0.1% (v / v) Tween-20 and 0.02 mol / L phosphate buffer (PBS, pH 7.2) thoroughly to prepare sample pad treatment solution; Immersion and Drying: The glass fiber is completely immersed in the sample pad treatment solution for 10 minutes. After removal, it is dried at 60 ℃ for 12 hours to prepare the sample pad. II. Preparation of the binding pad (a) Microsphere labeling Microsphere washing and activation: Fluorescent microspheres (PolyMicrospheres 200 nm) with carboxyl groups on the surface and filled with europium chelates of lanthanides were washed by centrifugation in 0.05 mol / L MES buffer (pH 5.0) at 5000 rpm for 15 minutes, and the washing was repeated 3 times. After washing, the microspheres were dispersed at 10 mg / mL in activation buffer (0.05 mol / L MES buffer, pH 5.0, containing 0.9% (w / v) NaCl), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.5 mg / mL N-hydroxysuccinimide (NHS) were added to a final concentration of 1.0 mg / mL. The mixture was shaken at room temperature for 20 minutes to activate the fluorescent microspheres. Antibody or quality control conjugation: Activated microspheres were washed by centrifugation using conjugation buffer (0.02 mol / L PBS buffer, pH 7.2, containing 0.0005% (w / v) BSA) at 5000 rpm for 10 minutes, repeated twice. After washing, trypsin-like protein secondary antibody, eosinophil cationic protein secondary antibody, chicken IgY antibody, or rabbit IgG antibody were added to each tube of microsphere solution (5 mg / mL) to achieve a final concentration of 0.3 mg / mL for each antibody and quality control. The mixture was incubated at room temperature with shaking for 3 hours to achieve conjugation between the antibody / quality control and the fluorescent microspheres. Washing and Storage: After the coupling reaction, the labeled fluorescent microspheres were washed by centrifugation in 0.02 mol / L phosphate-buffered saline (PBS, pH 7.2) containing 0.1% (v / v) Tween-20 at 5000 rpm for 10 minutes, and the washing was repeated 3 times. After washing, the microspheres were dispersed in microsphere storage solution (0.1% (w / v) bovine serum albumin (BSA), 0.01% (v / v) sodium azide, and 0.02 mol / L phosphate-buffered saline (PBS, pH 7.2)). (ii) Immersion and drying of the conjugate pad in treatment solution Preparation of conjugate pad treatment solution: Mix 1.0% (w / v) trehalose, 0.02% (v / v) polysorbate 80 and 0.02 mol / L Tris-HCl buffer (pH 7.5) evenly to prepare conjugate pad treatment solution. Immersion and Drying: Immerse the glass fiber in the composite pad treatment solution for 10 minutes. After removal, dry at 60°C for 12 hours. (III) Preparation of the binding pad Fluorescent microsphere solutions labeled with trypsin-like secondary antibody, eosinophil cationic protein secondary antibody, and chicken IgY antibody were mixed. The labeled fluorescent microspheres were diluted with microsphere preservation solution to a concentration of 0.3 mg / mL. The diluted fluorescent microsphere solutions were then sprayed onto dried glass fibers at a spraying rate of 3.5 μL / cm and dried at 60 °C for 12 hours to prepare the conjugate pad. III. Preparation of the coating membrane Preparation of coating buffer T1: 0.6 mg / mL of trypsin-like antibody was dissolved in 0.001% (v / v) Tween-20, 25% (v / v) methanol, and 0.02 mol / L phosphate-buffered saline (PBS, pH 7.2) to prepare coating buffer T1. The detection line T1 was run on a nitrocellulose membrane (NC membrane) using coating buffer T1 at a running speed of 1.5 μL / cm.

[0044] Preparation of coating buffer T2: 0.55 mg / mL of eosinophil cationic protein primary antibody was dissolved in phosphate-buffered saline (PBS, pH 7.2) containing 0.001% (v / v) Tween-20, 25% (v / v) methanol, and 0.02 mol / L to prepare coating buffer T2. The detection line T2 was then drawn on the NC membrane using coating buffer T2 at a speed of 1.5 μL / cm. Preparation of the quality control antibody coating solution: Goat anti-chicken IgY antibody was dissolved in phosphate buffer (PBS, pH 7.2) containing 0.001% (v / v) Tween-20, 25% (v / v) methanol, and 0.02 mol / L to obtain an antibody concentration of 0.7 mg / mL. The quality control antibody coating solution was used to draw control lines on an NC membrane at a speed of 1.5 μL / cm. The marked NC film was dried at 45 °C for 12 hours. IV. Assembly of the test strips Following the order from the sample application end to the absorbent end, the blood filtration membrane (glass fiber), sample pad (glass fiber), conjugate pad (glass fiber), coating membrane (nitrocellulose membrane), and absorbent paper (plant cellulose) are sequentially pasted onto the PVC base plate, with each component overlapping the others by 2-3 mm. The assembled test strip is then cut into strips 3-5 mm wide using a cutting machine to obtain the test strip.

[0045] V. Assembly of the test card Insert the cut test strips into the cartridge, ensuring that the sample application end of the test strip and the observation window for the test results correspond to the respective positions on the cartridge. Seal the cartridge to complete the assembly of the test card. Seal in an aluminum foil bag and refrigerate for later use.

[0046] This invention provides a detection method for the above-mentioned reagent kit, comprising the following steps: (a) Preparation before testing: Remove the test card from the packaging box and allow it to equilibrate at room temperature (10–30 ℃) for 30–60 minutes (keep the aluminum foil bag sealed to prevent moisture absorption). Allow the sample to be tested (serum, plasma, or whole blood) to reach room temperature. If the sample is refrigerated, it needs to be thawed and mixed in advance to avoid repeated freeze-thaw cycles. Scan the QR code on the kit and enter the reagent name, batch number, and other information into the matching testing device.

[0047] (ii) Sample dilution: Pipette 50 μL of the sample to be tested into a centrifuge tube containing 0.1 mL of sample dilution solution (0.5% (w / v) PEG10000, 0.05% (v / v) Tween-20, 0.1 mol / L Tris-HCl buffer (pH 7.5), 0.9% NaCl, 0.02% Proclin 300) and mix thoroughly.

[0048] (III) Sample addition procedure: Open the aluminum foil bag, take out the test card, and place it flat on a horizontal table. Use a micropipette to take 100 μL of the diluted sample and add it vertically to the sample well of the test card.

[0049] (iv) Display panel test: Let the test card stand at room temperature on the display panel for 15–20 minutes, then insert the test card into the card slot of the matching fluorescence immunoassay analyzer and select the “Tryptase+ECP combined test” item. The instrument will automatically read the fluorescence signal.

[0050] (v) Result calculation and interpretation: The instrument has a built-in standard curve (drawn based on known concentrations of Tryptase and ECP calibrators), which automatically substitutes the detected fluorescence signal into the equation to calculate the concentration of the target substance in the sample (unit: ng / mL).

[0051] Analytical performance evaluation of the fluorescence chromatography kit for combined quantitative detection of trypsin-like proteins and eosinophil cationic proteins of the present invention: (a) Blank limit and detection limit test Based on the basic information and market demand of trypsin and eosinophil cationic protein, the kit of this invention declares in the technical requirements that the LOB of trypsin is ≤0.50 ng / mL and the LOD is ≤0.80 ng / mL, and the LOB of eosinophil cationic protein is ≤1.00 ng / mL and the LOD is ≤1.60 ng / mL.

[0052] Two blank samples and two samples with detection limit concentrations were tested using the kit of this invention. Each sample was tested four times daily for five consecutive days, and the test values ​​were recorded. See Tables 1 and 2.

[0053] Calculate the percentage of blank sample test results with concentrations less than or equal to the declared LoB. If the percentage is greater than or equal to 88%, the blank limit validation is considered successful; if the percentage is less than 88%, the blank limit validation is unsuccessful. Calculate the percentage of detection limit concentration test results with concentrations greater than or equal to the declared LoB. If the percentage is greater than or equal to 88%, the detection limit validation is considered successful; if the percentage is less than 88%, the detection limit validation is unsuccessful.

[0054] Table 1: Results of Blank Limit and Detection Limit Tests for Trypsin

[0055] The results showed that 95% of the blank sample test results for trypsin had a concentration less than or equal to the declared LoB, indicating successful blank limit validation. 100% of the test results for the detection limit concentration samples had a concentration greater than or equal to the declared LoB, indicating that the detection limit validation met the requirements.

[0056] Table 2: Results of Blank Limit and Detection Limit Tests for Eosinophil Cationic Proteins

[0057] The results showed that in the blank sample test results of eosinophil cationic protein, the concentration was less than or equal to the declared LoB in 92.5% of the total, indicating successful blank limit verification. In the sample test results of the detection limit concentration, the concentration was greater than or equal to the declared LoB in 100% of the total, indicating that the detection limit verification met the requirements.

[0058] (ii) Linearity Test Based on the basic information and market demand for trypsin and eosinophil cationic protein, the kit of this invention declares in the technical requirements that the linear range of trypsin is 1.00-200.00 ng / mL and the linear range of eosinophil cationic protein is 2.00-200.00 ng / mL.

[0059] High-value samples with concentrations at the upper limit of the linear range were diluted to seven concentrations at a certain ratio, with the lowest linear evaluation sample concentration representing the lower limit of the linear range. Each concentration sample was tested three times using the kit of this invention, and the average concentration was calculated. The mean values ​​of each sample and their corresponding theoretical concentrations were then fitted to a straight line using the least squares method, and the linear correlation coefficient r was calculated. If the linear correlation coefficient r is not less than 0.9900 within the linear evaluation concentration range, the declared linear range can be considered basically reasonable. See Tables 3 and 4.

[0060] Table 3: Results of linear assay for trypsin

[0061] The results showed that the linear correlation coefficient r of trypsin was not less than 0.9900 in the concentration range of 1.00-200.00 ng / mL, and the declared linear range met the requirements.

[0062] Table 4: Results of linear assay for eosinophil cationic proteins

[0063] The results showed that the linear correlation coefficient r of eosinophil cationic protein was not less than 0.9900 in the concentration range of 2.00-200.00 ng / mL, and the declared linear range met the requirements.

[0064] (iii) Repeatability testing Based on the basic information and market demand of trypsin and eosinophil cationic protein, the kit of this invention declares in the technical requirements that the repeatability coefficient of variation of trypsin is no more than 15% and the repeatability coefficient of variation of eosinophil cationic protein is no more than 15%.

[0065] The kit of this invention was used to test high-concentration and low-concentration repeatable samples, with each sample tested 10 times. The mean M and standard deviation SD of the 10 measurements were calculated, and the repeatability coefficient of variation (CV) was calculated as SD / M × 100%. The repeatability coefficient of variation (CV) should be ≤ 15.0%. See Tables 5 and 6.

[0066] Table 5: Results of Repeatability Tests for Trypsin

[0067] The results showed that the reproducibility of trypsin met the acceptance criteria.

[0068] Table 6: Results of repeatability test of eosinophil cationic proteins

[0069] The results showed that the reproducibility of eosinophil cationic proteins met the acceptance criteria.

[0070] (iv) Accuracy test Based on the basic information and market demand for trypsin and eosinophil cationic protein, the accuracy of the kit of this invention is assessed by using a recovery experiment, as stated in the technical requirements. The recovery rate should be within the range of 85.0%-115.0%. The accuracy of eosinophil cationic protein is also assessed by using a recovery experiment, with a recovery rate within the range of 85.0%-115.0%.

[0071] A high-concentration sample A solution was added to a low-concentration sample B solution, with the volume ratio of A solution to B solution being 1:9. The kit of this invention was used to measure the concentrations of mixed sample A+B, sample A, and sample B, with each measurement repeated three times. The concentration values ​​of the three measurements were obtained, and the average value was calculated. The recovery rate was calculated according to formula (1): R = [C×(V0+V) - C0×V0] / (Cs×V) × 100% (1) In the formula: R is the recovery rate, V is the volume of high-concentration sample A, V0 is the volume of low-concentration sample B, C is the concentration of the mixed sample, C0 is the concentration of sample B, and Cs is the concentration of sample A. See Tables 7 and 8.

[0072] Table 7: Results of Trypsin Accuracy Test

[0073] The results showed that the recovery rate of trypsin was in the range of 85%-115%, and the accuracy met the acceptance criteria.

[0074] Table 8: Results of the accuracy test for eosinophil cationic proteins

[0075] The results showed that the recovery rate of eosinophil cationic proteins was in the range of 85%-115%, and the accuracy met the acceptance criteria.

[0076] (V) Clinical application of the fluorescent chromatography kit for the combined quantitative detection of trypsin-like proteins and eosinophil cationic proteins of this invention: (1) Research on constructing the best diagnostic performance prediction model based on patient and healthy population samples This study used a total of 300 serum samples, including 150 patient samples. All patients underwent sampling within 0.5-6 hours of the onset of suspected respiratory allergic reactions and were diagnosed with acute allergic respiratory disease by a hospital. The other 150 samples were from the general population undergoing routine physical examinations. All samples were from Guangdong Province.

[0077] By simultaneously detecting the levels of two biomarkers, trypsin and eosinophil cationic protein, a predictive model with optimal diagnostic performance was established based on the results of binary logistic regression analysis of the research variables (Table 9).

[0078] Table 9: Regression Analysis of Research Variables

[0079] The results show that by incorporating the two measured biomarkers, the final joint detection and diagnostic prediction model is as follows: The predicted value of the combined detection and diagnostic model is -5.112 + 0.291 × TRYPTASE + 0.179 × ECP.

[0080] In the formula: TRYPTASE represents the concentration of trypsin-like protein in the subject's serum; ECP represents the concentration of eosinophil cationic protein in the subject's serum.

[0081] ROC curves were plotted for trypsin, eosinophil cationic protein, and a diagnostic model based on the combined detection of the two. Figure 2 The diagnostic efficacy of the detection methods is presented in a visual way, and their respective areas under the curve (AUC) and Youden index are compared, as shown in Tables 10, 11, 12 and 13.

[0082] Table 10: Area Data under ROC Curve

[0083] Table 11: Relationship between specificity (TNR) and sensitivity (TPR) at different trypsin concentrations

[0084] Table 12: Relationship between specificity (TNR) and sensitivity (TPR) at different eosinophil cationic protein concentrations.

[0085] Table 13: Relationship between specificity (TNR) and sensitivity (TPR) measured under different diagnostic model prediction values

[0086] The results showed that the AUC of the single-analyte trypsin diagnostic model was 0.931 (95% CI, 0.905-0.956), and the maximum Youden index was 0.674 (sensitivity 84.7%, specificity 82.7%). The AUC of the single-analyte eosinophil cationic protein diagnostic model was 0.906 (95% CI, 0.875-0.937), and the maximum Youden index was 0.633 (sensitivity 76%, specificity 87.3%). The AUC of the combined detection model was 0.951 (95% CI, 0.93-0.972), and the maximum Youden index was 0.780 (sensitivity 94.7%, specificity 83.3%). The combined detection model's AUC and maximum Youden index were both greater than those of the single-analyte model, indicating that the diagnostic model established by combining trypsin and eosinophil cationic protein detection can effectively improve the diagnostic efficacy in acute allergic respiratory diseases.

[0087] (2) Research on constructing a diagnostic model for biomarker concentration changes based on dynamic monitoring In the diagnostic research of acute allergic respiratory diseases, the dynamic changes of two biomarkers, trypsin and eosinophil cationic protein, are of great significance. The previous part of the study established a predictive model based on these two biomarkers using 300 serum samples. This study further validates whether combining the dynamic changes in the concentrations of these two biomarkers over time can improve diagnostic performance.

[0088] The experimental sample consisted of 30 patients who underwent a second sampling within a specified time frame (20-28 hours after the suspected respiratory allergic reaction) from a pool of 150 patients initially identified. Simultaneously, 30 healthy individuals were selected as the control group. The concentrations of trypsin and eosinophil cationic protein were measured at time points T1, E1 (0.5-6 hours) and T2, E2 (20-28 hours) in both groups. The concentration ratios at the two time points were calculated, and the proportion of the reverse trend of "significantly decreased trypsin and significantly increased eosinophil cationic protein" observed in the patient and control groups was analyzed. This allowed for a comparison of the performance differences between single-time-point diagnosis and diagnosis combined with dynamic trend analysis.

[0089] 1) Comparison of dynamic changes in biomarkers between the patient group and the control group Table 14: Dynamic changes in biomarkers in the patient group

[0090] As shown in Table 14, among the 30 patients, except for one special case, the remaining 29 cases exhibited an inverse trend: the T2 / T1 ratio of trypsin was significantly less than 0.8 (i.e., a significant decrease in concentration), while the E2 / E1 ratio of eosinophil cationic protein was significantly greater than 1.2 (i.e., a significant increase in concentration). This concordance rate was as high as 96.7%. This indicates that in patients with acute allergic respiratory diseases, trypsin and eosinophil cationic protein exhibit a significant inverse relationship over time. This is closely related to the pathophysiological process of the disease; trypsin release increases in the early stages of the allergic reaction and then gradually decreases, while eosinophil cationic protein continues to rise and play a role in the later stages.

[0091] Table 15: Dynamic changes in biomarkers in the control group

[0092] As shown in Table 15, in the 30 healthy individuals in the control group, the T2 / T1 and E2 / E1 ratios of trypsin and eosinophil cationic protein were close to 1, with no obvious reverse trend. The proportion meeting the "reverse trend" criteria was 0%. This indicates that the concentrations of these two biomarkers in healthy individuals are relatively stable and do not exhibit the dynamic changes seen in patients, further highlighting the specificity of this reverse trend in the diagnosis of acute allergic respiratory diseases.

[0093] 2) Performance comparison of different diagnostic methods Table 16: Comparison of diagnostic performance between single time points (T1, E1) and dynamic trends (T1-T2, E1-E2)

[0094] Table 16 clearly demonstrates the performance difference between single-time-point (T1, E1) diagnosis and dynamic trend (T1→T2, E1→E2) diagnosis. The single-time-point diagnosis has a sensitivity of 93.3%, a specificity of 83.3%, and an overall accuracy rate of 88.3%, while the diagnosis method combining dynamic trends achieves a sensitivity of 96.7%, a specificity of 100%, and an overall accuracy rate as high as 98.3%.

[0095] This significant performance improvement fully demonstrates that combining the concentration trends of trypsin and eosinophil cationic proteins over time for diagnosis can greatly improve the accuracy of diagnosing acute allergic respiratory diseases. Compared to single-time-point detection, dynamic trend analysis is better able to capture the characteristic changes of biomarkers during disease development, reducing the errors and missed diagnoses that may occur with single-time-point detection.

[0096] This embodiment presents a novel method for diagnosing acute allergic respiratory diseases by combining the detection of trypsin and eosinophil cationic protein. This method exhibits high sensitivity and specificity. Furthermore, by incorporating the concentration trends of trypsin and eosinophil cationic protein over time, the diagnostic performance for acute allergic respiratory diseases can be further improved. Combining the measurement results with other clinical information, such as the clinical timing of the allergic reaction, this method holds promise as an important tool for the diagnosis or auxiliary diagnosis of allergic diseases.

[0097] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. A system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases, characterized in that, include: (1) Reagents and / or instruments for detecting trypsin concentration and reagents and / or instruments for detecting eosinophil cationic protein concentration, or reagents and / or instruments for detecting trypsin concentration and eosinophil cationic protein concentration. (2) Device 1, which includes a data input module 1, a data processing module 1, a data comparison module 1 and a conclusion output module 1; (3) Device 2, which includes a data input module 2, a data processing module 2, a data comparison module 2 and a conclusion output module 2; The data input module 1 is used to input the detection values ​​of trypsin concentration and eosinophil cationic protein concentration in the serum S1 of the test subject obtained by (1); the serum S1 is obtained by sampling the test subject within a time range of 0.5-6 hours after the suspected respiratory allergic reaction occurs; The data processing module 1 is used to substitute the detected value into the joint detection diagnostic prediction model to calculate the diagnostic model prediction value; the joint detection diagnostic prediction model is shown in the following formula: Diagnostic model prediction value = -5.112 + 0.291 × T1 + 0.179 × E1; where T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1; The data comparison module 1 is used to compare the predicted value of the diagnostic model with a threshold; the threshold is 0.

244. The conclusion output module 1 is used to output a conclusion. When the diagnostic model prediction value is ≤0.244, the conclusion "the subject is not or is not a candidate for acute allergic respiratory disease" is output; when the model prediction value is >0.244, the conclusion "the subject is or is a candidate for acute allergic respiratory disease" is output. The data input module 2 is used to input (1) the detection values ​​of trypsin concentration and eosinophil cationic protein concentration in serum S1 and S2 of the test subject; serum S1 is obtained by sampling the test subject within a time range of 0.5-6 hours after the suspected respiratory allergic reaction occurs; serum S2 is obtained by sampling the test subject within a time range of 20-28 hours after the suspected respiratory allergic reaction occurs. The data processing module 2 is used to substitute the detected values ​​into the concentration change diagnostic model to calculate M1 and M2; the concentration change diagnostic model is shown in the following formula: M1=T2 / T1, M2=E2 / E1; where T1 represents the concentration of trypsin in the subject's serum S1; E1 represents the concentration of eosinophil cationic protein in the subject's serum S1; T2 represents the concentration of trypsin in the subject's serum S2; E2 represents the concentration of eosinophil cationic protein in the subject's serum S2; The data comparison module 2 is used to compare M1 and M2 with thresholds; the threshold for M1 is 0.8; the threshold for M2 is 1.

2. The conclusion output module 2 is used to output a conclusion. When M1≤0.8 and M2≥1.2, the conclusion "the subject is or is a candidate for acute allergic respiratory disease" is output; when M1>0.8 and / or M2<1.2, the conclusion "the subject is or is not a candidate for acute allergic respiratory disease" is output.

2. The system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases according to claim 1, characterized in that, The diagnosis or auxiliary diagnosis of acute allergic respiratory disease refers to the diagnosis or auxiliary diagnosis of acute allergic respiratory disease in humans.

3. The system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases according to claim 1, characterized in that, The reagents used to detect trypsin concentration and eosinophil cationic protein concentration are detection cards for detecting trypsin concentration and eosinophil cationic protein concentration.

4. The system for diagnosing or assisting in the diagnosis of acute allergic respiratory diseases according to claim 3, characterized in that, The test card for detecting trypsin and eosinophil cationic protein concentrations consists of a card shell and a test strip. The test strip includes a PVC base plate, a blood filtration membrane assembled on the PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The conjugate pad is equipped with fluorescent microspheres-trypsin antibody, fluorescent microspheres-eosinophil cationic protein antibody, and fluorescent microspheres-quality control material. The nitrocellulose membrane has two detection lines and a quality control line. The two detection lines are coated with trypsin antibody and eosinophil cationic protein antibody, respectively, and the quality control line is coated with quality control antibody.

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