A reagent and kit for quantitative detection of egg white allergen-specific IgE

Abstract

The application provides a reagent and kit for quantitatively detecting chicken egg protein allergen specific IgE. The kit comprises biotin-labeled chicken egg protein allergen and a preservative solution, the biotin-labeled chicken egg protein allergen comprises Sulfo-NHS-biotin-labeled ovomucin, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin and Sulfo-NHS-PEG4-biotin-labeled lysozyme with a mass ratio of (0.5-1):1:(0.5-1):0.5, and the preservative solution is a phosphate buffer solution containing 1-2 wt% BSA, 1-3 wt% glycerol and 2-5 wt% mannitol. The reagent has higher detection rate, detection sensitivity and reliability, the detection result has higher consistency with clinical symptoms, and the batch consistency of the finished product is better.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics of biological medical devices, specifically relating to a quantitative detection kit for egg white allergen-specific IgE. Background Technology

[0002] Eggs are a common food allergen and one of the eight most common food allergens listed by the FDA. Eggs are often the first complementary food introduced to infants and young children, accounting for 35% of food allergies in infants and children. In my country, egg allergy is second only to milk allergy and is also prevalent among adults, accounting for 12% of adult food allergies. Epidemiological surveys have found that the prevalence of egg allergy continues to rise, making accurate detection and prevention of food allergies particularly important.

[0003] Egg allergy is primarily mediated by specific immunoglobulin E (IgE), which binds to mast cells or basophils, leading to the release of allergic mediators. This activates T cells, basophils, and eosinophils, resulting in a type I hypersensitivity reaction. Symptoms include vomiting, abdominal pain, diarrhea, and urticaria, severely impacting the patient's quality of life. Eggs are rich in vitamins, protein, and fatty acids; simply avoiding them can easily lead to malnutrition. Accurate diagnosis can correctly identify the allergen and allow for the appropriate selection of how to consume eggs.

[0004] Six major egg allergens have been identified. Egg white primarily contains ovomucoid (Gald1), ovalbumin (Gald2), ovotransferrin (Gald3), and lysozyme (Gald4), while egg yolk primarily contains α-vitilin (Gald5) and vitellin (Gald6). Studies have shown that the allergenicity of egg whites is significantly higher than that of egg yolks. Egg whites contain 23 allergenic or non-allergenic proteins, with ovomucoid, ovalbumin, ovotransferrin, and lysozyme accounting for 80.4% of the total protein content.

[0005] Currently, egg white allergen detection kits mainly include those using Western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, and magnetic particle chemiluminescence immunoassay. Among these, magnetic particle chemiluminescence immunoassay offers better detection sensitivity and enables fully automated, fully quantitative detection, making it the preferred method. Most egg white allergens used for in vitro diagnostics are naturally extracted allergens containing various sensitizing proteins. Ideally, when using these kits to prepare magnetic particle chemiluminescence immunoassay kits, the detection results should be more consistent with clinical symptoms and outcomes. However, in practice, it has been found that the consistency between the detection results and clinical outcomes of existing magnetic particle chemiluminescence immunoassay kits for detecting egg white allergens is still not ideal. Summary of the Invention

[0006] The purpose of this invention is to provide a quantitative detection kit for egg white allergen-specific IgE with higher consistency between detection results and clinical results, and more accurate detection results.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A quantitative detection kit for egg white allergen-specific IgE includes biotin-labeled egg white allergens and a preservation solution. The biotin-labeled egg white allergens include Sulfo-NHS-biotin-labeled ovomucoid, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin, and Sulfo-NHS-PEG4-Biotin-labeled lysozyme. The mass ratio of Sulfo-NHS-biotin-labeled ovomucoid, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin, and Sulfo-NHS-PEG4-Biotin-labeled lysozyme is (0.5-1):1:(0.5-1):0.5. The preservation solution is a phosphate buffer containing 1-2 wt% BSA, 1-3 wt% glycerol, and 2-5 wt% mannitol, and the pH of the preservation solution is 7.2-7.5.

[0009] Preferably, the mass ratio of Sulfo-NHS-biotin-labeled ovomucoid, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin and Sulfo-NHS-PEG4-Biotin-labeled lysozyme is (0.7-1):1:(0.5-0.8):0.5.

[0010] Preferably, the concentration of the biotin-labeled egg white allergen in the reagent is 0.5–5 μg / mL, for example, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, or 5 μg / mL.

[0011] Preferably, the labeling molar ratio of ovomucoid to Sulfo-NHS-biotin is 1:10 to 20, for example, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20.

[0012] Preferably, the labeling molar ratio of ovalbumin to Sulfo-NHS-LC-biotin is 1:10 to 20, for example, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20.

[0013] Preferably, the labeling molar ratio of ovotransferrin and Sulfo-NHS-biotin is 1:15 to 25, for example, 1:15, 1:18, 1:20, 1:22, 1:24, or 1:25.

[0014] Preferably, the labeling molar ratio of lysozyme and Sulfo-NHS-PEG4-Biotin is 1:15 to 25, for example, 1:15, 1:18, 1:20, 1:22, 1:24, or 1:25.

[0015] Preferably, ovomucoid and Sulfo-NHS-biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M CB buffer at pH 9.0-9.5 to obtain the Sulfo-NHS-biotin-labeled ovomucoid.

[0016] Preferably, ovalbumin and Sulfo-NHS-LC-biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M CB buffer at pH 9.0-9.5 to obtain the Sulfo-NHS-LC-biotin-labeled ovalbumin.

[0017] Preferably, ovalbumin and Sulfo-NHS-biotin are mixed and reacted at room temperature. After the reaction, the mixture is dialyzed with 0.05-0.2M PBS buffer at pH 7.2-7.5 to obtain the Sulfo-NHS-LC-biotin-labeled ovalbumin.

[0018] Preferably, the lysozyme and Sulfo-NHS-PEG4-Biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M PBS buffer at pH 7.2-7.5 to obtain the Sulfo-NHS-PEG4-Biotin-labeled lysozyme.

[0019] The present invention also provides a quantitative detection kit for egg white allergen-specific IgE, wherein the kit is a chemiluminescent kit and includes the above-mentioned reagents.

[0020] Preferably, the egg white allergen-specific IgE quantitative detection kit includes reagent 1, reagent 2, reagent 3, reagent 4 and luminescent substrate solution, wherein reagent 1 is a streptavidin-coated magnetic microparticle suspension; reagent 2 is the above-mentioned reagent; reagent 3 is an alkaline phosphatase-labeled mouse anti-human IgE antibody; reagent 4 is a biotin-labeled anti-human IgE antibody; and the luminescent substrate solution is an AMPPD solution.

[0021] Preferably, the concentration of reagent 1 is 0.4–1 mg / mL, more preferably 0.6–1 mg / mL.

[0022] Preferably, the solvent of reagent 1 is a phosphate buffer solution with a pH of 7.2 to 7.5 containing 1 to 2 wt% BSA, 1 to 3 wt% glycerol and 2 to 5 wt% mannitol.

[0023] Preferably, the concentrations of reagent 3 and reagent 4 are each independently 0.1 to 2.0 μg / mL.

[0024] More preferably, the concentration of reagent 4 is 0.1–0.5 μg / mL.

[0025] Preferably, the anti-human IgE antibody is mixed with Sulfo-NHS-LC-biotin and dialyzed with phosphate buffer containing 1-2 wt% BSA at a pH of 7.2-7.5 to obtain the alkaline phosphatase-labeled mouse anti-human IgE antibody.

[0026] Preferably, the egg white allergen-specific IgE quantitative detection kit further includes calibrators and quality control products.

[0027] According to some examples, the concentrations of the calibrators are 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, and 100 IU / mL.

[0028] According to some examples, the concentration of the quality control sample is 0.7 IU / ml and 17.5 IU / ml.

[0029] The present invention also provides the application of the above-mentioned reagents or egg white allergen-specific IgE quantitative detection kit in the detection of egg white allergen-specific IgE.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] This invention targets different allergenic proteins in egg white, specifically selecting biotin and labeling methods, and further combining them with specific compounding ratios to form a mixed antigen reagent for multiple allergenic proteins. This improves the detection rate, detection sensitivity, and reliability, resulting in higher consistency between detection results and clinical symptoms. By effectively controlling the batch-to-batch variation of raw materials, it is also beneficial to control the batch-to-batch variation of the finished egg white allergen-specific IgE quantitative detection kit, thus improving the batch-to-batch consistency of the quantitative detection kit. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0033] Currently, most egg white allergens used for in vitro diagnostics are naturally extracted allergens, containing various sensitizing proteins. Theoretically, the detection results should be more consistent with clinical symptoms. However, in clinical applications, it has been found that the consistency between the detection results of existing magnetic particle chemiluminescence kits for detecting egg white allergens and clinical results is still not ideal. After extensive research, the inventors believe that although naturally extracted allergens contain various types of sensitizing proteins, the extraction and purification methods for different sensitizing proteins vary due to different antigen sources and individual differences, leading to significant differences in antigen components between batches. Different sensitizing proteins also have different properties, and the same biotin labeling method is not suitable for all sensitizing proteins. This is the main reason why the sensitivity and accuracy of natural allergen detection are lower than theoretically predicted. Based on the above research, the inventors conducted extensive research and experimental verification, proposing to select different biochemical labeling methods for different sensitizing proteins and optimize the compounding ratio of different sensitizing proteins to obtain an egg white allergen-specific IgE quantitative detection kit with higher sensitivity, higher consistency between detection results and clinical results, and smaller batch-to-batch differences.

[0034] The technical solution of the present invention will be described below with reference to specific embodiments.

[0035] In the following examples, the implementation conditions not specified are standard conditions in the industry, and the reagents are all commercially available.

[0036] In the following examples, ovomucoid, ovalbumin, ovotransferrin and lysozyme were purchased from Indoor Biotechnologies, Biomay and SinoBiological, respectively.

[0037] Sulfo-NHS-biotin, Sulfo-NHS-LC-Biotin, and Sulfo-NHS-PEG4-Biotin were purchased from Thermo Fisher Scientific.

[0038] Streptavidin magnetic beads can be purchased from companies such as Thermo Fisher, Scientific, Roche, GE Healthcare, and Merck.

[0039] Natural egg white extract was purchased from Stallergenes Greer.

[0040] Example 1

[0041] This embodiment provides a quantitative detection kit for egg white allergen-specific IgE, which includes reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution.

[0042] Reagent 1 is a 0.6 mg / mL streptavidin-coated magnetic microparticle suspension, which is prepared as follows:

[0043] After mixing 10 mg / mL streptavidin magnetic beads, magnetic separation was performed. The mixture was then resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol. After washing three times, magnetic separation was performed again. Finally, the mixture was resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol to obtain a 0.6 mg / mL streptavidin-coated magnetic microparticle suspension.

[0044] Reagent 2 is a biotin-labeled egg white allergen mixture solution, which is prepared as follows:

[0045] Ovalbumin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the mixture was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled ovomucoid was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled ovomucoid solution.

[0046] Ovalbumin was added to the Sulfo-NHS-LC-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovalbumin was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-LC-biotin-labeled ovalbumin was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled ovalbumin solution.

[0047] Ovotransferrin was added to Sulfo-NHS-biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovotransferrin was dialyzed against 0.1M PBS buffer at pH 7.4. The prepared Sulfo-NHS-biotin-labeled ovotransferrin was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled ovotransferrin solution.

[0048] Lysozyme was added to Sulfo-NHS-PEG4-Biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the solution was dialyzed against 0.1M PBS buffer at pH 7.4. The prepared Sulfo-NHS-PEG4-Biotin-labeled lysozyme was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled lysozyme solution.

[0049] According to the mass ratio of ovomucoid, ovalbumin, ovotransferrin, and lysozyme of 0.8:1:0.7:0.5, the above biotin-labeled ovomucoid solution, biotin-labeled ovalbumin solution, biotin-labeled ovotransferrin solution, and biotin-labeled lysozyme solution were mixed to obtain a biotin-labeled egg white allergen mixed solution with a total allergen concentration of 5 μg / mL.

[0050] Reagent 3 is an alkaline phosphatase-labeled anti-human IgE antibody, which is prepared using conventional methods in the art: the alkaline phosphatase to be activated is dialyzed, then a coupling agent is added to react, the free coupling agent is separated and removed, then the activated alkaline phosphatase is added to the activated antibody solution to perform a coupling reaction, and then the free AP is separated and removed.

[0051] Reagent 4 is a biotin-labeled anti-human IgE antibody, which is prepared by adding the anti-human IgE antibody to Sulfo-NHS-LC-biotin dissolved in dimethylformamide, and then dialyzing with phosphate buffer containing 2 wt% BSA at pH 7.4.

[0052] Preparation of calibrators: IgE protein derived from WHO standards was diluted with 0.1M Tris-HCl buffer (pH 7.4) to prepare calibrators at concentrations of 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, and 100 IU / mL.

[0053] Preparation of quality control samples: IgE protein from WHO standards was used to prepare quality control samples of 0.7 IU / mL and 17.5 IU / mL using 0.1 M Tris-HCl buffer at pH 7.4.

[0054] The luminescent substrate solution is an alkaline phosphatase chemiluminescent substrate based on AMPPD luminescence, which is produced in-house. For details, please refer to the invention patent "An Enzymatic Chemiluminescent Substrate for Alkaline Phosphatase", authorized announcement number CN104990912B.

[0055] The detection method of the egg white allergen-specific IgE quantitative detection kit in this embodiment is as follows:

[0056] (1) The calibrator was mixed with streptavidin-coated magnetic microparticle suspension and biotin-labeled anti-human IgE antibody, respectively, and incubated at 37°C for 15 min. The supernatant was removed by magnetic separation and the mixture was washed three times.

[0057] (2) Add alkaline phosphatase-labeled anti-human IgE antibody, mix, incubate at 37°C for 15 min, remove supernatant by magnetic separation, and wash three times;

[0058] (3) Add the luminescent substrate solution, incubate at 37°C for 5 min, measure the chemiluminescence intensity, and plot a standard curve with the concentration of the calibrator as the x-axis and the chemiluminescence intensity as the y-axis.

[0059] (4) Mix the sample to be tested with streptavidin-coated magnetic microparticle suspension and biotin-labeled egg white allergen solution, incubate at 37°C for 15 min, remove the supernatant by magnetic separation, and wash three times.

[0060] (5) Add alkaline phosphatase-labeled anti-human IgE antibody, mix, incubate at 37°C for 15 min, remove supernatant by magnetic separation, and wash three times;

[0061] (6) Add the luminescent substrate solution, incubate at 37°C for 5 min, measure the chemiluminescence intensity, substitute it into the standard curve, and calculate the concentration of egg white allergen-specific IgE in the sample to be tested.

[0062] Example 2

[0063] This embodiment provides a quantitative detection kit for egg white allergen-specific IgE, which includes reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. Reagent 2, a biotin-labeled egg white allergen mixed solution, is basically the same as in Example 1, except that the mass ratio of ovomucoid, ovalbumin, ovotransferrin, and lysozyme in the biotin-labeled egg white allergen mixed solution is 0.5:1:0.5:0.5.

[0064] Example 3

[0065] This embodiment provides a quantitative detection kit for egg white allergen-specific IgE, which includes reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. Reagent 2, a biotin-labeled egg white allergen mixed solution, is basically the same as in Example 1, except that the mass ratio of ovomucoid, ovalbumin, ovotransferrin, and lysozyme in the biotin-labeled egg white allergen mixed solution is 1:1:1:0.5.

[0066] Comparative Example 1

[0067] A quantitative detection kit for egg white allergen-specific IgE is provided, comprising reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. Reagent 2, the biotin-labeled egg white allergen mixed solution, is a biotin-labeled natural egg white extract solution.

[0068] The egg white-specific IgE content in clinical samples (20 positive samples and 10 negative samples) was quantitatively detected using the egg white allergen-specific IgE quantitative detection kits of Examples 1-3 and Comparative Example 1. The results were compared with those obtained using a substitute egg white allergen mixture solution and the results obtained using Phadia's Immuno CAP assay. The results are shown in Table 1, unit: IU / mL.

[0069] Table 1

[0070] Sample number Phadia Comparative Example 1 Example 1 Example 2 Example 3 P1 0.42 0.15 0.33 0.21 0.28 P2 0.67 0.04 0.59 0.33 0.86 P3 0.96 0.28 0.89 0.77 1.34 P4 1.23 0.55 1.19 0.81 1.67 P5 1.33 0.28 1.37 1.14 1.92 P6 2.92 3.51 3.04 2.34 3.8 P7 3.69 9.12 3.94 2.89 4.79 P8 5.06 2.04 6.46 5.35 5.8 P9 7.56 10.36 7.93 6.69 5.77 P10 9.81 0.22 10.26 8.95 14.15 P11 12.56 13.26 13.41 13.64 16.14 P12 17.47 10.31 18.57 15.01 21.01 P13 21.89 32.19 22.13 17.47 29.69 P14 28.37 10.82 31.21 23.7 36.88 P15 38.95 35.13 35.06 27.27 46.74 P16 43.48 66.23 44.27 43.83 62.94 P17 69.2 13.54 69.2 55.36 83.04 P18 80.57 70.48 64.46 56.4 >100 P19 91.13 >100 88.9 63.79 >100 P20 >100 >100 >100 >100 >100 N1 0.12 0.11 0.16 0.15 0.16 N2 <0.1 0.44 <0.1 <0.1 0.16 N3 <0.1 <0.1 <0.1 <0.1 <0.1 N4 0.23 0.12 0.26 0.17 0.39 N5 0.1 0.14 <0.1 <0.1 <0.1 N6 <0.1 0.12 <0.1 <0.1 0.29 N7 0.31 0.13 0.26 0.25 0.39 N8 0.32 0.31 0.3 0.13 0.25 N9 0.28 <0.1 0.26 <0.1 0.35 N10 <0.1 <0.1 <0.1 <0.1 <0.1

[0071] In Table 1, P1 to P20 are positive samples, and N1 to N10 are negative samples.

[0072] Table 1 shows that the quantitative detection results of the egg white allergen-specific IgE quantitative detection kits in Examples 1-3 are closer to the Phadia detection data than those of natural egg white extracts, with Example 1 showing the best results.

[0073] The positive and negative concordance rates of Example 1 and Comparative Example 1 with Phadia are shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] Table 2 shows that the egg white allergen-specific IgE quantitative detection kit of Example 1 has significantly improved detection sensitivity and specificity for clinical samples compared with the kit of Comparative Example 1.

[0078] Comparative Example 2

[0079] This comparative example provides a quantitative detection kit for egg white allergen-specific IgE, comprising reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. The preparation method of the biotin-labeled egg white allergen mixed solution of reagent 2 is as follows:

[0080] Ovalbumin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the ovum was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled ovum was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 5 wt% mannitol, which is the biotin-labeled ovum solution.

[0081] Ovalbumin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovalbumin was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-LC-biotin-labeled ovalbumin was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 5 wt% mannitol, which is the biotin-labeled ovalbumin solution.

[0082] Ovotransferrin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovotransferrin was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled ovotransferrin was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 5 wt% mannitol, which is the biotin-labeled ovotransferrin solution.

[0083] Lysozyme was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:15, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the solution was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled lysozyme was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 5 wt% mannitol, which is the biotin-labeled lysozyme solution.

[0084] According to the mass ratio of ovomucoid, ovalbumin, ovotransferrin, and lysozyme of 0.8:1:0.7:0.5, the above biotin-labeled ovomucoid solution, biotin-labeled ovalbumin solution, biotin-labeled ovotransferrin solution, and biotin-labeled lysozyme solution were mixed to obtain a biotin-labeled egg white allergen mixed solution with a total allergen concentration of 5 μg / mL.

[0085] Comparative Example 3

[0086] This comparative example provides a quantitative detection kit for egg white allergen-specific IgE, comprising reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. The preparation method of the biotin-labeled egg white allergen mixed solution of reagent 2 is as follows:

[0087] Ovomucin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the ovomucoid was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-LC-biotin-labeled ovomucoid was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 3 wt% glycerol, which is the biotin-labeled ovomucoid solution.

[0088] Ovalbumin was added to the Sulfo-NHS-LC-biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the ovalbumin was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-LC-biotin-labeled ovalbumin was stored in phosphate buffer (pH 7.4) containing 2wt% BSA and 3wt% glycerol, which is the biotin-labeled ovalbumin solution.

[0089] Ovotransferrin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the mixture was dialyzed against 0.1 MCB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled ovotransferrin was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 3 wt% glycerol, which is the biotin-labeled ovotransferrin solution.

[0090] Lysozyme was added to the Sulfo-NHS-LC-biotin solution at a molar ratio of 1:20, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the solution was dialyzed against 0.1M PBS buffer at pH 7.4. The prepared Sulfo-NHS-LC-biotin-labeled lysozyme was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA and 3 wt% glycerol, which is the biotin-labeled lysozyme solution.

[0091] The biotin-labeled ovomucoid, ovalbumin, ovotransferrin, and lysozyme solutions were mixed in a mass ratio of 1:1:1:0.5 to obtain a biotin-labeled egg white allergen mixed solution with a total allergen concentration of 5 μg / mL.

[0092] Comparative Example 4

[0093] This comparative example provides a quantitative detection kit for egg white allergen-specific IgE, comprising reagent 1, reagent 2, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution. Reagent 1, reagent 3, reagent 4, calibrator, quality control, and luminescent substrate solution are the same as in Example 1. The preparation method of the biotin-labeled egg white allergen mixed solution of reagent 2 is as follows:

[0094] Ovalbumin was added to the Sulfo-NHS-biotin solution at a molar ratio of 1:30, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the mixture was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-biotin-labeled ovomucoid was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled ovomucoid solution.

[0095] Ovalbumin was added to the Sulfo-NHS-LC-biotin solution at a molar ratio of 1:30, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovalbumin was dialyzed against 0.1M CB buffer at pH 9.5. The prepared Sulfo-NHS-LC-biotin-labeled ovalbumin was stored in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, which is the biotin-labeled ovalbumin solution.

[0096] Ovotransferrin was added to a Sulfo-NHS-PEG4-Biotin solution at a molar ratio of 1:30, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction, the ovotransferrin was dialyzed against a 0.1M Tris-HCl buffer at pH 7.4. The prepared Sulfo-NHS-PEG4-Biotin-labeled ovotransferrin was stored in a phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol, which is the biotin-labeled ovotransferrin solution.

[0097] Lysozyme was added to the Sulfo-NHS-PEG4-Biotin solution at a molar ratio of 1:30, with DMSO as the solvent. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the solution was dialyzed against 0.1M Tris-HCl buffer at pH 7.4. The prepared Sulfo-NHS-PEG4-Biotin-labeled lysozyme was stored in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol and 5 wt% mannitol, which is the biotin-labeled lysozyme solution.

[0098] A biotin-labeled egg white allergen mixed solution with a total allergen concentration of 5 μg / mL was obtained by mixing the above-mentioned biotin-labeled ovomucoid, biotin-labeled ovalbumin, biotin-labeled ovalbumin, and biotin-labeled lysozyme solutions in a mass ratio of 0.5:1:0.5:0.5.

[0099] Table 3

[0100]

[0101]

[0102] In Table 3, P21 to P44 are positive samples, and N11 to N26 are negative samples.

[0103] Table 3 shows that reagent 2 in the egg white allergen-specific IgE quantitative detection kits of Comparative Examples 2-4 resulted in detection results that were not as good as those in Example 1.

[0104] Other performance verifications:

[0105] Accuracy analysis of the detection results of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0106] Experimental method: A high-concentration sample A (accuracy reference H) of known concentration is added to a low-concentration sample B (accuracy reference L) at a volume ratio not exceeding 1:19 to obtain the test sample. The concentration of the test sample should be within the reagent detection range. The recovery rate is calculated according to the formula:

[0107]

[0108] In the formula:

[0109] R – Recovery rate;

[0110] V—Volume of the accuracy reference H;

[0111] V0 — Volume of the accuracy reference material L;

[0112] C—Detection concentration of the mixed sample;

[0113] C0—Concentration of accuracy reference material L;

[0114] C S —The concentration of the accuracy reference material H.

[0115] The accuracy analysis data is shown in Table 4.

[0116] Table 4

[0117]

[0118]

[0119] The results showed that the recovery rate was in the range of 85% to 115%, which met the requirements.

[0120] Analysis of the detection limit of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0121] Experimental Method: Five samples with concentrations close to the detection limit (detection limit references 1-5) were tested. Each sample was tested five times. The results were sorted by magnitude. The blank limit and detection limit were considered to be reasonably set if the following conditions were met:

[0122] a) The number of test results below the blank limit should be less than or equal to 3;

[0123] b) No test results were found that were higher than the lower limit of the reference range.

[0124] Using the calibration point CAL1 (zero calibration point) as the sample, conduct at least 20 tests. Calculate the luminescence value Mean+2SD, and back-calculate the concentration using linear regression of the luminescence values ​​at the CAL1 and CAL2 calibration points from at least 5 results to obtain the blank limit.

[0125] The results of the detection limit analysis are shown in Tables 5 and 6:

[0126] Table 5

[0127]

[0128]

[0129] Table 6

[0130]

[0131] The detection limits showed that the detection limits were set reasonably, all not exceeding 0.1 IU / mL, which met the requirements.

[0132] Linear analysis of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0133] Experimental Method: Dilute high-value samples (linear reference standards) approaching the upper limit of the linear interval with low-value samples approaching the lower limit of the linear interval, and dilute proportionally to at least 5 concentrations, where the low-value concentration samples must be close to 0.1 IU / mL. Each concentration is measured three times, and the average value of each concentration point is calculated. The average value of the measured concentrations is then fitted to the theoretical concentration using the least squares method to form a linear relationship, and the linear correlation coefficient r is calculated.

[0134] The experimental results are shown in Table 7, in IU / mL:

[0135] Table 7

[0136]

[0137] The results show that the linear correlation coefficient r is greater than 0.9900, which meets the requirements.

[0138] Repeatability analysis of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0139] The samples were tested 10 times each at two concentration levels: 0.35 IU / mL to 0.7 IU / mL (repeatability reference RP1) and 3.5 IU / mL to 17.5 IU / mL (repeatability reference RP2). The mean M and standard deviation SD of the test results were calculated using the following formula to obtain the coefficient of variation CV:

[0140] CV = SD / M × 100%

[0141] In the formula:

[0142] CV — Coefficient of Variation;

[0143] M — the average value of 10 concentration measurements;

[0144] SD — the standard deviation of 10 concentration measurements.

[0145] The experimental results are shown in Table 8:

[0146] Table 8

[0147]

[0148] The results showed that the intra-batch CV of repeatability results was no higher than 10%, which met the requirements.

[0149] Specificity analysis of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0150] Experimental methods: Samples containing total IgE at a concentration of not less than 60 IU / mL and without specific IgE antibodies, and IgA samples with a concentration of not less than 700 μg / mL, IgM samples with a concentration of 500 μg / mL, and IgG samples with a concentration of 7000 μg / mL were tested once. The results are shown in Table 9.

[0151] Table 9

[0152]

[0153] The results showed that all measurements were no higher than 0.1 IU / mL, which met the requirements.

[0154] Inter-batch variation analysis of the egg white allergen-specific IgE quantitative detection kit in Example 1

[0155] Experimental method: The batch-to-batch differences of three batches of finished reagent kits were detected using the clinical samples used above.

[0156] The experimental results are shown in Table 10, in IU / mL:

[0157] Table 10

[0158]

[0159]

[0160] In Table 10, P1 to P20 are positive samples, and N1 to N10 are negative samples.

[0161] The results showed that after producing three batches of finished reagent kits, the CV (volume variation) for positive samples was no greater than 8%, and the CV for negative samples was no greater than 10%, which is far lower than the industry standard of 15% inter-batch variation. This indicates that the raw material source of the egg white allergen-specific IgE quantitative detection kit of the present invention is more controllable, the inter-batch variation of the reagents is small, and the performance is more stable.

[0162] In summary, the egg white allergen-specific IgE quantitative detection kit of Example 1 is the most accurate kit for detection results, and has excellent other performance characteristics, making it suitable for large-scale production and utilization.

[0163] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A reagent for the quantitative detection of egg white allergen-specific IgE, characterized in that, The reagents include biotin-labeled egg white allergens and a preservation solution. The biotin-labeled egg white allergens include Sulfo-NHS-biotin-labeled ovomucoid, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin, and Sulfo-NHS-PEG4-Biotin-labeled lysozyme. The mass ratio of Sulfo-NHS-biotin-labeled ovomucoid, Sulfo-NHS-LC-biotin-labeled ovalbumin, Sulfo-NHS-biotin-labeled ovotransferrin, and Sulfo-NHS-PEG4-Biotin-labeled lysozyme is (0.7~1):1:(0.5~0.8):0.

5. The preservation solution is a phosphate buffer containing 1-2 wt% BSA, 1-3 wt% glycerol and 2-5 wt% mannitol, and the pH value of the preservation solution is 7.2-7.

5.

2. The reagent for quantitative detection of egg white allergen-specific IgE according to claim 1, characterized in that, The concentration of the biotin-labeled egg white allergen in the reagent is 0.5~5 μg / mL; And / or, the labeling molar ratio of ovomucoid to Sulfo-NHS-biotin is 1:10~20; And / or, the labeling molar ratio of ovalbumin to Sulfo-NHS-LC-biotin is 1:10~20; And / or, the labeling molar ratio of ovotransferrin and Sulfo-NHS-biotin is 1:15~25; And / or, the labeling molar ratio of lysozyme and Sulfo-NHS-PEG4-Biotin is 1:15~25.

3. The reagent for quantitative detection of egg white allergen-specific IgE according to claim 1, characterized in that, Ovalbumin and Sulfo-NHS-biotin were mixed and reacted at room temperature. After the reaction was completed, the mixture was dialyzed with 0.05-0.2M CB buffer at pH 9.0-9.5 to obtain the Sulfo-NHS-biotin-labeled ovumbumin. And / or, ovalbumin and Sulfo-NHS-LC-biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M CB buffer at pH 9.0-9.5 to obtain the Sulfo-NHS-LC-biotin-labeled ovalbumin. And / or, ovalbumin and Sulfo-NHS-biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M PBS buffer at pH 7.2-7.5 to obtain the Sulfo-NHS-LC-biotin-labeled ovalbumin and the Sulfo-NHS-biotin-labeled ovalbumin. And / or, lysozyme and Sulfo-NHS-PEG4-Biotin are mixed and reacted at room temperature. After the reaction is completed, the mixture is dialyzed with 0.05-0.2M PBS buffer at pH 7.2-7.5 to obtain the Sulfo-NHS-PEG4-Biotin-labeled lysozyme.

4. A quantitative detection kit for egg white allergen-specific IgE, characterized in that, The egg white allergen-specific IgE quantitative detection kit is a chemiluminescent kit, which includes the reagents described in any one of claims 1 to 3.

5. The egg white allergen-specific IgE quantitative detection kit according to claim 4, characterized in that, The aforementioned egg white allergen-specific IgE quantitative detection kit includes reagent 1, reagent 2, reagent 3, reagent 4, and a luminescent substrate solution. Reagent 1 is a streptavidin-coated magnetic microparticle suspension; The reagent 2 is the reagent according to any one of claims 1 to 4; Reagent 3 is an alkaline phosphatase-labeled mouse anti-human IgE antibody; Reagent 4 is a biotin-labeled anti-human IgE antibody. The luminescent substrate solution is an AMPPD solution.

6. The egg white allergen-specific IgE quantitative detection kit according to claim 5, characterized in that, The concentration of reagent 1 is 0.4~1 mg / mL; And / or, the solvent of reagent 1 is a phosphate buffer solution with a pH of 7.2 to 7.5 containing 1 to 2 wt% BSA, 1 to 3 wt% glycerol and 2 to 5 wt% mannitol; And / or, the concentrations of reagent 3 and reagent 4 are independently 0.1~2.0 μg / mL.

7. The egg white allergen-specific IgE quantitative detection kit according to claim 5, characterized in that, The anti-human IgE antibody was mixed with Sulfo-NHS-LC-biotin and dialyzed with phosphate buffer containing 1-2 wt% BSA at pH 7.2-7.5 to obtain the alkaline phosphatase-labeled mouse anti-human IgE antibody.

8. The egg white allergen-specific IgE quantitative detection kit according to claim 5, characterized in that, The egg white allergen-specific IgE quantitative detection kit also includes calibrators and quality control products.

Images