Preparation method and application of a preservation solution for inhaled mixed allergens

Abstract

The application discloses a preparation method of an inhaled mixed allergen storage solution. The storage solution is used for storing mixed allergens composed of house dust mites, dust mites, cat dander, horse dander, dog hair, timothy grass, cladosporium, common birch, olive, artemisia and medicinal wall grass, and the storage solution comprises the following components in the form of mass fractions: (1) 5mM-50mM PBS buffer solution, pH 7.0-8.0, 0.5%-5% salt, 5%-30% human serum albumin, 0.05%-0.2% preservative, and 0.1%-1% protein stabilizer. The preparation method and application of the inhaled mixed allergen storage solution effectively solve the cross reaction between various proteins, and realize more sensitive detection of common inhaled allergens in samples.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for preparing and applying a stable inhaled mixed allergen reagent preservation solution. Background Technology

[0002] Allergic diseases have been listed by the WHO as a major global health problem, and in developed countries, they are considered one of the main factors affecting health. Clinically, allergic reactions often manifest as symptoms affecting the skin, digestive tract, respiratory tract, and cardiovascular system; in severe cases, they can lead to shock or even death.

[0003] Immunoglobulin E (IgE) is associated with type I hypersensitivity reactions. Different allergens induce the production of different specific IgEs, and excessively high levels often indicate an allergic constitution or the presence of type I hypersensitivity reactions. Monitoring the levels of specific IgE antibodies in human samples provides important information for allergen screening, disease monitoring, clinical diagnosis, and prevention.

[0004] Antigens that trigger allergic reactions are called allergens. There are 2,000-3,000 common antigens that can cause allergic reactions, and medical literature records nearly 20,000. They can cause allergic reactions in the body through inhalation, ingestion, injection, or contact.

[0005] Existing allergen-specific testing reagents are based on a combination of multiple methods such as immunoblotting and enzyme-linked immunosorbent assay (ELISA), and are usually used for single-item testing. Single-item testing has good stability, higher specificity and sensitivity, but it is expensive. Accurate combined testing can effectively improve testing efficiency and reduce testing costs for patients.

[0006] Currently, there is no technology for quantitative detection of mixed inhaled allergen screening. Test reagents for screening multiple allergen mixtures require the preparation of suitable preservation solutions. Preparing a mixture of several allergens reduces the testing cost to a fraction of that of a single test. However, allergen proteins are composed of proteins whose spatial structure is mainly composed of chemical elements such as carbon, hydrogen, oxygen, and nitrogen. They are polymers formed by different amino acids linked by different peptide bonds. Cross-reactions between similar proteins can lead to the formation of another similar protein, resulting in decreased stability of the mixed reagent. Therefore, it is necessary to select suitable preservation solutions to effectively ensure the stability of each allergen protein, allowing it to be preserved for a long time under storage conditions. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the inventors provide a method for preparing a stable inhaled mixed allergen reagent preservation solution. Common inhaled allergen proteins are selected and mixed. Using a fluorescent magnetic microparticle chemiluminescent immunoassay quantitative immunoassay platform, the cross-reaction between various proteins can be effectively resolved through the screening and preparation of the preservation solution and the combination of various allergen proteins. This method can more sensitively detect common inhaled allergens in the sample and effectively ensure the stability of the mixed inhaled allergen proteins during preservation.

[0008] Specifically, one of the technical solutions involved in this invention is:

[0009] The present invention provides an inhalable mixed allergen preservation solution, wherein the inhalable mixed allergen is a combination of eleven substances: house dust mite, flour dust mite, cat dander, horse dander, dog dander, timothy hay, Cladosporium, common birch, olive, artemisia, and medicinal wall grass.

[0010] The preservation solution used for the above eleven inhaled mixed allergens includes the following components by mass fraction: (1) 5mM-50mM PBS buffer, pH 7.0-8.0, 0.5%-5% salt, 5%-30% human serum albumin (HSA), 0.05%-0.2% preservative, and 0.1%-1% protein stabilizer;

[0011] Preferably, the concentration of the PBS buffer is 10 mM and the pH is 7.4.

[0012] Preferably, the HSA is 10%.

[0013] Preferably, the salt is 0.9% sodium chloride.

[0014] Preferably, the preservative is 0.1% proclin950.

[0015] Preferably, the protein stabilizer is 0.1% arginine.

[0016] The second technical solution involved in this invention is:

[0017] A method for preparing an inhaled mixed allergen preservation solution, which is prepared using the aforementioned formula, specifically includes the following steps;

[0018] (1) Prepare PBS buffer solution with a concentration of 5mM-50mM and pH 7.0-8.0;

[0019] (2) Add 0.5% to 5% by mass of salt to the buffer solution;

[0020] (3) In step 2), add 5%-30% human serum albumin, 0.05%-0.2% preservative, and 0.1%-1% protein stabilizer in sequence for later use.

[0021] Preferably, it includes the following steps;

[0022] (1) Prepare PBS buffer solution with a concentration of 10 mM and pH 7.4;

[0023] (2) Add 0.9% by mass sodium chloride to the buffer solution;

[0024] (3) In step 2), add 10% by weight of human serum albumin, 0.1% by weight of proclin950, and 0.1% by weight of arginine in sequence, and set aside.

[0025] The third technical solution involved in this invention is: the application of an inhaled mixed allergen preservation solution, which is used to prepare a reagent containing inhaled mixed allergens. The preparation method of the reagent includes the following steps: selecting multiple allergen proteins for mixing; biotinylating the allergen proteins; identifying the potency of each biotinylated allergen; screening and compatibility of the preservation solution, screening and combining according to the proportion of individual allergens, and preparing according to the final potency in a 1:1 ratio.

[0026] Preferably, the application of the inhaled mixed allergen preservation solution involves preparing a reagent containing inhaled mixed allergens, the preparation method of which includes the following steps:

[0027] (1) Prepare PBS buffer with a concentration of 10 mM and pH 7.4;

[0028] (2) Add 0.9% sodium chloride to the buffer solution;

[0029] (3) In step 2), add 10% human serum albumin, 0.1% proclin950, 0.1% arginine and 0.02% of each biotinylated allergen protein in sequence;

[0030] (4) After fully dissolving and mixing, adjust the pH value to 7.40±0.05, and make up to volume.

[0031] Beneficial effects of this invention:

[0032] The inhaled mixed allergen reagent provided by this invention, by preparing a suitable preservation solution in a PBS buffer system at pH 7-8, adds 0.5%-5% salt, 0.05%-0.2% preservative, 5%-30% human serum albumin, and 0.1%-1% arginine protein stabilizer. The combination of human serum albumin and arginine has a synergistic effect. The addition of a certain amount of human serum albumin effectively coats and protects the various mixed proteins, isolating the protein linkages. Simultaneously, the addition of arginine makes the albumin protective layer more stable. Under long-term storage, the reagent will not deteriorate or react with each other, stably protecting multiple mixed allergen proteins. Experiments have shown that this preservation solution allows the eleven mixed allergen proteins of this invention to be stored for a long period, with a shelf life of over 36 months.

[0033] This invention precisely selects common inhaled allergen proteins and utilizes a fluorescent magnetic microparticle chemiluminescent immunoassay platform. Through the combination of preservation solution and various allergen proteins, it effectively solves the cross-reaction between different proteins, and more sensitively detects common inhaled allergens in samples. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0035] The present invention will be further illustrated with specific examples. The examples below are only for explaining the present invention and do not limit its content.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0037] In a typical embodiment of the present invention, a stable inhaled mixed allergen reagent is provided. The inhaled mixed allergen is a combination of eleven species, including house dust mite, dry dust mite, cat dander, horse dander, dog dander, timothy hay, mycorrhiza, common birch, olive, artemisia, and medicinal wall grass. The reagent is prepared with 5mM-50mM PBS buffer, pH 7.0-8.0, 0.5%-5% salt, 5%-30% human serum albumin (HSA), 0.05%-0.2% preservative, 0.1%-1% protein stabilizer, and 0.02% of each biotinylated allergen protein.

[0038] Experiments have shown that, using a standard PBS buffer system with a pH of 7-8, a PBS buffer concentration of 5mM-50mM, 0.5%-5% sodium chloride, 0.05%-0.2% proclin950, and the addition of 5%-30% human serum albumin (HSA) and 0.1%-1% arginine protein stabilizer, the system exhibits good stability for 0.02% of various biotinylated allergen proteins.

[0039] If the mass fraction of human serum albumin is less than 5%, a significant matrix effect will occur in the system's reaction. This is mainly reflected in lower test values ​​near negative results, leading to false negatives. Similarly, in samples with higher concentrations, test values ​​will be higher, and these falsely high values ​​will not provide effective guidance for clinical quality. If the mass fraction is greater than 30%, the matrix effect will also occur. In the low-concentration stage, it is easy to cause false positives, while in high-concentration samples, it will lead to lower values. In addition to affecting the test matrix, high concentrations of human serum albumin are also detrimental to cost control. In short, both excessively high and excessively low concentrations are detrimental to stability.

[0040] If the mass fraction of arginine is less than 0.1%, the effective component of arginine cannot fully form a protective layer with each allergen protein. Under human serum albumin conditions, it is easily destroyed and cannot play a protective role. If it is greater than 1%, the protective layer is too thick, and each individual allergen cannot fully bind with the antibody in the sample, resulting in a falsely low result and easy to cause missed detection.

[0041] Experiments show that 5%-30% human serum albumin (HSA) and 0.1%-1% arginine protein stabilizer can achieve the best synergistic effect. Too much or too little of any component will not be conducive to achieving the synergistic effect, so as to stabilize and protect inhaled mixed allergen proteins and prevent their degradation.

[0042] As an optional implementation, the human serum albumin has a mass fraction of 10%.

[0043] As an optional implementation, the arginine has a mass fraction of 0.1%.

[0044] As a preferred embodiment, the preservation solution prepared with 10 mM PBS buffer, pH 7.4, 0.9% sodium chloride, 10% human serum albumin, 0.1% proclin 950, and 0.1% arginine exhibits the most stable preservation time and efficacy. This preservation solution is used in the preparation of reagents for the eleven inhaled mixed allergen combinations of this invention as follows:

[0045] (1) Prepare PBS buffer with a concentration of 10 mM and a pH of 7.4;

[0046] (2) Add sodium chloride to the buffer solution to a concentration of 0.9%;

[0047] (3) Add human serum albumin (HSA) at a concentration of 10%;

[0048] (4) Add preservative proclin 950 at a concentration of 0.1%;

[0049] (5) Add arginine, a protein stabilizer, at a concentration of 0.1%.

[0050] (6) Biotinylated allergen proteins, 0.02%.

[0051] (7) After fully dissolving and mixing, adjust the pH to 7.40±0.05 and bring the volume to a final volume.

[0052] The reagent preservation solution of this application will be described in detail below with reference to embodiments, comparative examples and experimental data.

[0053] Table 1 shows the embodiments of the present invention.

[0054]

[0055]

[0056] Table 2 shows the comparative examples of the present invention.

[0057]

[0058] Performance was evaluated using the preservation solutions of each example and comparative example, and reagents prepared with 0.02% of each biotinylated allergen protein. The stability was assessed by storage at 2-8°C for 40 months and by testing the same sample after storage at 37°C for 7 days. The results are shown in Table 4.

[0059] Table 4

[0060] Group 40-month measurement value 7-day measurement values Example 1 38.5 36.4 Example 2 40.5 39.8 Example 3 37.9 36.8 Example 4 37.4 36.2 Example 5 35.8 35.3 Example 6 33.7 32.5 Example 7 31.1 30.2 Comparative Example 1 25.3 25.9 Comparative Example 2 27.2 26.7 Comparative Example 3 22.8 23.1

[0061] Furthermore, the average test value on the first day for Examples 1 to 7 was 40.8, which serves as the theoretical value. The data from the above experimental examples indicate that Example 2 exhibits the best stability, closely matching the theoretical value. Replacing the protein stabilizer with other materials affects the reagent's stability. The concentrations of human serum albumin and arginine also influence reagent stability; human serum albumin levels above 30% or below 5% have an impact, while arginine levels above 1% or below 0.1% do. Therefore, the experimental data show that a human serum albumin concentration of 10% and an arginine concentration of 0.1% can extend the shelf life of the reagent containing 11 mixed inhaled allergens.

[0062] This invention precisely selects common inhaled allergen proteins and utilizes a fluorescent magnetic microparticle chemiluminescent immunoassay platform. Through the combination of preservation solution and various allergen proteins, it effectively solves the cross-reaction between different proteins, and more sensitively detects common inhaled allergens in samples.

[0063] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing an inhalable mixed allergen preservation solution, characterized in that, This preservation solution is used to preserve a mixture of allergens consisting of house dust mites, cat dander, horse dander, dog dander, timothy hay, Cladosporium, common birch, olive, artemisia, and medicinal wall grass. The preservation solution consists of the following components by mass fraction: 5 mM-50 mM PBS buffer, pH 7.0-8.0, 0.5%-5% salt, 5%-30% human serum albumin, 0.05%-0.2% preservative, and 0.1%-1% protein stabilizer, wherein the protein stabilizer is arginine.

2. The method for preparing the inhaled mixed allergen preservation solution according to claim 1, characterized in that, The PBS buffer solution has a concentration of 10 mM and a pH of 7.

4.

3. The method for preparing the inhaled mixed allergen preservation solution according to claim 2, characterized in that, The mass fraction of the human serum albumin is 10%.

4. The method for preparing the inhaled mixed allergen preservation solution according to claim 3, characterized in that, The salt is 0.9% sodium chloride.

5. The method for preparing the inhaled mixed allergen preservation solution according to claim 4, characterized in that, The preservative is 0.1% proclin950.

6. The method for preparing the inhaled mixed allergen preservation solution according to claim 5, characterized in that, The protein stabilizer is 0.1% arginine.

7. The method for preparing the inhaled mixed allergen preservation solution according to claim 6, characterized in that, Specifically, it includes the following steps; (1) Prepare PBS buffer with a concentration of 10 mM and a pH of 7.4; (2) Add 0.9% sodium chloride to the buffer solution; (3) In step (2), add 10% human serum albumin, 0.1% proclin950 and 0.1% arginine in sequence and mix for later use.

8. The application of the inhaled mixed allergen preservative solution as described in claim 1, characterized in that, The preservation solution was formulated into a reagent with a mixture of allergens consisting of house dust mites, dust mites, cat dander, horse dander, dog dander, timothy hay, Cladosporium, common birch, olive, artemisia, and medicinal wall grass. The preparation method of the reagent included biotinylation of the screened allergen proteins.

9. The application of the inhaled mixed allergen preservative solution according to claim 8, characterized in that, The reagent preparation includes the following specific steps: (1) Prepare PBS buffer with a concentration of 10 mM and a pH of 7.4; (2) Add 0.9% sodium chloride to the buffer solution; (3) In step 2), add 10% human serum albumin, 0.1% proclin950, 0.1% arginine and 0.02% of each biotinylated allergen protein in sequence; (4) After fully dissolving and mixing, adjust the pH value to 7.40±0.05 and make up to volume.

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