Food allergen visual detection chip and preparation method thereof

Abstract

The invention discloses a food allergen visual detection chip preparation method, which comprises: using a nitrocellulose membrane as a carrier, coating an allergen component protein antibody dissolved in a PBS buffer solution system on the nitrocellulose membrane by using a chip spotting instrument, and drying to form a detection reagent strip; preparing a liquid reagent required by a reaction system, and subpackaging to form a liquid detection reagent; and assembling the detection reagent strip and the liquid detection reagent component into a complete kit. The invention has the following technical effects: high sensitivity and specificity: multiple allergens can be detected at the same time, and cross reaction is reduced; compared with a traditional method, the method has the advantages that the detection time is remarkably shortened, and the working efficiency is improved. Through a visual result, non-professionals can easily understand a detection result, and the attention of the public to food safety is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a food allergen visual detection chip and a preparation method thereof. Background Art

[0002] The detection of food allergens is an important research topic in the current public health field. With the increasing prevalence of food allergy symptoms, especially in developed countries, about 1% to 2% of adults and more than 8% of children worldwide are affected by food allergies. [1] Existing detection methods mainly include immunochemical tests (such as IgE inhibition test and immunoblotting test) and mediator release tests (such as histamine release rate test and β-hexosaminidase release rate test). Although these methods can detect allergens to a certain extent, they have some defects. For example, the determination results of specific IgE may be affected by the same specific IgG and may not be directly related to clinical symptoms. [1] .

[0003] Currently, the detection technology for food allergens is constantly developing, but the problem of cross-sensitization still needs to be solved, and the sensitivity and accuracy of detection need to be improved. Existing technologies often face challenges when processing complex samples, so there is an urgent need to develop new detection devices and methods to improve the ability to identify food allergens. Protein component allergen chip detection technology can help doctors understand the specific conditions of patients and provide correct treatment and preventive measures, which plays a vital role in the chronic disease management of allergic diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a food allergen visual detection chip and preparation method to efficiently and accurately detect allergens in food, specifically including: improving the ability to detect multiple allergens simultaneously. Reducing detection costs and shortening detection time. Enhancing the visualization of detection results to facilitate user understanding and application in order to improve the ability to identify food allergens.

[0005] According to a first aspect of the present invention, a method for preparing a food allergen visual detection chip comprises:

[0006] Using nitrocellulose membrane as a carrier, the allergen component protein antibodies dissolved in PBS buffer system are coated on the nitrocellulose membrane using a chip spotter, and after drying, a detection reagent strip is formed;

[0007] Prepare the liquid reagents required for the reaction system and package them to form liquid detection reagents;

[0008] The detection reagent strip and the liquid detection reagent are assembled into a complete test kit.

[0009] Preferably, the allergen component protein antibodies include: one or more of rabbit anti-milk, egg or peanut allergen component protein antibodies.

[0010] Preferably, the egg allergen components are Gald 1, Gald 2, Gald 3, and Gald 4; the milk allergen components are Bosd 4, Bosd 5, Bosd 6, and Bosd 8; and the peanut allergen components are Arah 2, Arah 6, Arah 8, and Arah 9; and their purities are all greater than 95%.

[0011] Preferably, the allergen component protein antibody further comprises: shrimp allergen component protein antibody or crab allergen component protein antibody; the shrimp allergen component is Mete 1, and the crab allergen component is Chaf 1.

[0012] Preferably, the drying refers to drying at a temperature of 37±10%°C for more than 24 hours.

[0013] Preferably, the liquid detection reagent comprises biotin-labeled anti-rabbit IgE antibody, streptavidin-alkaline phosphatase solution, BCIP / NBT substrate solution, and washing solution.

[0014] Preferably, the preparation of biotin-labeled anti-human IgE antibody comprises: dissolving anti-rabbit IgE antibody in NaHCO3 (0.1 mol / L); dissolving biotin in 0.5 mL DMSO to obtain a biotin solution;

[0015] The prepared biotin DMSO solution was slowly added to the solution in which the anti-rabbit IgE was dissolved, and the mixture was mixed and incubated at room temperature for 1 hour. The reactant was then dialyzed to obtain the biotin-labeled anti-human IgE antibody.

[0016] According to a second aspect of the present invention, a food allergen visual detection chip comprises:

[0017] Using nitrocellulose membrane as a carrier, the allergen component protein antibodies dissolved in PBS buffer system are coated on the nitrocellulose membrane using a chip spotter and then dried to form a detection reagent strip;

[0018] Prepare liquid reagents required for the reaction system and subpackage the resulting liquid detection reagents;

[0019] A test kit is formed by grouping the detection reagent strip and the liquid detection reagent.

[0020] Preferably, the allergen component protein antibodies include: one or more of rabbit anti-milk, egg or peanut allergen component protein antibodies.

[0021] The egg allergen components are Gald 1, Gald 2, Gald 3, and Gald 4; the milk allergen components are Bosd 4, Bosd 5, Bosd 6, and Bosd 8; and the peanut allergen components are Arah 2, Arah 6, Arah 8, and Arah 9; and their purities are all >95%.

[0022] The present invention has the following technical effects: high sensitivity and specificity: it can detect multiple allergens at the same time and reduce cross-reactions; fast and convenient: compared with traditional methods, it significantly shortens the detection time and improves work efficiency. By visualizing the results, non-professionals can also easily understand the test results, which promotes the public's attention to food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of a method for preparing a food allergen visual detection chip of the present invention. DETAILED DESCRIPTION

[0024] like Figure 1 As shown, a method for preparing a food allergen visual detection chip of the present invention comprises: using a nitrocellulose membrane as a carrier, coating the allergen component protein antibody dissolved in a PBS buffer system onto the nitrocellulose membrane by a chip spotter, and forming a detection reagent strip after drying; configuring the liquid reagent required by the reaction system and packaging it to form a liquid detection reagent; and packaging the detection reagent strip and the liquid detection reagent into a complete test kit.

[0025] The above-mentioned allergen component protein antibodies include: one or more of rabbit anti-milk, egg or peanut allergen component protein antibodies.

[0026] The egg allergen components are Gald 1, Gald 2, Gald 3, and Gald 4; the milk allergen components are Bosd 4, Bosd 5, Bosd 6, and Bosd 8; and the peanut allergen components are Arah 2, Arah 6, Arah 8, and Arah 9; and their purities are all greater than 95%.

[0027] The above-mentioned allergen component protein antibodies also include: shrimp allergen component protein antibodies or crab allergen component protein antibodies; the shrimp allergen component is Mete 1, and the crab allergen component is Chaf 1.

[0028] The above-mentioned drying refers to drying at a temperature of 37±10%°C for more than 24 hours.

[0029] The liquid detection reagent includes biotin-labeled anti-rabbit IgE antibody, streptavidin-alkaline phosphatase solution, BCIP / NBT substrate solution, and washing solution.

[0030] The preparation of the biotin-labeled anti-human IgE antibody comprises: dissolving the anti-rabbit IgE antibody in NaHCO3 (0.1 mol / L); dissolving biotin in 0.5 mL DMSO to obtain a biotin solution;

[0031] The prepared biotin DMSO solution was slowly added to the solution in which the anti-rabbit IgE was dissolved, and the mixture was mixed and incubated at room temperature for 1 hour. The reactant was then dialyzed to obtain the biotin-labeled anti-human IgE antibody.

[0032] The present invention also provides a food allergen visual detection chip, which comprises: a detection reagent strip formed by coating allergen component protein antibodies dissolved in a PBS buffer system onto a nitrocellulose membrane using a nitrocellulose membrane as a carrier using a chip spotter and then drying the coated antibodies; a liquid detection reagent formed by configuring and packaging liquid reagents required for the reaction system; and a test kit formed by packaging the detection reagent strip and the liquid detection reagent.

[0033] The details of the above contents of the present invention are described in detail below.

[0034] Allergen protein gene sequence synthesis, expression and purification

[0035] The present invention first determines the allergenic molecules of common allergens and retrieves the reference sequences in GenBank, and then optimizes the sequences for expression in yeast and Escherichia coli. A variety of protein purification methods are used, and the allergen activity is detected by agarose immunodiffusion method and enzyme-linked immunosorbent assay. The gel excision purification method is selected to maintain the protein antigenicity. The recombinant plasmid is extracted and double-enzyme digested, transformed into Escherichia coli and Pichia pastoris for positive clone screening, and finally the purity of the allergen protein is detected by SDS-PAGE. The protein test sample is analyzed by SDS-PAGE, and the purity percentage is calculated by the signal intensity of the target band to the signal intensity of all bands. The report results show that the purity of egg allergen components Gald 1, Gald 2, Gald 3, Gald 4, shrimp allergen components Mete 1, crab Chaf 1, milk allergen components Bosd 4, Bosd 5, Bosd 6, Bosd 8, peanut Arah 2, Arah 6, Arah 8, Arah 9 is all greater than 95%.

[0036] Preparation and optimization of allergen component chip carrier

[0037] Protein chip is a high-throughput protein function analysis technology, which requires efficient fixation of proteins and maintenance of their activity. The chip using glass slides as carriers and agarose as a three-dimensional structure has advantages in performance, but there are problems of incompatibility with glass surfaces and easy shrinkage and deformation. In order to improve stability and reactivity, the present invention uses coupling agents such as affinity silanes, mercaptosilanes, and aminosilanes to modify glass slides to enhance the binding ability of the agarose layer. The surface characteristics of the chip are analyzed by atomic force microscopy and X-ray photoelectron spectroscopy, the effects of different modifications on chip performance are evaluated, and its application in protein detection is explored. By comparing with the ImmunoCAP fluorescence system, the sensitivity and accuracy of the allergen-specific IgE antibody protein chip test sample are evaluated. The accuracy is analyzed using Bland-altman, and the analysis shows that the test results of the test sample allergen-specific IgE antibody protein chip are significantly linearly correlated with the test results of the ImmunoCAP fluorescence system. The detection limit of the test allergen-specific IgE antibody protein chip was <0.1 IU / mL, and the sensitivity and accuracy of the test allergen-specific IgE antibody protein chip were consistent with the ImmunoCAP fluorescence system.

[0038] Preparation of detection probes for allergen component-specific slide-loaded antibodies and fluorescently labeled anti-IgE antibodies

[0039] The present invention focuses on the use of immunological technology to detect allergen proteins, using polyclonal and monoclonal antibodies. Polyclonal antibodies target multiple epitopes, have high activity, are suitable as capture antibodies, and enhance the binding efficiency of the detected antigen on the protein chip; while monoclonal antibodies have high specificity and can accurately reflect the amount of the detected antigen. By preparing these two types of antibodies separately, an important material basis is provided for the establishment of an immune chip method for detecting allergens. The antibodies are indirectly tested by the corresponding antigen on the ELISA platform, and the antibody titer is determined by the signal value. The report shows that the titers of mouse anti-human IgE antibody, rabbit anti-Gald 1 antibody, rabbit anti-Gald 2 antibody, rabbit anti-Gald 3 antibody, rabbit anti-Gald 4 antibody, rabbit anti-Pena 1 antibody, rabbit anti-Bosd 4 antibody, rabbit anti-Bosd5 antibody, rabbit anti-Bosd 6 antibody, rabbit anti-Bosd 8 antibody, rabbit anti-Arah 2 antibody, rabbit anti-Arah 6 antibody, rabbit anti-Arah 8 antibody, and rabbit anti-Arah 9 antibody are all greater than 10^5.

[0040] Preparation and quality control of allergen component chips

[0041] The production of allergen component chips mainly consists of multiple parts such as substrate processing, microarray spotting, antibody fixation, and chip sealing. Proteins with high purity and intact biological activity are dissolved in PBS buffer containing 40% glycerol to prevent protein denaturation and evaporation of droplets formed in the subsequent spotting step. Use a commercial contact spotter or non-contact inkjet method to spot protein microarrays, and directly fix the ligands to be fixed by chemical bonds through chemical methods to complete the antibody fixation link. Finally, use appropriate blocking agents such as BSA, Gly, etc. to block the unreacted aldehyde groups on the substrate to reduce the nonspecific adsorption of the substrate to the sample to form false positives and remove unnecessary background interference. After completing the blocking step, use a PBS solution containing 0.1% Tween-20 to repeatedly wash the chip to remove excess blocking solution.

[0042] The protein chip has high fixation efficiency and can be used as a carrier for subsequent diagnostic products. Three batches of trial production were carried out, and the analytical performance test results of the three batches of products all met the enterprise standards, proving that the production process is reasonable and the quality control meets the requirements, ensuring that the products produced are of qualified quality.

[0043] Clinical efficacy validation of allergen component chips

[0044] A convenience sampling method was used to collect data from about 300 patients with suspected food allergies, drug allergies, allergic rhinitis, asthma, eczema, etc. who visited Peking University First Hospital in 2022. The allergen protein chip and ImmunoCAP detection system developed by our team were used to detect sIgE of allergens in patient serum samples.

[0045] The sera used were all from patients with suspected food allergies, drug allergies, allergic rhinitis, asthma, eczema and other allergies. Fresh peripheral blood was drawn from the patients, and serum was obtained by centrifugation and frozen at -80°C for use. The specific methods were as follows: ① 5 mL of peripheral venous blood was drawn from the patients aseptically and stored in a yellow non-anticoagulant tube; ② Centrifuged at 2500rpm for 5 minutes; ③ Gently aspirate the upper serum, transfer it to an EP tube, and freeze it in a -80°C refrigerator for use.

[0046] The data of the quantitative test results were statistically analyzed and the following statistical data were given: the test results were statistically analyzed using a four-cell table to obtain the positive compliance rate, negative compliance rate, total compliance rate and Kappa value; the correlation coefficient r, determination coefficient R2, regression equation Y=a+bX, scatter plot, and significance analysis of the correlation coefficient and the slope of the regression equation were given.

[0047] The invention results show that the allergen protein chip developed by our team has good consistency with the ImmunoCAP detection system, and the clinical invention performance indicators can better meet the requirements of in vitro clinical testing. It can be clinically used for in vitro detection of allergen component-specific antibody IgE concentration in human serum, and can be clinically used for in vitro auxiliary diagnosis of type I hypersensitivity diseases.

[0048] Although the present invention is described in detail above, the present invention is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made based on the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A method for preparing a food allergen visual detection chip, comprising: Using nitrocellulose membrane as a carrier, the allergen component protein antibodies dissolved in PBS buffer system are coated on the nitrocellulose membrane using a chip spotter, and after drying, a detection reagent strip is formed; Prepare the liquid reagents required for the reaction system and package them to form liquid detection reagents; The detection reagent strip and the liquid detection reagent are assembled into a complete test kit.

2. The preparation method according to claim 1, wherein the allergen component protein antibody comprises: One or more of rabbit antibodies against components of milk, egg or peanut allergens.

3. The preparation method according to claim 2, wherein The egg allergen components are Gald 1, Gald2, Gald 3, and Gald 4; the milk allergen components are Bosd 4, Bosd 5, Bosd 6, and Bosd 8; the peanut allergen components are Arah 2, Arah 6, Arah 8, and Arah 9; and their purities are all >95%.

4. The preparation method according to claim 2, wherein the allergen component protein antibody further comprises: Antibodies to shrimp allergen component proteins or antibodies to crab allergen component proteins; The shrimp allergen component is Mete 1 and the crab allergen component is Chaf 1.

5. The preparation method according to claim 1, wherein The drying refers to drying at a temperature of 37±10%°C for more than 24 hours.

6. The preparation method according to claim 1, wherein The liquid detection reagent includes biotin-labeled anti-rabbit IgE antibody, streptavidin-alkaline phosphatase solution, BCIP / NBT substrate solution, and washing solution.

7. The preparation method according to claim 1, wherein The preparation of biotin-labeled anti-human IgE antibody includes: The anti-rabbit IgE antibody was dissolved in NaHCO3 (0.1 mol / L); Dissolve biotin in 0.5 mL DMSO to obtain a biotin solution; The prepared biotin DMSO solution was slowly added to the solution in which the anti-rabbit IgE was dissolved, and the mixture was mixed and incubated at room temperature for 1 hour. The reactant was then dialyzed to obtain the biotin-labeled anti-human IgE antibody.

8. A food allergen visual detection chip, comprising: Using nitrocellulose membrane as a carrier, the allergen component protein antibodies dissolved in PBS buffer system are coated on the nitrocellulose membrane using a chip spotter and then dried to form a detection reagent strip; Prepare liquid reagents required for the reaction system and subpackage the resulting liquid detection reagents; A test kit is formed by grouping the detection reagent strip and the liquid detection reagent.

9. The chip according to claim 8, wherein the allergen component protein antibodies include: One or more of rabbit antibodies against components of milk, egg or peanut allergens.

10. The chip according to claim 9, wherein: The egg allergen components are Gald 1, Gald 2, Gald 3, and Gald 4; the milk allergen components are Bosd 4, Bosd 5, Bosd 6, and Bosd 8; the peanut allergen components are Arah 2, Arah 6, Arah 8, and Arah 9; and their purities are all >95%.

Images