A single-cell microarray-based reagent kit and method for in situ quantitative detection of receptor-binding allergen-specific IgE.

Abstract

This invention discloses a single-cell microarray-based kit and method for in situ quantitative detection of receptor-binding allergen-specific IgE. The kit includes: a single-cell microcavity array chip, an antigen barcode chip, a plastic clamp, IgE standards, fluorescently modified IgE detection antibodies, and auxiliary reagents. During detection, basophil single cells are loaded into the microcavity array chip, lysis buffer is added, and the cells are clamped together with the antigen barcode chip. Low-temperature incubation allows the allergen-specific IgE released from cell lysis to be captured in situ by the antigen on the chip. A sandwich immunoassay is then performed using a fluorescently labeled antibody, and absolute quantification is achieved using a standard curve. This invention is the first to achieve high-throughput, high-sensitivity, multi-component parallel in situ quantitative detection of basophil membrane receptor-binding sIgE at the single-cell level. It can directly reflect the functional sensitization state of effector cells and has advantages such as simple operation, low sample requirement, and no need for complex valve devices.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a single-cell chip-based receptor-binding allergen-specific IgE in situ quantitative detection kit and method. Background Technology

[0002] Serum receptor-binding allergen-specific IgE (sIgE) testing is currently a core method for diagnosing the etiology of allergic diseases. However, traditional sIgE testing methods are mostly based on allergen extracts, which cannot accurately identify specific components and cross-reactions, affecting diagnostic accuracy. While allergen component diagnostic (CRD) technology has improved accuracy, it still relies on serum free sIgE levels. Clinical studies have shown that serum free sIgE levels are not entirely correlated with the severity of patient symptoms and the efficacy of allergen-specific immunotherapy (AIT). As the only etiological treatment, AIT has poor or no effect on approximately 22%-35% of patients, and it can take months or even years to determine ineffectiveness, leading to unnecessary treatment and risks. Therefore, there is an urgent need to discover novel biomarkers that more directly reflect the potential of allergic reactions.

[0003] Mast cells and basophils are the main effector cells in IgE-mediated allergic reactions, and their surfaces highly express the IgE high-affinity receptor FcεRI. In allergic patients, most receptor sites are occupied by sIgE, leaving cells in a "ready-to-be-activated" state. Therefore, quantitative detection of sIgE bound to effector cell membrane receptors (i.e., receptor-bound sIgE) can directly reflect the functional readiness and degranulation potential of effector cells. Basophils can be obtained from peripheral blood, and quantifying their receptor-bound sIgE is expected to improve the accuracy of disease risk prediction, explain clinical heterogeneity, and provide a mechanism-guided detection tool for predicting AIT efficacy. Its theoretical value and clinical application potential are superior to simple serum sIgE diagnosis. However, current technologies can only detect total IgE bound to cells through flow cytometry or ELISA, and cannot accurately distinguish and quantify basophil receptor-bound sIgE, limiting its clinical significance.

[0004] The basophil activation assay (BAT) assesses cellular response by detecting markers such as CD63 / CD203c. However, its results are influenced by multiple factors, including stimulation conditions, sample processing, gating thresholds, and cellular background status. Furthermore, it primarily provides population proportion or distribution readings, making it difficult to trace response differences back to upstream mechanisms such as sIgE load within the same cell at the single-cell scale. Given the significant heterogeneity of basophils and the potential for certain high-functioning subsets to dominate disease progression, there is an urgent need to establish a quantitative single-cell-level method for detecting receptor-binding sIgE, complementing BAT.

[0005] In recent years, single-cell microfluidic chip technology has provided a new approach for membrane protein detection. Its basic principle is to enclose a single cell in a nano-scale microchamber, lyse it, and then capture the target protein using a bottom antibody array, followed by quantification via sandwich immunoassay. This technology offers high sensitivity and requires small sample volumes, making it potentially applicable to clinical allergy testing. However, existing platforms have the following limitations: to support parallel detection of multiple indicators, longer chambers are required, leading to increased volume and decreased sensitivity, particularly unfavorable for detecting trace targets such as membrane receptor-binding proteins; throughput is low, yielding only a few thousand single-cell chambers, resulting in insufficient data reliability; and if cell lysis is required, a valve device must be introduced to introduce the lysis buffer, which is complex to design, technically demanding, and prone to instability.

[0006] In summary, the key factors determining the occurrence and intensity of allergic reactions depend not only on serum sIgE levels but also directly on the functional sIgE loading on the surface of effector cells. Quantitative detection of basophil receptor-binding sIgE has significant clinical value; however, currently, there is a lack of kits and methods capable of accurate, high-throughput, and multi-component parallel detection at the single-cell level. Therefore, there is an urgent need to develop a clinically applicable solution to address these issues. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a single-cell chip-based reagent kit and method for in situ quantitative detection of receptor-binding allergen-specific IgE. This method enables high-throughput, multi-component, and high-sensitivity in situ quantitative detection of receptor-binding allergen-specific IgE bound to basophil membrane receptors at the single-cell level. The aim is to accurately assess the functional sensitization status of effector cells in patients, assist in predicting the risk of allergic diseases, and evaluate the efficacy of immunotherapy.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A single-cell microarray-based receptor-binding allergen-specific IgE in situ quantitative detection kit, comprising: Single-cell microcavity array chip for capturing and containing individual basophils; An antigen barcode chip, the surface of which is printed with one or more allergen components, is used to capture in situ receptor-binding allergen-specific IgE that binds to a single basophil membrane receptor and is released from the microcavity array chip. Plastic clamps are used to clamp the single-cell microcavity array chip and the antigen barcode chip to form a tight contact interface; IgE standard is used to establish a concentration-quantitative standard curve. Fluorescently modified IgE detection antibodies are used to bind to captured receptor-binding allergen-specific IgE to generate a detectable fluorescent signal. Auxiliary reagents include antibody buffer, antigen blocking solution, cell lysis buffer, and cell washing solution.

[0009] In the above scheme, the single-cell microcavity array chip is prepared by polydimethylsiloxane and curing agent, and its upper surface contains multiple microcavities for accommodating single cells.

[0010] In the above scheme, the antigen barcode chip contains multiple different antigen channels, with a channel width of 5-100μm and a spacing of 10-200μm.

[0011] In the above scheme, the allergen components printed on the antigen barcode chip include: Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Art v 1, Art v 3, Art v 4, Fel d 1, Can f 1, Bet v 1, Phl p 1.

[0012] In the above scheme, the antibody buffer is a BSA solution with a mass concentration of 0.5-3% prepared by sterile phosphate buffered saline (PBS) and sterile BSA solution, which is used to dilute the fluorescently modified IgE detection antibody stock solution.

[0013] In the above scheme, the antigen blocking solution is a 2-5% BSA solution prepared with sterile phosphate buffered saline (PBS), sterile BSA solution, and sterile deionized water.

[0014] In the above scheme, the cell lysis buffer is a solution prepared with cell lysis buffer, protease inhibitor, and phosphatase inhibitor, and the cell washing buffer is a solution prepared with sterile PBS and sterile FBS.

[0015] A method for in situ quantitative detection of receptor-binding allergen-specific IgE based on single-cell microarrays, using the kit described above, includes the following steps: (1) Preprocessing: The single-cell microcavity array chip and the antigen barcode chip are preprocessed; (2) Single-cell loading: The separated basophil suspension is loaded onto the microcavity array chip, so that single cells fall into the microcavities and cells that do not enter the microcavities are washed away; (3) In situ lysis and capture: Cell lysis buffer is dropped onto the microcavity array chip, and then the antigen barcode chip is attached to the microcavity array chip and clamped with a plastic clamp. The chip is incubated at low temperature so that the receptor-binding receptor-binding allergen-specific IgE released by the lysis of a single basophil is captured in situ by the corresponding allergen component on the antigen barcode chip. (4) Signal detection: Remove the antigen barcode chip, add the fluorescently modified IgE detection antibody to the surface of the antigen barcode chip for incubation, then rinse and scan the chip to obtain the fluorescence signal; (5) Quantitative analysis: Using the quantitative formula established by the IgE standard, the fluorescence signal is converted into the absolute concentration of receptor-binding allergen-specific IgE.

[0016] In the above scheme, the pretreatment method is as follows: take the microcavity array chip out of the vacuum bag, wet the microcavity with cell washing solution, remove air bubbles in the microcavity, and keep the microcavity moist; take the antigen barcode chip out of the dry vacuum packaging and soak it in antigen blocking solution for 5-20 minutes before use.

[0017] In the above scheme, in step (3), the temperature for low-temperature incubation is 4℃ and the incubation time is 0.5-5 hours; in step (4), the incubation time is 20-50 minutes.

[0018] Through the above technical solution, the receptor-binding allergen-specific IgE in situ quantitative detection kit and method based on single-cell chip provided by the present invention has the following beneficial effects: 1. In-situ quantification at the single-cell level directly reflects the functional sensitization status of effector cells. This invention is the first to achieve in situ capture and absolute quantification of receptor-binding allergen-specific IgE (sIgE) on the membrane receptor of a single basophil. The detection results directly reflect the functional sIgE loading on the surface of effector cells that can be cross-linked by allergens, overcoming the defect that traditional serum sIgE detection cannot reflect the true sensitization potential of cells, and helping to explain the problem of inconsistency between serological indicators and clinical phenotypes.

[0019] 2. Achieving both high throughput and high sensitivity By employing a separate coupling design of the "high-density single-cell microcavity array chip" and the "barcode chip," the sensitivity loss caused by increasing the microcavity volume to enhance detection metrics is avoided. This invention achieves a single-cell detection throughput of over 30,000 cells and a detection sensitivity of 1 pg / mL for IgE, thus unifying high throughput with high sensitivity and meeting the needs for detecting rare clinical samples and low-abundance membrane proteins.

[0020] 3. Parallel detection of multiple allergens for precise assessment of sensitization spectrum. Antigen barcode chips can simultaneously print multiple allergen components (such as Derp1, Derp2, Feld1, etc.), enabling parallel reading of multiple receptor-binding sIgE on the surface of a single basophil, supporting the extension of the allergen component diagnosis (CRD) concept at the single-cell level, and providing patients with a more refined sensitization spectrum assessment.

[0021] 4. Simple operation, no complicated valve devices required. This invention employs a one-step lysis capture process of "dropping lysis buffer + bonding and clamping + low-temperature incubation", which eliminates the valve structure required for guiding the lysis buffer in traditional microfluidic chips. This significantly reduces the difficulty of chip manufacturing and the complexity of operation, improves the repeatability and batch consistency of detection, and facilitates clinical application.

[0022] 5. Small sample requirements, suitable for valuable clinical samples Each microcavity chip requires only tens of thousands of single-cell data points, making it particularly suitable for clinical samples with a low percentage of basophils in peripheral blood, thus reducing the burden of blood collection for patients.

[0023] 6. Complements the formation mechanism of BAT, providing single-cell retrospective capability. The absolute quantitative results of single-cell receptor-binding sIgE provided by this invention can serve as an upstream mechanism anchor for the basophil activation assay (BAT), tracing the differences in cellular responses back to the sIgE load level of the same cell, thus compensating for the inability of BAT population readings to resolve cellular heterogeneity.

[0024] 7. The results are reliable and support absolute quantification and cross-batch comparisons. Establish a quantitative curve using IgE standards (R) 2 >0.98), uniformly converting fluorescence signals into absolute concentrations, achieving quantitative comparability between different experimental batches and samples, and providing a standardized tool for clinical risk assessment and monitoring of immunotherapy efficacy.

[0025] 8. Outstanding clinical application value This invention directly detects functional sIgE in effector cells, which is expected to be used to predict the early efficacy of allergen-specific immunotherapy (AIT), identify highly sensitized subgroups, guide personalized treatment decisions, and make up for the shortcomings of existing biomarkers, thus having broad prospects for clinical translation.

[0026] In summary, this invention provides a simple, cost-effective, and high-performance single-cell level receptor-binding sIgE detection method, solving the long-standing clinical problem of "inaccurate assessment of the true sensitization status of effector cells". Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0028] Figure 1 This is a schematic diagram of a single-cell microcavity array chip and an antigen barcode chip, as shown in the embodiment. Figure 2The flowchart illustrates the in situ quantitative detection of single basophil receptor-binding sIgE as an example. Figure 3 The example kit shows the sensitivity graph for detecting the concentration gradient of IgE standards. Figure 4 The following is a clustering diagram of basophils based on the expression of receptor-binding sIgE as an example. Figure 5 This is a four-dimensional distribution diagram of receptor-binding sIgE expression in a single basophil cell, as shown in the example. Figure 6 This example illustrates the correlation analysis between different allergens corresponding to a single basophil receptor-binding sIgE.

[0029] In the figure, 1. Single-cell microcavity array chip; 2. Antigen barcode chip; 3. Microcavity; 4. Antigen channel. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0031] Example 1 A single-cell chip-based reagent kit and method for in situ quantitative detection of receptor-binding allergen-specific IgE.

[0032] 1. Screening of relevant allergens (antigens) in clinical allergy patients Based on literature review and preliminary clinical sample analysis, this embodiment selects the following 12 common or representative allergen components as capture antigens: Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Art v 1, Art v 3, Art v 4, Fel d 1, Can f 1, Bet v 1, and Phl p 1. These antigens are derived from major allergens such as house dust mites, artemisia, cats, dogs, birch, and timothy hay, covering the spectrum of common inhaled allergens in clinical practice.

[0033] 2. Components and preparation of the reagent kit The kit provided in this embodiment contains the following components (see Table 1): Table 1. Kit Components like Figure 1As shown, the single-cell microcavity array chip 1 was fabricated as follows: polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, poured into a mold, baked at 65°C for 2 hours, and then demolded. The chip has a single-layer structure, with 84,829 microcavities 3 on the upper surface, each microcavity having dimensions of 500 μm (length) × 40 μm (width) × 30 μm (depth).

[0034] Fabrication of antigen barcode chip 2: Ten parallel channels are fabricated on a glass substrate using microfluidic channel printing technology. Each channel is coated with one or more of the above 12 allergen components (combinations can be selected as needed). The channel width is 50 μm and the channel spacing is 100 μm. This ensures that each microcavity corresponds to multiple antigen channels 4, enabling parallel capture of multiple indicators.

[0035] Antibody buffer: Prepare a 1% (w / w) BSA solution using sterile phosphate-buffered saline (PBS) and sterile BSA solution.

[0036] Antigen blocking solution: Prepare a 3% (w / w) BSA solution using sterile PBS, sterile BSA solution, and sterile deionized water.

[0037] Cell lysis buffer: Use mild RIPA lysis buffer, add 1:1000 diluted protease inhibitor and 1:1000 diluted phosphatase inhibitor, and mix well.

[0038] Cell washing solution: Prepare a solution containing 2% FBS using sterile PBS and sterile FBS.

[0039] Fluorescently modified IgE detection antibody: Dilute the fluorescently labeled anti-human IgE antibody with antibody buffer at a ratio of 1:200 to ensure a final concentration greater than 100 μg / mL.

[0040] 3. Detection Method The detection process in this embodiment is as follows: Figure 2 As shown, the specific steps are as follows: (1) Preprocessing of single-cell microcavity array chip Remove the single-cell microcavity array chip from the vacuum packaging and perform hydroxylation treatment using a Plasma instrument (50 W power, 1 minute). Then immediately wet the microcavity with cell washing solution, gently shake or vacuum degas to remove residual air bubbles in the microcavity, and keep the microcavity moist for later use.

[0041] (2) Antigen barcode chip preprocessing Remove the antigen barcode chip from the dry vacuum packaging, soak it in the antigen blocking solution at room temperature for 12 minutes, and then spin dry or blow dry the surface liquid with nitrogen for later use.

[0042] (3) Basophil extraction Five mL of peripheral blood was drawn from patients with clinical allergies, and basophils were extracted using density gradient centrifugation or negative immunomagnetic bead sorting. The cell suspension was collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and a white cell pellet was visible at the bottom of the tube. The cells were resuspended in cell washing buffer and washed once, then centrifuged again and the supernatant was discarded. Finally, the cells were resuspended in 1 mL of RPMI 1640 medium containing 10% FBS and counted using a hemocytometer or automated cell counter, adjusting the cell density to approximately 8 × 10⁶ cells / mL. 4 per mL.

[0043] (4) Single-cell loading The pretreated microcavity array chip was placed on a horizontal platform with the cavity side facing upwards. 1 mL of cell suspension was evenly spread on the chip surface using a pipette, ensuring complete coverage of the microcavity area. The chip was allowed to settle under gravity for 6 minutes, allowing individual cells to fall into the microcavities. The chip surface was then gently rinsed three times with cell washing buffer to remove any excess cells that had not entered the microcavities. The microcavity filling rate was observed under a microscope, typically exceeding 85%.

[0044] (5) In situ cleavage and capture of receptor-binding sIgE The microcavity array chip loaded with single cells was placed on a 4°C cooling stage. 40 μL of pre-cooled cell lysis buffer was added to the edge of the chip surface, on the side without microcavities. Then, the antigen barcode chip (coated side down) was slowly tilted down from the side where the lysis buffer was added towards the microcavity side, allowing the lysis buffer to evenly saturate all microcavities through capillary action. The two chips were clamped together with plastic clamps, ensuring tight contact and no air bubbles. The assembled chip was incubated at 4°C for 1 hour. During this process, basophils within the microcavities were lysed, and the released membrane receptor-binding sIgE was captured in situ by the corresponding allergen components on the antigen barcode chip.

[0045] (6) Immunofluorescence detection of receptor-binding sIgE After incubation, carefully disassemble the clamp and remove the antigen barcode chip. Gently rinse the chip surface once with antibody buffer to remove unbound material. Take 400 μL of fluorescently labeled anti-human IgE detection antibody working solution diluted with antibody buffer and evenly drop it onto the surface of the antigen barcode chip. Incubate at room temperature in the dark for 45 minutes. After incubation, rinse the chip twice with antibody buffer for 1 minute each time, then rinse briefly with deionized water and dry by shaking or blowing with nitrogen. Place the chip in a fluorescence scanner, set the appropriate channel according to the excitation / emission wavelength of the fluorescent dye, and scan to obtain fluorescence images and signal intensity.

[0046] (7) Quantitative calculation Beforehand, IgE standards (concentration gradient: 1000 μg / mL to 0.1 pg / mL, 10-fold serial dilution) were used to perform detection under the same conditions to obtain a standard curve of fluorescence signal intensity versus IgE concentration (see [link to data]). Figure 3 The standard curve equation obtained in this embodiment is: Y = 145.4 lgX + 145.4, and the correlation coefficient R0 is... 2 > 0.98, the detection limit reaches 1 pg / mL. Substitute the fluorescence signal value of the sample into the above equation to convert it into the absolute concentration (pg / mL or fg / cell) of receptor-binding sIgE on the surface of each single cell.

[0047] 4. Results Analysis (1) Detection sensitivity and throughput like Figure 3 As shown, the kit of this invention achieves a detection sensitivity of 1 pg / mL for IgE, exhibiting good linearity across a concentration range of 10 orders of magnitude. The single-cell microcavity array chip has an effective single-cell throughput of 31,386 (based on 84,829 microcavities minus empty and multi-cell cavities), enabling the simultaneous acquisition of receptor-binding sIgE expression data from tens of thousands of single cells, meeting the requirements for high-throughput statistical analysis.

[0048] (2) Heterogeneity analysis of single-cell receptor-binding sIgE expression Cluster analysis, four-dimensional distribution analysis, and correlation analysis were performed on the sIgE signals corresponding to different antigens in each single cell using specialized analysis software. The results are as follows: Figure 4 , Figure 5 , Figure 6 As shown.

[0049] Clustering analysis ( Figure 4 Based on the expression profiles of sIgE specific to 12 receptor-binding allergens, basophils can be divided into multiple subsets. Significant differences in sIgE expression patterns exist among the different subsets, with some highly expressed subsets dominating, reflecting significant intercellular heterogeneity and demonstrating the necessity of single-cell level detection.

[0050] Four-dimensional distribution analysis Figure 5 Four representative allergens (Der p1, Fel d1, Bet v1, and Phl p1) were selected to demonstrate the sIgE expression distribution of individual cells across these four dimensions. The results showed that different cells exhibited significant differences in their reactivity to different allergens; some cells showed a high response to only a single allergen, while others showed co-sensitization to multiple allergens.

[0051] Correlation analysis ( Figure 6Correlation analysis of receptor-binding sIgE expression among the main components of house dust mites, Der p1, Der p2, Der p5, Der p7, and Der p10, revealed a high positive correlation between Der p1 and Der p2 (R=0.78), and a high correlation between Der p7 and Der p2 (R=0.69). However, Der p10 showed a low correlation with other components, suggesting differences in cross-sensitization or co-sensitization patterns among different allergen components, providing single-cell-level evidence for clinical component diagnosis.

[0052] The above results indicate that the kit and detection method provided by this invention can successfully achieve in situ capture, quantitative detection, and multi-dimensional data analysis of single basophil membrane receptor-binding sIgE. It has the advantages of high sensitivity, high throughput, parallel operation of multiple indicators, simple operation, and small sample requirement. It can accurately reflect the true sensitization status of effector cells and is expected to be used for risk assessment, efficacy prediction, and personalized diagnosis and treatment of allergic diseases.

[0053] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A single cell chip-based receptor-binding type allergen-specific IgE in situ quantitative detection kit, characterized in that, The application relates to a single-cell microcavity array chip, an antigen bar code chip, a plastic clamp, an IgE standard, a fluorescently modified IgE detection antibody and an auxiliary reagent. The single-cell microcavity array chip is used for capturing and containing single basophils. The antigen bar code chip is used for in-situ capturing of receptor-binding allergen-specific IgE combined with the membrane receptors of single basophils released from the microcavity array chip. The plastic clamp is used for clamping the single-cell microcavity array chip and the antigen bar code chip to form a closely contacted interface. The IgE standard is used for establishing a concentration quantitative standard curve. The fluorescently modified IgE detection antibody is used for combining with the captured receptor-binding allergen-specific IgE to generate a detectable fluorescent signal. The auxiliary reagent includes an antibody buffer, an antigen blocking solution, a cell lysis solution and a cell washing solution.

2. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The single-cell microcavity array chip is prepared from polydimethylsiloxane and a curing agent, and the upper surface of the single-cell microcavity array chip comprises a plurality of microcavities for containing single cells.

3. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The antigen bar code chip comprises a plurality of different antigen channels, the channel width is 5-100 mu m, and the interval is 10-200 mu m.

4. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The allergen components printed on the antigen bar code chip include Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Art v 1, Art v 3, Art v 4, Fel d 1, Can f 1, Betv 1 and Phl p 1.

5. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The antibody buffer is a BSA solution with a mass concentration of 0.5-3% prepared from sterile phosphate buffer PBS and sterile BSA solution, and is used for diluting the fluorescently modified IgE detection antibody stock solution.

6. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The antigen blocking solution is a BSA solution with a mass concentration of 2-5% prepared from sterile phosphate buffer PBS, sterile BSA solution and sterile deionized water.

7. The single cell chip-based receptor binding type allergen specific IgE in situ quantitative detection kit according to claim 1, characterized in that, The cell lysis solution is a solution prepared from a cell lysis solution, a protease inhibitor and a phosphatase inhibitor, and the cell washing solution is a solution prepared from sterile PBS and sterile FBS.

8. A method for in situ quantitative detection of allergen-specific IgE based on a single cell chip, using the kit according to any one of claims 1 to 7, characterized in that, The application further discloses a single-cell microcavity array chip antigen bar code chip method for detecting receptor-binding allergen-specific IgE. The method comprises the following steps: (1) pretreatment: pretreating the single-cell microcavity array chip and the antigen bar code chip; (2) single-cell loading: loading the separated basophil cell suspension onto the microcavity array chip, allowing single cells to fall into the microcavities, and flushing away the cells not entering the microcavities; (3) in-situ lysis and capture: adding the cell lysis solution on the microcavity array chip, then pasting the antigen bar code chip to the microcavity array chip and clamping with the plastic clamp, and incubating at low temperature, so that the receptor-binding allergen-specific IgE released by the single basophils is in-situ captured by the corresponding allergen components on the antigen bar code chip; (4) signal detection: removing the antigen bar code chip, adding the fluorescently modified IgE detection antibody to the surface of the antigen bar code chip for incubation, then flushing and scanning the chip to obtain a fluorescent signal; (5) quantitative analysis: converting the fluorescent signal into the absolute concentration of the receptor-binding allergen-specific IgE by using the quantitative formula established by the IgE standard.

9. The method according to claim 8, wherein the method is a single cell-chip based in situ quantitative detection method for specific IgE of allergen receptor binding type, characterized in that, The pretreatment method is as follows: Take the microcavity array chip out of the vacuum bag, wet the microcavity with cell washing solution, remove air bubbles in the microcavity, and keep the microcavity moist; take the antigen barcode chip out of the dry vacuum packaging and soak it in antigen blocking solution for 5-20 minutes before use.

10. The method according to claim 8, wherein the method is a single cell-chip based in situ quantitative detection method for specific IgE of allergen receptor binding type. In step (3), the temperature for low-temperature incubation is 4℃ and the incubation time is 0.5-5 hours; in step (4), the incubation time is 20-50 minutes.

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