A total IgE quantitative fluorescence immunochromatography kit, detection card and detection method suitable for micro tear fluid samples

Abstract

The application relates to a total IgE quantitative fluorescence immunochromatography kit, a detection card and a detection method suitable for a trace tear sample. The detection card comprises a bottom plate and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad arranged in sequence along the liquid migration direction; the combination pad is pre-coated with fluorescent microsphere labeled mouse anti-human IgE monoclonal antibody and fluorescent microsphere labeled rabbit anti-DNP monoclonal antibody; the nitrocellulose membrane is provided with a detection line coated with mouse anti-human IgE monoclonal antibody and a quality control line coated with DNP-BSA; the kit further comprises a sample buffer containing HEPES, sodium chloride, Tween20 and Proclin300 and a batch information carrier storing calibration curve information. The application adopts a fluorescent microsphere double antibody sandwich method, and realizes stable quantitative detection of 1-5 muL trace tear samples through the synergistic optimization of chromatography structure, pad material system, buffer system and batch individual calibration, the detection range is 2.01-352.9 IU / mL, and the application is suitable for the auxiliary diagnosis of ocular surface allergy related diseases.

Description

Technical Field

[0001] This invention relates to a quantitative fluorescence immunochromatographic kit, detection card, and detection method for total IgE in trace tear samples, belonging to the field of in vitro diagnostic technology. Background Technology

[0002] Allergic conjunctivitis is one of the most common ocular surface immune-related diseases in clinical ophthalmology, and its pathogenesis is mainly based on IgE-mediated type I hypersensitivity reactions. Epidemiological data shows that allergic conjunctivitis affects approximately 15%–40% of the global population, and its incidence is showing an increasing trend year by year. The main clinical manifestations of this disease include itchy eyes, tearing, conjunctival hyperemia, eyelid edema, and increased secretions, which seriously affect patients' visual quality and daily life.

[0003] Immunoglobulin E (IgE) is a core effector molecule in type I hypersensitivity reactions. When the body is exposed to a specific allergen, B lymphocytes, under the regulation of T helper cells (Th2), undergo class switching and secrete IgE antibodies. IgE binds to high-affinity receptors (FcεRI) on the membrane surfaces of mast cells and basophils on the ocular surface. Upon re-exposure to the allergen, it mediates the massive release of inflammatory mediators such as histamine, leukotrienes, and prostaglandins, triggering a local inflammatory response on the ocular surface and producing the typical allergic symptoms described above. Therefore, IgE levels are an important biomarker reflecting the local allergic state of the ocular surface.

[0004] Tears are physiological secretions covering the ocular surface, composed of an aqueous layer, a mucin layer, and a lipid layer. Their composition directly reflects the local immune microenvironment of the ocular surface. In patients with allergic conjunctivitis, the total IgE level in tears is usually significantly elevated, and the degree of elevation is correlated with the severity of the local allergic reaction on the ocular surface. Compared with serum total IgE or specific IgE testing, tear total IgE testing has the following advantages: First, tear IgE is directly derived from the ocular surface, more accurately reflecting the local immune microenvironment, while serum IgE reflects systemic allergic levels, leading to inconsistencies between the two. Second, some patients have normal serum IgE levels but significant ocular allergic symptoms; tear IgE testing has higher diagnostic value in such cases. Third, changes in tear IgE levels can be used for disease assessment, classification, and treatment follow-up of allergic conjunctivitis. Therefore, tear total IgE is an important indicator for the diagnosis and management of ocular surface allergy-related diseases.

[0005] However, tears are typical micro-volume biological samples, facing multiple challenges in clinical testing applications: First, the basal tear secretion is extremely limited, with a normal person's basal tear secretion being approximately 1–2 μL / min, and the volume collected in a single clinical session typically not exceeding 5–10 μL, far lower than the sample requirements for routine blood or urine tests; Second, tear samples have a complex composition, influenced by factors such as sampling method, handling technique, the condition of the subject's ocular surface, tear dilution, and residual eye drops, resulting in significant individual and batch-to-batch variations; Third, the IgE concentration range in tears is wide, with a low basal IgE concentration in healthy individuals and a significantly higher concentration in allergic patients, requiring the detection system to maintain good quantitative stability over a wide dynamic range; Fourth, the low volume of tear samples places higher demands on the sensitivity, repeatability, and anti-interference capabilities of the detection system.

[0006] Currently, the main methods for detecting total IgE in tears include the following categories: (I) Laboratory Immunoassay Methods Enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA) are currently the commonly used methods for laboratory detection of tear IgE, offering high sensitivity and quantitative accuracy. However, both methods rely on specialized large-scale instruments and professional operators, involve cumbersome procedures, are time-consuming (usually several hours), and require large sample volumes (typically tens to hundreds of microliters), making them unsuitable for point-of-care testing (POCT) in outpatient clinics or primary healthcare institutions. Furthermore, the centralized laboratory testing model is not conducive to the rapid diagnosis and disease assessment of allergic conjunctivitis.

[0007] (II) Colloidal gold immunochromatography Colloidal gold immunochromatography (CICH) has been widely used in various point-of-care testing scenarios due to its advantages such as ease of operation, no need for specialized instruments, and rapid results. While some existing tear IgE-related detection products utilize CICH, this technology has the following inherent limitations: First, the optical signal of the colloidal gold labeling system relies on visual inspection or a reflectance photometer for grayscale reading, resulting in a narrow signal dynamic range. This typically only enables qualitative or semi-quantitative detection, failing to meet the clinical need for precise quantification. Second, under conditions of micro-volume tear samples (1–5 μL), the release efficiency of colloidal gold conjugates is limited, resulting in weak signals at low concentrations of the target analyte, poor repeatability, and a high coefficient of variation (CV), making it unreliable to distinguish different degrees of allergic states. Third, the colloidal gold system is susceptible to fluctuations in the sample matrix, non-specific adsorption, and chromatographic migration, leading to insufficient signal stability in low-concentration sample detection, making it unsuitable for precise quantitative analysis.

[0008] (III) Other detection technologies While tear IgE detection schemes based on microfluidic chips and surface plasmon resonance (SPR) technologies have shown some application potential in the research stage, these technologies have low commercialization rates, high costs, and complex operations, and no commercial products suitable for clinical application have yet been developed.

[0009] In summary, existing tear total IgE detection technologies exhibit significant contradictions in terms of adaptability to micro-samples, quantitative accuracy, detection speed, and clinical applicability, making it difficult to simultaneously meet the following requirements: high sensitivity detection of micro-volume tear samples (1–5 μL); stable and reliable quantitative output over a wide concentration range; batch calibration management capabilities to control batch-to-batch variations; suitability for outpatient point-of-care testing scenarios; and ease of operation and rapid results.

[0010] Fluorescent microsphere immunochromatography uses fluorescent microspheres as a signal labeling system. Fluorescent microspheres offer advantages such as high fluorescence intensity, good signal stability, and precise quantitative reading, providing a wider signal dynamic range and a lower detection limit compared to colloidal gold systems. However, when conventional fluorescent immunochromatography systems are directly applied to micro-volume tear samples, problems such as insufficient release of fluorescent conjugates, unstable chromatographic migration, large fluctuations in low-concentration signals, and unstable quality control signals can easily arise due to the extremely small sample size, low target analyte concentration, and unique matrix composition of the tear sample, leading to unreliable detection results.

[0011] Therefore, there is an urgent need for a quantitative fluorescence immunochromatographic detection system specifically optimized for the characteristics of trace tear fluid samples. By systematically and collaboratively optimizing the test card structure, padding material system, fluorescent microsphere coupling system, buffer system, quality control system, and batch calibration method, a stable and accurate quantitative detection of total IgE in trace tear fluid samples can be achieved while ensuring detection speed and ease of operation, so as to meet the actual clinical needs of auxiliary diagnosis of ocular surface allergy-related diseases. Summary of the Invention

[0012] This invention provides a quantitative total IgE fluorescence immunochromatographic kit, detection card, and detection method suitable for trace tear fluid samples, which can effectively solve the above-mentioned problems.

[0013] (I) Technical Solution The present invention provides a fluorescent immunochromatographic assay card for quantitative detection of total immunoglobulin IgE in tears. The assay card includes a base plate (1) and a sample pad (2), a conjugation pad (3), a nitrocellulose membrane (4) and an absorbent pad (5) arranged sequentially on the base plate (1) along the liquid migration direction. The conjugate pad (3) is coated with a first fluorescent microsphere conjugate and a second fluorescent microsphere conjugate. The first fluorescent microsphere conjugate is a fluorescent microsphere-labeled mouse anti-human IgE monoclonal antibody, and the second fluorescent microsphere conjugate is a fluorescent microsphere-labeled rabbit anti-DNP monoclonal antibody. The nitrocellulose membrane (4) is provided with a detection line (41) and a control line (42) in sequence along the migration direction of the liquid sample. The detection line (41) is coated with mouse anti-human IgE monoclonal antibody, and the control line (42) is coated with DNP-BSA.

[0014] In some embodiments, the edge of the nitrocellulose membrane (4) near the conjugate pad (3) is used as the starting reference line, the center of the detection line (41) is 8-10 mm away from the starting reference line, and the center of the quality control line (42) is 14-18 mm away from the starting reference line.

[0015] In some embodiments, the sample pad is 15-25 mm long, the conjugation pad is 8-12 mm long, the nitrocellulose membrane is 20-30 mm long, and the absorbent pad is 26-30 mm long.

[0016] In some embodiments, the coating concentration of mouse anti-human IgE monoclonal antibody in the detection line (41) is 1-2 mg / mL, and the coating concentration of DNP-BSA conjugate in the control line (42) is 0.2-0.8 mg / mL; The coating buffer used to coat the test line and the control line is PBS buffer, and the coating buffer contains 1.5-2.5% sucrose and 0.05-0.5% bovine serum albumin by mass.

[0017] In some embodiments, the fluorescent microspheres are fluorescent microspheres with carboxyl groups modified on their surface, and their particle size is 100 nm to 500 nm; Both the first fluorescently labeled conjugate and the second fluorescently labeled conjugate were prepared using a carbodiimide / N-hydroxysuccinimide crosslinking method; The components and concentrations of the spray buffer for the conjugation pad are: 0.01-0.05 mol / L Tris, 15-25 g / L sucrose, 8-12 g / L trehalose, 5-10 g / L bovine serum albumin, and 0.5-1.5 g / L Proclin 300, with water as the solvent.

[0018] In some embodiments, the mixing ratio of the first fluorescent microsphere conjugate to the second fluorescent microsphere conjugate is in the range of 5:1 to 15:1.

[0019] This invention provides a fluorescent immunochromatographic kit for the quantitative detection of total immunoglobulin E in tears, comprising: Such as the fluorescence immunochromatographic assay card mentioned above; The sample buffer, comprising and containing the following components and concentrations: 0.02-0.1 mol / L HEPES, 0.2-0.5 mol / L sodium chloride, 0.05-0.5% (v / v) Tween 20, and 0.01-0.05% (v / v) Proclin 300, with water as the solvent; and The batch information carrier contains pre-written four-parameter fitting calibration curve parameters corresponding to the batch of reagent kits, which can be read by the fluorescence immunoassay analyzer for automatic batch calibration.

[0020] In some embodiments, the batch information carrier is a non-volatile storage medium, preferably an SD memory card, NFC chip, or RFID tag; the sample buffer is individually aliquoted according to single-use volume, with each aliquot having a volume of 80-120 μL; the kit also includes a capillary tear collector for non-invasive tear collection. This non-invasive collection involves siphoning the sample from the outer edge of the conjunctival sac, causing no trauma to the body.

[0021] In some embodiments, the sample volume applicable to the kit is 1 μL to 5 μL, and the linear range for quantitative detection of total immunoglobulin E in tears is 2.01 IU / mL to 352.9 IU / mL.

[0022] This invention provides a method for quantitatively detecting total immunoglobulin E in tears using the kit described above. The method is an in vitro detection method for non-disease diagnostic purposes and includes the following steps: S1. Equilibrate the fluorescent immunochromatographic detection card, the sample buffer, and the tear sample to be tested to room temperature. S2. Connect the batch information carrier to the fluorescence immunoassay analyzer and read the four-parameter fitting calibration curve parameters of the current batch. S3. Elute the collected tear sample to be tested into the sample buffer solution and mix thoroughly to form a sample mixture; S4. Take a quantitative volume of the sample mixture and add it to the sample pad of the test card; S5. After the chromatography reaction has been pre-set for a certain time, insert the detection card into the fluorescence immunoassay analyzer and collect the fluorescence signal intensity of the detection line and the control line. S6. The fluorescence immunoassay analyzer calculates the ratio of fluorescence signal intensity of the detection line to that of the control line, and substitutes the ratio into the parameters of the four-parameter fitting calibration curve read in step S2 to convert and output the absolute concentration value of total immunoglobulin E in the tear sample to be tested; if the fluorescence signal intensity of the control line is lower than the preset threshold or there is no signal, it indicates that the test is invalid. In some embodiments, in step S3, the inlet volume of the tear sample to be tested is 2-3 μL; in step S4, the quantitative volume of the sample mixture added is 60-100 μL; and in step S5, the preset time for the chromatography reaction is 10-20 min.

[0023] (ii) Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: 1. Achieve stable quantitative detection of trace tear fluid samples This invention addresses the challenges of detecting small-volume tear samples and low-concentration target analytes. By optimizing the chromatographic structure, padding material system, and buffer system, it improves the release efficiency and migration stability of micro-volume tear samples during chromatography. Simultaneously, by employing a fluorescent microsphere labeling system, batch-by-batch calibration, and a four-parameter fitting quantitative model, it enhances the detection sensitivity and quantitative stability of low-concentration IgE samples, thus achieving stable quantitative detection of 1–5 μL of tear samples. Compared to existing colloidal gold tear IgE products, which primarily focus on qualitative or semi-quantitative detection, this invention maintains excellent signal repeatability and quantitative stability even under micro-volume sample conditions, with a detection range of 2.01–352.9 IU / mL.

[0024] 2. Achieve rapid quantitative detection This invention uses fluorescence immunochromatography technology, which can complete the chromatographic reaction and output quantitative results within 15 minutes after the sample is loaded onto the card, balancing detection speed and result objectivity, and is suitable for point-of-care testing (POCT) scenarios.

[0025] 3. More targeted treatment for localized ocular allergies. This invention directly detects total IgE in tears, which better reflects the local allergic state of the ocular surface compared to serum IgE. It has stronger specificity for ocular surface diseases and can be used for auxiliary diagnosis, classification reference and follow-up observation of ocular surface allergy-related diseases such as allergic conjunctivitis.

[0026] 4. The chromatographic structure is well-suited for trace samples. This invention designs the material types, lengths, overlap relationships, and positional layouts of the sample pad (2), conjugate pad (3), nitrocellulose membrane (4), and absorbent pad (5) in a matched manner, so that a small amount of tear sample can still achieve stable release, migration, and signal formation under limited liquid volume conditions, which is beneficial to improving the detection stability and repeatability under low sample volume conditions.

[0027] 5. The quality control system is independent and reliable. Tears contain high concentrations of lysozyme, lactoferrin, and secretory IgA, and some patients with ocular surface inflammation have rheumatoid factor (RF) or heterophile antibodies in their tears. Traditional 'chicken IgY / goat anti-chicken IgY' or 'rabbit IgG / goat anti-rabbit IgG' quality control systems are easily affected by tear matrix interference, leading to C-line signal fluctuations. This invention uses a small molecule hapten DNP system, completely avoiding the non-specific binding of endogenous antibodies in the human body, ensuring the accuracy of quality control for micro-sample testing, and helping to improve the controllability and reliability of test results.

[0028] 6. The conjugate pad and buffer system is beneficial for improving repeatability. This invention introduces sugar protectants such as sucrose and trehalose and BSA protein stabilizers into the conjugate pad (3) spray membrane system, and sets an appropriate salt concentration (NaCl 0.385 mol / L) and surfactant (Tween20) in the sample buffer, which is beneficial to improve the release of fluorescent conjugates, chromatographic migration and the stability of low concentration sample detection.

[0029] 7. Possesses good analytical performance and a foundation for productization. This invention can combine batch-specific calibration, T / C quantification, and four-parameter fitting to achieve stable output, making it suitable for forming a product system with good linearity, precision, blank limit, detection limit, quantitation limit, specificity, and anti-interference capabilities. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the structure of the detection card of the present invention, wherein 1 is the base plate, 2 is the sample pad, 3 is the conjugate pad, 4 is the nitrocellulose membrane, 41 is the detection line (T line), 42 is the quality control line (C line), and 5 is the absorbent pad.

[0032] Figure 2This is a schematic diagram of the detection principle of the present invention, showing the reaction principle at the detection line (41) and the quality control line (42).

[0033] Figure 3 This is a schematic diagram of the detection process of the reagent kit of the present invention.

[0034] Figure 4 This is a schematic diagram of the linear relationship for quantitative detection in the kit of the present invention, showing the correlation between the measured value and the labeled concentration of the reference sample. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a 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 those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0036] Example 1: Preparation of the test card like Figure 1 As shown, a base plate (1) with a total length of 8 cm was used, and the sample pad (2), conjugate pad (3), nitrocellulose membrane (4), and absorbent pad (5) were attached sequentially according to the direction of liquid migration. The specific attachment parameters are as follows: Sample pad (2): Made of JG64 glass fiber material, 20 mm in length, attached starting 4 mm from the bottom of the base plate; Bonding pad (3): Made of JG888 glass fiber material, 10.5 mm in length, attached starting 21 mm from the bottom of the base plate; Nitrocellulose membrane (4): CN140, 25 mm in length, attached starting 30 mm from the bottom of the base plate; Absorbent pad (5): AN3, 28.5 mm in length, attached starting 55 mm from the bottom of the base plate.

[0037] A detection line (41) and a control line (42) were sprayed onto the nitrocellulose membrane (4): the detection line (41) was located 9 mm from the lower edge of the membrane and coated with mouse anti-human IgE monoclonal antibody (catalog number: B58113) at a concentration of 1.6 mg / mL; the control line (42) was located 16 mm from the lower edge of the membrane and coated with DNP-BSA at a concentration of 0.5 mg / mL. The coating buffer system for the sprayed lines consisted of PBS, 2% sucrose, and 0.1% BSA. After spraying, the membrane was dried overnight in a 37°C oven for later use.

[0038] Example 2: Preparation of fluorescent microsphere conjugates Two conjugates were prepared using the same carboxyl fluorescent microspheres (model: A.04, particle size: 300 nm, surface group: carboxyl). (1) Preparation of the first fluorescent microsphere conjugate: 1 mg of the above carboxyl fluorescent microspheres were dispersed in MES buffer (0.05 mol / L, pH 6.0), and 200 μg each of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (n-hydroxysuccinimide) were added sequentially. The mixture was activated at room temperature for 30 min. After activation, excess activator was removed by centrifugation. The activated fluorescent microspheres were resuspended in PBS buffer (0.01 mol / L, pH 7.4), and 100 μg of mouse anti-human IgE monoclonal antibody (catalog number: nbp1-44928) was added. The mixture was coupled at 37 °C with shaking for 2 h. After coupling, 100 μL of 10% BSA was added for blocking for 30 min. The mixture was centrifuged and washed 3 times. Finally, it was resuspended in PBS buffer containing 0.1% BSA to obtain the first fluorescent microsphere conjugate.

[0039] (2) Preparation of the second fluorescent microsphere conjugate: The operation steps are the same as above, except that the conjugate antibody is replaced with rabbit anti-DNP monoclonal antibody (BIOBRIDGE, M-9402), and the other conditions are kept the same to obtain the second fluorescent microsphere conjugate.

[0040] The first fluorescent microsphere conjugate and the second fluorescent microsphere conjugate are mixed in a ratio of 10:1 and then used for coating the conjugate pad (3).

[0041] Example 3: Preparation of conjugate pad spray film solution and conjugate pad preparation Prepare the conjugation pad spray membrane buffer with the following formula: Tris 0.025 mol / L, sucrose 20 g / L, trehalose 10 g / L, BSA 6 g / L, and proclin300 1 g / L. Make up the volume to 1 L with ultrapure water, dissolve and mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use.

[0042] The first and second fluorescent microsphere conjugates prepared in Example 2 were added to the above-mentioned spraying buffer at a predetermined ratio. After thorough mixing, the mixture was uniformly sprayed onto the JG888 conjugate pad (3) using an automatic spraying device. The spraying amount was the amount suitable for the buffer system. After spraying, the mixture was dried overnight in a 37°C drying oven and then sealed and stored in a dry environment for later use.

[0043] Example 4: Preparation of Sample Buffer Prepare sample buffer according to the following formula: Hepes 0.05 mol / L, sodium chloride 0.385 mol / L, Tween 20 1 mL / L, Proclin 300 0.2 mL / L. Make up to a total volume of 1 L with ultrapure water, dissolve and mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and aliquot into 100 μL sample dilution tubes. Store at 4°C protected from light for later use.

[0044] In the sample buffer solution, Hepes serves as a biocompatible buffer component, helping to maintain the stability of the appropriate buffer system; sodium chloride (0.385 mol / L) is used to maintain the osmotic pressure and ionic strength of the solution; Tween20, as a nonionic surfactant, helps to reduce the nonspecific adsorption of trace tear samples during chromatography, improving the release efficiency of low-concentration target substances and the repeatability of detection; and Proclin300 serves as a preservative to prevent microbial contamination.

[0045] Example 5: Assembly of the reagent kit The test card prepared in Example 1 (with the prepared conjugate pad (3) and the nitrocellulose membrane (4) that has been sprayed) is finally assembled and packaged together with the sample buffer (100 μL / sample) prepared in Example 4, the batch information carrier (SD card, which contains the four-parameter fitting calibration curve parameters of the corresponding batch product), the tear collection device and the instruction manual to obtain the fluorescent immunochromatographic kit for quantitative detection of total IgE in tears as described in this invention.

[0046] The four-parameter fitting calibration curve parameters pre-written in the batch information carrier (SD card) are obtained by batch-by-batch calibration using enterprise reference materials before the corresponding batch of products leaves the factory. The parameters include the upper asymptote parameter a, the lower asymptote parameter d, the midpoint concentration parameter c, and the slope parameter b of the four-parameter logistic equation (4pl), which can be directly read by the analyzer and used for concentration conversion.

[0047] Example 6: Method for detecting total IgE in tears like Figure 2 and Figure 3 As shown, the method for detecting total IgE in tears using the kit prepared in Example 5 of this invention includes the following steps: (1) Equilibrate the reagents and subject samples to room temperature (18–25°C) for no less than 30 min; (2) Insert the batch information carrier (SD card) into the matching analyzer so that the analyzer can read the corresponding batch calibration curve parameters; (3) Using the matching tear collection device, a tear sample was collected from the lower eyelid margin of the subject, with a collection volume of approximately 2.2 μL; (4) Add the collected tear sample to a sample dilution tube containing 100 μL of sample buffer, mix thoroughly, and allow the tear to be fully released into the buffer. (5) Take 80 μL of the mixed sample solution and add it to the sample well of the test card; (6) After allowing the chromatography reaction to stand at room temperature (18–25℃) for 15 min, insert the detection card into the matching analyzer; (7) The analyzer collects the fluorescence signals of the detection line (41) and the quality control line (42), and calculates and outputs the total IgE concentration value of the tear fluid (unit: IU / mL) based on the T / C value and the four-parameter fitting calibration curve parameters (4pl equation) stored in the SD card. (8) Result interpretation: If there is a fluorescence signal on the control line (42), the detection is valid and the concentration value output by the analyzer is the final result; if there is no fluorescence signal on the control line (42), the detection is invalid and needs to be re-detected.

[0048] Example 7: Validation of repeatability and quantitative performance The repeatability and quantitative performance of the kit prepared in Example 5 of this invention were verified using internal reference standards at different concentration levels (a total of 9 concentration levels, covering a detection range of 2.01–352.9 IU / mL). Each concentration level was tested multiple times, and the average detection concentration, coefficient of variation (CV), and bias were recorded. The results are shown in Table 1.

[0049] Table 1. Repeatability verification results of the kit of the present invention at different concentration levels.

[0050] The results show that the kit described in this invention can achieve stable quantitative output in the concentration range of 2.01–352.9 IU / mL, and the CV values ​​of each concentration level are within the acceptable range, demonstrating good repeatability and quantitative stability. It is suitable for the quantitative detection of total IgE in trace tear samples.

[0051] Figure 4 The correlation between the measured values ​​of the detection method of the present invention and the labeled concentration of the reference sample is shown.

[0052] like Figure 4As shown, within the detection range, the two exhibit a good linear relationship, indicating that the present invention has good quantitative detection capability.

[0053] Comparative Example 1 (Colloidal Gold System): The colloidal gold-labeled antibody is used instead of the fluorescent microsphere-labeled antibody in this invention, and a suitable conjugate pad spray solution for the colloidal gold-labeled antibody is used. The other structural conditions of the test card remain consistent with those of this invention.

[0054] The colloidal gold labeling method is as follows: The pH of the colloidal gold solution was adjusted to near the isoelectric point (pI) of mouse anti-human IgE monoclonal antibody (catalog number: nbp1-44928) using 0.2 mol / L potassium carbonate. Then, an appropriate amount of mouse anti-human IgE monoclonal antibody was added for conjugation, and the mixture was incubated at room temperature for 15 min. After conjugation, 10% BSA was added to a final concentration of 1% for blocking, and the mixture was centrifuged at 12000 rpm for 30 min. The supernatant was discarded, and the resulting colloidal gold conjugate was resuspended in PBS buffer containing 1% BSA and 0.25% Tween 20. This was then replaced with a conjugate pad spraying buffer (20 mmol / L phosphate buffer, pH 7.4, containing 5% sucrose and 1% BSA) suitable for colloidal gold systems. The buffer was then sprayed onto the conjugate pad using an automated spraying device and dried for later use. The remaining steps (including the setting of the detection line and control line, sample buffer, and detection procedure) are consistent with this invention.

[0055] The stability of the detection signals of the two systems under low-concentration tear IgE sample conditions was compared, and the results are shown in Table 2.

[0056] Table 2 Comparison of response results of colloidal gold system and fluorescent microsphere system for low-concentration samples

[0057] The results showed that as the sample concentration decreased, the fluctuation of the detection signal of the colloidal gold system further increased. Under the condition of low-concentration micro-tear samples (≤25.44 IU / mL), the CV value of the colloidal gold system exceeded 30%, reaching a maximum of 61%, making it difficult to obtain a stable quantitative response relationship and unsuitable for the quantitative detection of micro-tear samples. In contrast, this invention, through the synergistic optimization of the fluorescent microsphere labeling system, specific chromatographic structure, and sample buffer system, achieved significantly lower CV values ​​than the colloidal gold system under the same low-concentration sample conditions. It obtained a more stable detection signal and quantitative output result under micro-tear conditions, verifying the superiority of the fluorescent microsphere system in the detection of low-concentration IgE in micro-tears.

[0058] Comparative Example 2: Comparison between conventional PBS buffer system and the buffer system of the present invention The standard PBS buffer (10 mmol / L PBS, pH 7.4, NaCl concentration 0.137 mol / L) was used as the sample diluent, without HEPES and Tween20. The rest of the test card structure, fluorescent microsphere coupling system, conjugate pad spray membrane system and detection procedure were consistent with those in Example 6.

[0059] Low concentrations of internal reference standards (6.73 IU / mL, 3.66 IU / mL, and 2.01 IU / mL) were used for detection, and each concentration was tested four times. The average detection concentration and CV value were calculated, and the results are shown in Table 3.

[0060] Table 3 Comparison of detection results for different sample buffer systems

[0061] The results showed that when using the conventional PBS buffer system, the repeatability of low-concentration sample detection decreased and the fluctuation of detection results increased. However, the buffer system of the present invention, which contains HEPES, high-salt-concentration NaCl and Tween 20, can improve the release efficiency of target substances and the chromatographic migration stability in micro-tear samples, thereby improving the repeatability and quantitative stability of detection.

[0062] Comparative Example 3: Optimization and Comparison of Combined Pad Spray Film System Conjugate pads were prepared using different conjugate pad spray-film buffer systems, with all other conditions remaining consistent with Example 3. Comparative Example 3A: no trehalose added; Comparative Example 3B: no sucrose added; Comparative Example 3C: no BSA added; the example group used a spray-film system consisting of 20 g / L sucrose, 10 g / L trehalose, and 6 g / L BSA. A 25.44 IU / mL reference standard was used for detection, and the fluorescence signal intensity and repeatability were measured. The results are shown in Table 4.

[0063] Table 4 Comparison of test results for different bonding pad spray film systems

[0064] The results showed that the protective system composed of sucrose, trehalose, and BSA was beneficial in maintaining the activity of the fluorescent microsphere conjugate, improving the release efficiency of the conjugate pad, and enhancing detection repeatability. The absence of any one component led to a decrease in fluorescence signal or poorer detection repeatability.

[0065] Comparative Example 4: Comparison of different sampling sizes The reference standard (25.44 IU / mL) was tested using 1.0 μL, 2.2 μL, and 5.0 μL, respectively. The test results and repeatability were compared. Simultaneously, actual tear fluid samples were collected using siphon tubes of different volumes (1.0 μL, 2.2 μL, and 5.0 μL), and the difficulty of clinical sampling was evaluated. The results are shown in Table 5.

[0066] Table 5. Impact of different sampling volumes on detection results

[0067] The results showed that when the sampling volume was less than 2 μL, the detection signal intensity decreased and repeatability declined; when the sampling volume increased to more than 5 μL, although the detection signal further increased, the difficulty of clinical sampling increased significantly. Considering both detection performance and clinical operability, this invention preferably uses approximately 2.2 μL of tear fluid samples for detection.

[0068] Example 8: Clinical Application Implementation The kit of this invention was used for tear sample testing in outpatient ophthalmology subjects. Tears were collected by trained medical personnel, and the testing method described in Example 6 was followed. Rapid quantitative output of total IgE concentration in tears can be achieved within 15 minutes, providing auxiliary evidence for assessing the state of ocular surface allergic reactions. Combined with clinical symptoms (itching, tearing, conjunctival hyperemia, increased secretions, etc.), physical signs, and other auxiliary examination results, this invention can be used for the auxiliary diagnosis, classification reference, and follow-up observation of ocular surface allergy-related diseases such as allergic conjunctivitis, showing promising clinical application prospects.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A fluorescent immunochromatographic test card for quantitatively detecting total immunoglobulin IgE in tear fluid, characterized by, The detection card includes a base plate (1), and a sample pad (2), a conjugate pad (3), a nitrocellulose membrane (4), and an absorbent pad (5) arranged sequentially on the base plate (1) along the liquid migration direction. The conjugate pad (3) is coated with a first fluorescent microsphere conjugate and a second fluorescent microsphere conjugate. The first fluorescent microsphere conjugate is a fluorescent microsphere-labeled mouse anti-human IgE monoclonal antibody, and the second fluorescent microsphere conjugate is a fluorescent microsphere-labeled rabbit anti-DNP monoclonal antibody. The nitrocellulose membrane (4) is provided with a detection line (41) and a control line (42) in sequence along the migration direction of the liquid sample. The detection line (41) is coated with mouse anti-human IgE monoclonal antibody, and the control line (42) is coated with DNP-BSA.

2. The test card of claim 1, wherein The starting baseline is the edge of the nitrocellulose membrane (4) near the conjugate pad (3). The center of the detection line (41) is 8-10 mm away from the starting baseline, and the center of the quality control line (42) is 14-18 mm away from the starting baseline.

3. The test card according to claim 1 or 2, characterized in that The sample pad is 15-25 mm long, the conjugation pad is 8-12 mm long, the nitrocellulose membrane is 20-30 mm long, and the absorbent pad is 26-30 mm long.

4. The test card of claim 1, wherein The concentration of mouse anti-human IgE monoclonal antibody in the detection line (41) is 1-2 mg / mL, and the concentration of DNP-BSA conjugate in the control line (42) is 0.2-0.8 mg / mL. The coating buffer used to coat the test line and the control line is PBS buffer, and the coating buffer contains 1.5-2.5% sucrose and 0.05-0.5% bovine serum albumin by mass.

5. The detection card according to claim 1, characterized in that, The fluorescent microspheres are fluorescent microspheres with carboxyl groups modified on their surface, and their particle size is 100 nm to 500 nm. Both the first fluorescently labeled conjugate and the second fluorescently labeled conjugate were prepared using a carbodiimide / N-hydroxysuccinimide crosslinking method; The components and concentrations of the spray buffer for the conjugation pad are: 0.01-0.05 mol / L Tris, 15-25 g / L sucrose, 8-12 g / L trehalose, 5-10 g / L bovine serum albumin, and 0.5-1.5 g / L Proclin 300, with water as the solvent.

6. A fluorescent immunochromatographic kit for the quantitative detection of total immunoglobulin E in tears, characterized in that, include: The fluorescent immunochromatographic detection card as described in any one of claims 1 to 5; The sample buffer comprises the following components and concentrations: 0.02-0.1 mol / L HEPES, 0.2-0.5 mol / L sodium chloride, 0.05-0.5% Tween 20 (volume fraction), and 0.01-0.05% Proclin 300 (volume fraction), with water as the solvent. as well as The batch information carrier contains pre-written four-parameter fitting calibration curve parameters corresponding to the batch of reagent kits, which can be read by the fluorescence immunoassay analyzer for automatic batch calibration.

7. The reagent kit according to claim 6, characterized in that, The batch information carrier is a non-volatile storage medium; the sample buffer is individually aliquoted according to the single-use volume, with each aliquot having a volume of 80-120 μL; the kit also includes a capillary tear collector for non-invasive tear collection.

8. The reagent kit according to claim 6, characterized in that, The sample volume applicable to the kit is 1 μL to 5 μL, and the linear range for quantitative detection of total immunoglobulin E in tears is 2.01 IU / mL to 352.9 IU / mL.

9. A method for quantitatively detecting total immunoglobulin E in tears using a kit as described in any one of claims 6 to 8, wherein the method is an in vitro detection method for non-disease diagnostic purposes, characterized in that... Includes the following steps: S1. Equilibrate the fluorescent immunochromatographic detection card, the sample buffer, and the tear sample to be tested to room temperature. S2. Connect the batch information carrier to the fluorescence immunoassay analyzer and read the four-parameter fitting calibration curve parameters of the current batch. S3. Elute the collected tear sample to be tested into the sample buffer solution and mix thoroughly to form a sample mixture; S4. Take a quantitative volume of the sample mixture and add it to the sample pad of the test card; S5. After the chromatography reaction has been pre-set for a certain time, insert the detection card into the fluorescence immunoassay analyzer and collect the fluorescence signal intensity of the detection line and the control line. S6. The fluorescence immunoassay analyzer calculates the ratio of fluorescence signal intensity of the detection line to that of the control line, and substitutes the ratio into the parameters of the four-parameter fitting calibration curve read in step S2 to convert and output the absolute concentration value of total immunoglobulin E in the tear sample to be tested; if the fluorescence signal intensity of the control line is lower than the preset threshold or there is no signal, it indicates that the test is invalid.

10. The method according to claim 9, characterized in that, In step S3, the inlet volume of the tear sample to be tested is 2-3 μL; in step S4, the quantitative volume of the sample mixture added is 60-100 μL; in step S5, the preset time of the chromatography reaction is 10-20 min.

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