A kit for detecting allergens

Abstract

The application belongs to the field of biological medicine, and specifically discloses a kit for allergen detection, which comprises a reaction film strip, a sealing liquid, a chromogenic liquid, a washing buffer, a sample diluent and a termination liquid. The washing buffer is improved in the application, and the non-ionic surfactant is combined with the anionic surfactant, so that the cleaning liquid has good stability, the influence of non-specific interfering substances on the detection result is greatly reduced, and the detection accuracy is improved. Different kinds of bacteriostatic agents are combined, and the preservation effect is good. Through the selection of specific components, the problem of high price and high detection cost of imported cleaning liquid is solved. The kit is proved to have good repeatability and high accuracy through repeatability test and positive and negative coincidence rate test, and provides serological basis for clinical diagnosis of allergens and comprehensive guidance for disease course detection and post-treatment of allergic patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a reagent kit for allergen detection. Background Technology

[0002] In recent years, the number of allergy sufferers has been increasing, and the incidence of allergies has been rising year by year. It is reported that 22% of the world's population has varying degrees of allergy symptoms. More than 90% of allergic reactions are caused by eight categories of food: eggs, peanuts, dairy products, soybeans, wheat, tree nuts (nuts), shellfish (including crustaceans and mollusks), and fish. Food allergy refers to a specific immune response that occurs in the body after ingesting a particular food. Food allergies are classified into IgE-mediated hypersensitivity reactions and cell-mediated hypersensitivity reactions (non-IgE-mediated hypersensitivity reactions). Most food allergies are IgE-mediated. In IgE-mediated allergic reactions, the allergen stimulates the mononuclear phagocytic system in organs such as lymph nodes, liver, and spleen, triggering plasma cell reactions to produce specific IgE (sIgE) antibodies. Secondly, there are inhaled allergens, such as pollen, willow catkins, dust mites, and animal dander; and then there are contact allergens, such as molds (Penicillium, Alternaria, Aspergillus fumigatus, Cladosporium, Candida albicans), ultraviolet light, and radiation.

[0003] Clinically, allergen detection for IgE-mediated allergic reactions can be performed through skin prick tests, intradermal tests, skin patch tests, and serum-specific IgE detection. Among these, immunoblotting offers high sensitivity and allows for high-throughput detection. Researchers typically focus on screening for optimal main raw materials (antigens and antibodies) and optimizing the reaction process, often neglecting the role of the washing buffer. Currently, research and reports on washing buffers are relatively limited, and the few patents available demonstrate limited washing effectiveness, failing to meet the requirements of immunoblotting diagnostic reagents. However, in practical use, the washing effect of the washing buffer directly affects the accuracy of the test results, frequently resulting in false positives and false negatives in clinical practice. This application aims to simultaneously detect multiple antibodies against allergens (using immunoblotting), facilitating timely diagnosis and appropriate treatment for patients. Summary of the Invention

[0004] The primary objective of this invention is to provide an allergen detection kit that improves the washing buffer, thereby increasing its solubility and ensuring long-term stability.

[0005] A second objective of this invention is to provide a washing buffer for the above-mentioned kit and a method for preparing the same.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] According to one aspect of the present invention, an allergen detection kit is provided, comprising a reaction membrane strip, a blocking solution, a chromogenic solution, a washing buffer, a sample diluent, and a stop solution; wherein the washing buffer comprises a buffer, a surfactant, a composite antibacterial agent, a stabilizer, and a salt; wherein the composite antibacterial agent is prepared by mixing nano zinc oxide, chitosan, isothiazolinones, magnesium nitrate, a stabilizer, and a solvent.

[0008] As an improvement, the buffer solution is one of phosphate buffer, Tris buffer, acetate buffer, and citrate buffer.

[0009] As an improvement, the surfactant includes anionic surfactants and nonionic surfactants, wherein the nonionic surfactant is an amide oxy acid, and the anionic surfactant is an alcohol ether phosphate or coconut oil diethanolamide.

[0010] As an improvement, the isothiazolinones are methylchloroisothiazolinone or methylisothiazolinone;

[0011] As an improvement, the stabilizer is sodium citrate, and the solvent is isopropanol or acetic acid;

[0012] As an improvement, the salt is one of NaCl and KCl.

[0013] As a further improvement, the final concentration of the compound antibacterial agent is 0.1–2% w / v.

[0014] As a further improvement, the concentration of the surfactant is 0.3%-1.9% w / v.

[0015] As a further improvement, the concentration of the stabilizer is 50-500 mM.

[0016] As a further improvement, the concentration of NaCl is 150-250 mM.

[0017] As a further improvement, the buffer solution is a Tris-phosphate buffer with a concentration of 100-1000 mM.

[0018] As a further improvement, the buffer solution is a Tris-phosphate buffer with a concentration of 100 mM.

[0019] As a further improvement, the buffer solution is a Tris-phosphate buffer with a concentration of 200 mM.

[0020] As a further improvement, the final concentration of the compound antibacterial agent is 0.1% w / v.

[0021] As a further improvement, the final concentration of the compound antibacterial agent is 0.5% w / v.

[0022] As a further improvement, the final concentration of the compound antibacterial agent is 1.5% w / v.

[0023] As a further improvement, the concentration of the surfactant is 0.5% w / v.

[0024] As a further improvement, the concentration of the surfactant is 1.5% w / v.

[0025] As a further improvement, the concentration of the stabilizer is 50 mM.

[0026] As a further improvement, the concentration of the stabilizer is 100 mM.

[0027] As a further improvement, the concentration of NaCl is 150 mM.

[0028] As a further improvement, the washing buffer comprises 100 mM Tris-phosphate buffer, 1.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8.

[0029] As a further improvement, the washing buffer comprises 100 mM Tris-phosphate buffer, 0.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8.

[0030] As a further improvement, the washing buffer comprises 100 mM Tris-phosphate buffer, 1.5% w / v compound antibacterial agent, 0.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8.

[0031] As a further improvement, the washing buffer comprises 100 mM Tris-phosphate buffer, 0.5% w / v compound antibacterial agent, 0.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8.

[0032] As an improvement, the reaction membrane strip is provided with a detection strip and a quality control strip;

[0033] As an improvement, the detection band is coated with allergen protein antigens;

[0034] As an improvement, the allergen protein antigens include one or more of the following: willow and / or poplar and / or elm, common ragweed, artemisia, house dust mite and / or flour dust mite, house dust, cat dander, dog epidermis, cockroach, Penicillium and / or Cladosporium and / or Aspergillus fumigatus and / or Alternaria alternata, hop, egg white and / or egg white, milk, peanut, soybean, beef, mutton, cod and / or lobster and / or scallop, shrimp and crab;

[0035] As an improvement, the quality control band includes a first quality control band, a second quality control band, and a third quality control band, wherein the first quality control band is a strongly positive quality control band, the second quality control band is a moderately positive quality control band, and the third quality control band is a weakly positive quality control band;

[0036] The first quality control band was coated with human IgG at a concentration of 30-50 μg / mL.

[0037] The second quality control band is coated with human IgG at a concentration of 11-29 μg / mL;

[0038] The third quality control band is coated with human IgG at a concentration of 1-10 μg / mL.

[0039] As an improvement, the method for preparing the washing buffer includes the following steps:

[0040] (1) After washing and drying the crab shells, put them into a pulverizer for pulverization; put the pulverized crab shells into an acetic acid solution, heat and stir, filter and wash, add sodium hydroxide solution, heat and stir, add acetic acid solution, heat and stir; filter and dry to obtain a solid, then add ethanol and methanol, heat and wash, and put it into a dryer for drying to obtain chitosan solid; dissolve the chitosan solid in an acetic acid solution, stir and mix to obtain a chitosan solution; add isopropanol, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone, and nano zinc oxide to the chitosan solution in sequence, stir evenly, add sodium citrate, stir, and obtain a composite antibacterial agent;

[0041] (2) Add surfactant and salt to buffer solution, stir in a mixer to obtain first mixture, adjust the pH of buffer solution to 6-6.9 with boric acid or acetic acid, add compound antibacterial agent to first mixture, stir to obtain second mixture, finally add sodium citrate, stir, adjust the pH of second mixture to 6.5 with boric acid or acetic acid to obtain washing buffer.

[0042] As a further improvement, the method for preparing the washing buffer includes the following steps:

[0043] (1) Preparation of the compound antibacterial agent: After washing and drying crab shells, they are crushed in a pulverizer; the crushed crab shells are placed in an acetic acid solution, heated to 80-100℃, and stirred in a mixer for 1-2 hours; then filtered, rinsed with water, and the rinsed material is added to a sodium hydroxide solution, heated to 30-60℃, and stirred for 2 hours. Then, it is added to an acetic acid solution, heated to 30-60℃, and stirred in a mixer; after filtration, it is dried to obtain a solid, which is then heated and cleaned with ethanol and methanol, and then placed in a mixer. The chitosan solid was obtained by drying in a dryer. The chitosan solid was dissolved in a 1% (w / w) acetic acid solution and stirred at 30°C for 20 min to obtain a chitosan solution. The chitosan solution was heated to 40°C, and isopropanol, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone, and nano zinc oxide were added to the solution in sequence. The mixture was stirred at 2500-3000 r / min for 30-40 min to ensure complete dispersion. After cooling to room temperature, sodium citrate was added and stirred for 10-30 min to obtain a composite antibacterial agent.

[0044] (2) Add surfactant and salt to buffer solution, stir in a mixer for 1-2 hours to obtain first mixture. Adjust the pH of buffer solution to 6-6.9 with boric acid or acetic acid. Add compound antibacterial agent to first mixture and stir for 1-2 hours to obtain second mixture. Finally, add sodium citrate and stir for 1-2 hours. Adjust the pH of second mixture to 6.5 with boric acid or acetic acid to obtain washing buffer.

[0045] The concentration of the acetic acid solution is 0.1% w / v.

[0046] The third objective of this invention is to provide a method for preparing the above-mentioned reagent kit, wherein antigens and quality control materials are coated on a nitrocellulose membrane using a sample applicator, the nitrocellulose membrane is fixed on a PVC plate, and cut into single-use reaction membrane strips. Enzyme conjugates, chromogenic substrates, blocking solutions, washing buffers, sample diluents, and stop solutions are also prepared and placed together in a container for later use.

[0047] A fourth objective of this invention is to provide the application of the above-mentioned reagent kit in allergen detection, as follows:

[0048] Wet the reaction membrane strip coated with allergen protein antigen with washing buffer, place it in the incubation plate holder, and incubate at room temperature on a shaker (shaking 20 times per minute, the same below) for 1 to 2 minutes. Pour out the washing buffer.

[0049] Add 10 μl of the diluted sample to the incubation plate, cover the plate, and incubate overnight at room temperature on a shaker. Add 2 mL of wash buffer to the incubation plate, shake for 2 minutes, repeat 3 times, and discard the wash buffer. Add 2 mL of HRP-labeled mouse anti-human secondary antibody, cover the plate, and incubate at room temperature on a shaker for 30 minutes. Add 5 mL of wash buffer, shake for 2 minutes, repeat 3 times, and discard the wash buffer. Add 2 mL of TMB chromogenic solution, cover the plate, and incubate at room temperature for 10 minutes. Open the plate, aspirate the substrate solution, and terminate the reaction with the stop solution.

[0050] The test results are determined based on the presence or absence of bands. Examine the control bands and test bands. If the first, second, and third control bands appear, and their intensity decreases sequentially, the test results are valid. Examine the test bands for interpretation.

[0051] The present invention has the following beneficial effects:

[0052] This invention utilizes a combination of nonionic and anionic surfactants, resulting in improved stability of the cleaning solution and significantly reducing the impact of nonspecific interfering substances on detection results, thus enhancing accuracy. The combined use of different antibacterial agents also provides excellent preservative effects. The washing buffer solution provided by this invention, through the selection of specific components, solves the problems of high prices and high testing costs associated with imported cleaning solutions. The cleaning solution provided by this invention is low-cost and reduces domestic dependence on imported cleaning solutions. Furthermore, it has been verified that this washing buffer solution is suitable for allergen detection using immunoblotting.

[0053] This kit can detect multiple allergens simultaneously from a single sample, providing serological evidence for the clinical diagnosis of allergens. It features strong positive, moderate positive, and weak positive control bands, making the results more intuitive and accurate. Through repeatability tests, positive concordance rate tests, and negative concordance rate tests, the kit has been shown to have good repeatability, with both positive and negative concordance rates at 100%, indicating high accuracy. Detailed Implementation

[0054] The following embodiments illustrate the present invention in more detail, but they do not constitute a limitation thereof. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the reagents used in the following embodiments are commercially available common reagents.

[0055] The typical pH range for antigen-antibody reactions is 6-9. Excessive acidity or alkalinity can damage the covalent and hydrogen bonds between the antigen-antibody immune complexes, and may even denature the antigen and antibody, leading to the dissociation of the antigen-antibody complexes and affecting the accuracy of the detection. Since the specific binding force of normal antigens and antibodies is stronger than their non-specific binding force, the weakly acidic buffer solution used in this application does not cause the dissociation of normal antigen-antibody complexes, but rather the dissociation of non-specific weak bindings, thereby reducing the non-specificity of the test reagent.

[0056] Isothiazolinones have a strong inhibitory effect on various bacteria, molds, yeasts, and algae, with a usage concentration of 0.1%–0.02% (active ingredient approximately 1.5%). Their mechanism of action involves the two active ingredients penetrating the cell membrane within minutes of contact and inhibiting intracellular enzyme activity, thereby suppressing cell growth, metabolism, and macromolecular synthesis, leading to a rapid decline in intracellular energy levels. Due to the collapse of the energy system, the cell cannot synthesize compounds needed for daily metabolism, resulting in cell death. These preservatives are miscible with chlorine and most anionic, cationic, and nonionic surfactants, and do not affect enzyme activity at higher concentrations. They have a wide pH range (pH 2–8.5) and good chemical stability. Laboratory tests show that after two years of storage at 25°C, the active component of this preservative is only 3% lost, and it is miscible with water in any proportion. This series of preservatives has multiple target sites, thus significantly reducing the drug resistance of microorganisms due to mutation, making it an ideal alternative to thimerosal, sodium azide, and gentamicin as a biological preservative. However, its activity decreases when exposed to ammonium, sulfites, strong reducing agents, and strong alkalinities. Excessive use can cause irritation and sensitization. Isothiazolinones are mainly composed of 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). Isothiazolinones exert their biocidal effect by breaking the bonds in bacterial and algal proteins. Upon contact with microorganisms, isothiazolinones rapidly and irreversibly inhibit their growth, leading to cell death. Therefore, they have a strong inhibitory and bactericidal effect on common bacteria, fungi, and algae. They exhibit high biocidal efficiency, good degradability, and are characterized by no residue, safe operation, good compatibility, strong stability, and low cost. They are miscible with chlorine and most anionic, cationic, and nonionic surfactants. At high doses, isothiazolinones have a significant effect on the removal of biological slime.

[0057] Chitosan possesses strong antifungal properties, effectively inhibiting Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, and Bacillus subtilis. It also inhibits physiological changes in fresh foods and is naturally non-toxic. Antimicrobial polymers can inhibit the growth of certain microorganisms. Chitosan exhibits low toxicity, biodegradability, biocompatibility, low cost, and unique antimicrobial properties.

[0058] Chitosan is soluble in acetic acid, and isopropanol can be used as a dispersant.

[0059] Nano zinc oxide exhibits strong inhibitory effects against Escherichia coli and Staphylococcus aureus. In vitro, it also inhibits the growth of prokaryotic cells, eukaryotic cells, and Escherichia coli, with its antibacterial activity increasing with increasing nano zinc oxide content. The interaction time between nano zinc oxide and bacteria also affects its bactericidal ability.

[0060] Alcohol ether phosphates possess excellent water solubility, detergency, emulsification, lubrication, cleaning, dispersing, antistatic, and rust-preventing properties. They also exhibit excellent electrolyte compatibility and stability to heat and alkalis. They are resistant to acids, alkalis, hard water, inorganic salts, and high temperatures, while also demonstrating good biodegradability and low irritation. Furthermore, they possess strong degreasing power and exhibit high activity and stability at pH values ​​of around 6–7. They demonstrate strong compatibility and miscibility with other surfactants and can be used in the preparation of various chemicals.

[0061] Amide-type nonionic surfactants have no cloud point and, due to the presence of amide bonds in their molecules, exhibit strong resistance to hydrolysis and stability. Furthermore, they show better compatibility with anionic surfactants when the surfactants are acidic, which enhances the washing effect and further improves the repeatability and specificity of the test reagents.

[0062] Sodium citrate, acting as an emulsifier and stabilizer, ensures the stability of the entire system.

[0063] Salts are used to reduce electrostatic interactions. These include, but are not limited to, phosphates, citrates, calcium, magnesium, sulfates, sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, and sodium sulfate. Preferably, the salt is NaCl, preferably 100 mM NaCl.

[0064] A buffer solution is a buffer solution that resists pH changes through the action of its acid-base binding components. Suitable buffers include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and / or citrate buffers. The concentration of such buffers is preferably between about 100 mM and 1000 mM.

[0065] Buffer solutions contain reagents that reduce electrostatic interactions, such as sodium, potassium, ammonium, citrate, calcium, and magnesium salts.

[0066] Example 1

[0067] This invention provides a kit for allergen detection, comprising a reaction membrane strip, a blocking solution, a chromogenic solution, a washing buffer, a sample diluent, and a stop solution; the washing buffer comprises a buffer, a surfactant, a composite antibacterial agent, a stabilizer, and a salt; the composite antibacterial agent is prepared by mixing nano zinc oxide, chitosan, isothiazolinones, magnesium nitrate, a stabilizer, and a solvent.

[0068] The buffer solution is one of phosphate buffer, Tris buffer, acetate buffer, and citrate buffer.

[0069] The surfactants include anionic surfactants and nonionic surfactants. The nonionic surfactants are amide-oxy acids, and the anionic surfactants are alcohol ether phosphates or coconut oil diethanolamide.

[0070] The isothiazolinones are methylchloroisothiazolinone and methylisothiazolinone;

[0071] The stabilizer is sodium citrate, and the solvent is isopropanol and acetic acid.

[0072] The salt is one of NaCl and KCl.

[0073] This embodiment provides a washing buffer comprising 100 mM Tris-phosphate buffer, 1.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8.

[0074] The reaction membrane strip is equipped with a detection strip and a quality control strip;

[0075] The detection strip is coated with allergen protein antigens;

[0076] The allergen protein antigens include one or more of the following: willow and / or poplar and / or elm, common ragweed, artemisia, house dust mite and / or flour dust mite, house dust, cat hair, dog epidermis, cockroach, Penicillium and / or Cladosporium and / or Aspergillus fumigatus and / or Alternaria alternata, hop, egg white, milk, peanut, soybean, beef, mutton, cod and / or lobster and / or scallop, shrimp and crab;

[0077] The quality control bands include a first quality control band, a second quality control band, and a third quality control band. The first quality control band is a strongly positive quality control band, the second quality control band is a moderately positive quality control band, and the third quality control band is a weakly positive quality control band.

[0078] The first quality control band was coated with human IgG at a concentration of 30-50 μg / mL.

[0079] The second quality control band is coated with human IgG at a concentration of 11-29 μg / mL;

[0080] The third quality control band is coated with human IgG at a concentration of 1-10 μg / mL.

[0081] The method for preparing the washing buffer includes the following steps:

[0082] (1) Preparation of the compound antibacterial agent: After washing and drying crab shells, they are crushed in a pulverizer; the crushed crab shells are placed in an acetic acid solution, heated to 80-100℃, and stirred in a mixer for 1-2 hours; then filtered, rinsed with water, and the rinsed material is added to a sodium hydroxide solution, heated to 30-60℃, and stirred for 2 hours. Then, it is added to an acetic acid solution, heated to 30-60℃, and stirred in a mixer; after filtration, it is dried to obtain a solid, which is then heated and cleaned with ethanol and methanol, and then placed in a mixer. The chitosan solid was obtained by drying in a dryer. The chitosan solid was dissolved in a 1% (w / w) acetic acid solution and stirred at 30°C for 20 min to obtain a chitosan solution. The chitosan solution was heated to 40°C, and isopropanol, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone, and nano zinc oxide were added to the solution in sequence. The mixture was stirred at 2500-3000 r / min for 30-40 min to ensure complete dispersion. After cooling to room temperature, sodium citrate was added and stirred for 10-30 min to obtain a composite antibacterial agent.

[0083] (2) Add surfactant and salt to buffer solution, stir in a mixer for 1-2 hours to obtain first mixture, adjust the pH of buffer solution to 6-6.9 with boric acid or acetic acid, add compound antibacterial agent to first mixture, stir for 1-2 hours to obtain second mixture, finally add sodium citrate, stir for 1-2 hours, adjust the pH of second mixture to 6.5 with boric acid or acetic acid to obtain washing buffer.

[0084] The concentration of the acetic acid solution is 0.1% w / v.

[0085] The present invention also provides a method for preparing the above-mentioned reagent kit, wherein antigens and quality control materials are coated on a nitrocellulose membrane using a sample applicator, the nitrocellulose membrane is fixed on a PVC plate and cut into single-use reaction membrane strips, and enzyme conjugates, chromogenic substrates, blocking solutions, washing buffers, sample diluents and stop solutions are prepared and placed together in a container for later use.

[0086] This invention also provides the application of the above-mentioned reagent kit in allergen detection, as follows:

[0087] Wet the reaction membrane strip coated with allergen protein antigen with washing buffer, place it in the incubation plate holder, and incubate at room temperature on a shaker (shaking 20 times per minute, the same below) for 1 to 2 minutes. Pour out the washing buffer.

[0088] Add 10 μl of the diluted sample to the incubation plate, cover the plate, and incubate overnight at room temperature on a shaker. Add 2 mL of wash buffer to the incubation plate, shake for 2 minutes, repeat 3 times, and discard the wash buffer. Add 2 mL of HRP-labeled mouse anti-human secondary antibody, cover the plate, and incubate at room temperature on a shaker for 30 minutes. Add 5 mL of wash buffer, shake for 2 minutes, repeat 3 times, and discard the wash buffer. Add 2 mL of TMB chromogenic solution, cover the plate, and incubate at room temperature for 10 minutes. Open the plate, aspirate the substrate solution, and terminate the reaction with the stop solution.

[0089] The test results are determined based on the presence or absence of bands. Examine the control bands and test bands. If the first, second, and third control bands appear, and their intensity decreases sequentially, the test results are valid. Examine the test bands for interpretation.

[0090] Example 2

[0091] The difference from Example 1 is that an alternative washing buffer is provided, comprising 100 mM Tris-phosphate buffer, 0.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8. The surfactant is a 1:1 mixture of amide oxy acid and cocoyl diethanolamide.

[0092] Example 3

[0093] The difference from Example 1 is that another washing buffer is provided, comprising 100 mM Tris-phosphate buffer, 0.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8. The surfactant is a 1:1 mixture of amide oxy acid and cocoyl diethanolamide.

[0094] Example 4

[0095] The difference from Example 1 is that another washing buffer is provided, comprising 100 mM Tris-phosphate buffer, 0.5% w / v compound antibacterial agent, 0.5% w / v surfactant, 150 mM NaCl, 100 mM stabilizer, and pH 6.8. The surfactant is a 1:1 mixture of amide oxy acid and cocoyl diethanolamide.

[0096] Comparative Example 1

[0097] The difference from Example 1 is that this comparative example provides a washing buffer comprising: 100 mM phosphate buffer, 1% w / v proclin 300, 1.5% w / v surfactant, 150 mM NaCl, and pH 6.8.

[0098] Comparative Example 2

[0099] The difference from Example 1 is that this comparative example provides a washing buffer comprising: 1.5M phosphate buffer solution.

[0100] Example 5

[0101] To compare and verify the sensitivity of the reagent kits in Examples 1-4 and Comparative Examples 1-2, blood samples were drawn from 68 allergy sufferers for testing. The tests included tests for dust mites, milk, protein, shrimp, mango, mold, pollen, peanuts, and willow catkins. The results were compared with those of a commercially available reagent and the actual causes of allergies, as follows:

[0102] Table 1. Sensitivity Comparison Results

[0103] allergens dust mites milk protein shrimp mango mold pollen peanut Willow catkins Example 1 Positive Detection Rate 11 10 9 6 9 3 9 5 6 Example 2 11 10 9 6 9 3 9 5 6 Example 3 11 10 9 6 9 3 9 5 6 Example 4 11 10 9 6 9 3 9 5 6 Comparative Example 1 10 10 10 6 9 3 8 5 6 Comparative Example 2 11 9 9 6 8 3 9 5 6 Actual cause of allergy 11 10 9 6 9 3 9 5 6

[0104] As can be seen from Table 1, the detection methods provided in Examples 1-4 of the present invention have high detection sensitivity and the detection results are comparable to the actual causes of allergies. In contrast, Comparative Examples 1-2 all showed that some items were not detected.

[0105] Example 6

[0106] The washing buffer solutions prepared in Examples 1-4 were incubated at 40°C for 90 days, at 24°C for 180 days, or at 4°C for 360 days, respectively. The results are shown in Table 2.

[0107] Table 2. Stability of Examples 1-4 at different temperatures

[0108] Days temperature Appearance Sedimentation 90 days 4℃ colorless No sediment 90 days 24℃ colorless No sediment 90 days 40℃ colorless No sediment 180 days 4℃ colorless No sediment 180 days 24℃ colorless No sediment 180 days 40℃ colorless No sediment 360 days 4℃ colorless No sediment 360 days 24℃ colorless No sediment 360 days 40℃ colorless No sediment

[0109] Example 7

[0110] To verify the reproducibility of this kit, the same patient serum samples (allergic to dust mites, protein, shrimp, and mango) were tested 12 times.

[0111] Prepare 15 reaction membrane strips prepared in Example 1, with 9 detection strips, and coat them sequentially with dust mites, milk, egg white, shrimp, mango, mold, pollen, peanut, and willow catkins, respectively. Wet them with washing buffer, place them in the incubation plate tank, and incubate them on a shaker at room temperature for 1 to 2 minutes. Pour out the washing buffer.

[0112] Add 10 μl of the serum to be tested diluted with diluent to the incubation plate, cover the incubation plate, and incubate overnight at room temperature on a shaker. Add 2 mL of wash buffer to the incubation plate, shake for 2 minutes, repeat 3 times, and pour out the wash buffer. Add 2 mL of HRP-labeled mouse anti-human secondary antibody, cover the incubation plate, and incubate at room temperature on a shaker for 30 minutes. Add 5 mL of wash buffer, shake for 2 minutes, repeat 3 times, and pour out the wash buffer. Add 2 mL of TMB chromogenic solution, cover the incubation plate, and incubate at room temperature for 10 minutes. Open the incubation plate, aspirate the substrate solution, and terminate the reaction with the stop solution.

[0113] The detection results were interpreted based on the appearance of bands. The results are shown in Table 3. The 12 reaction membrane strips showed consistent results, indicating good reproducibility of this kit.

[0114] Table 3. Repeatability test results of this kit

[0115]

[0116] Example 8

[0117] To test the positive concordance rate of Examples 1-4 of this kit, 50 pollen antigen-positive samples were tested, and all results were positive. That is, the positive concordance rate was 100%, and the false negative rate was 0.

[0118] Example 9

[0119] Twenty negative samples were tested. The samples were clinical samples. The test results of Examples 1-4 were all negative, that is, the negative concordance rate was 100% and the false positive rate was 0.

[0120] In summary, this application utilizes immunoblotting to simultaneously detect multiple allergens, facilitating timely diagnosis and appropriate treatment for patients. The combined use of nonionic and anionic surfactants in this invention improves the stability of the washing solution and significantly reduces the impact of nonspecific interfering substances on the test results, thereby enhancing accuracy. The combined use of different antibacterial agents provides excellent preservative effects. The washing buffer solution provided by this invention, through the selection of specific components, solves the problems of high prices and high testing costs associated with imported washing solutions. The washing solution provided by this invention is low-cost and reduces domestic dependence on imported washing solutions. Furthermore, it has been verified that this washing buffer solution is suitable for allergen detection using immunoblotting.

[0121] This kit can detect multiple allergens simultaneously from a single sample, providing serological evidence for the clinical diagnosis of allergens. It features strong positive, moderate positive, and weak positive control bands, making the results more intuitive and accurate. Through repeatability tests, positive concordance rate tests, and negative concordance rate tests, the kit has been shown to have good repeatability, with both positive and negative concordance rates at 100%, indicating high accuracy.

Claims

1. A kit for allergen detection, characterized in that, This includes reaction membrane strips, blocking solution, colorimetric solution, washing buffer, sample dilution solution, and stop solution; The washing buffer comprises 100 mM Tris-phosphate buffer, 1.5% w / v compound antibacterial agent, 1.5% w / v surfactant, 150 mM NaCl and 100 mM stabilizer; The composite antibacterial agent is made by mixing nano zinc oxide, chitosan, isothiazolinones, magnesium nitrate, stabilizers and solvents; The surfactants include anionic surfactants and nonionic surfactants, wherein the nonionic surfactants are amide-oxy acids, and the anionic surfactants are alcohol ether phosphates or coconut oil diethanolamide; The isothiazolinones are methylchloroisothiazolinone and methylisothiazolinone; The stabilizer is sodium citrate, and the solvent is isopropanol and acetic acid.

2. The allergen detection kit as described in claim 1, characterized in that, The reaction membrane strip is equipped with a detection strip and a quality control strip; The test strip is coated with allergen protein antigens; The allergen protein antigens include one or more of the following: willow and / or poplar and / or elm, common ragweed, artemisia, house dust mite and / or flour dust mite, house dust, cat dander, dog epidermis, cockroach, Penicillium and / or Cladosporium and / or Aspergillus fumigatus and / or Alternaria alternata, hop, egg white and / or egg yolk, milk, peanut, soybean, beef, mutton, cod and / or lobster and / or scallop, shrimp, and crab.

3. The allergen detection kit as described in claim 2, characterized in that, The quality control bands include a first quality control band, a second quality control band, and a third quality control band. The first quality control band is a strongly positive quality control band, the second quality control band is a moderately positive quality control band, and the third quality control band is a weakly positive quality control band. The first quality control band was coated with human IgG at a concentration of 30-50 μg / mL. The second quality control band is coated with human IgG at a concentration of 11-29 μg / mL; The third quality control band is coated with human IgG at a concentration of 1-10 μg / mL.

4. The allergen detection kit as described in claim 3, characterized in that, The washing buffer solution is prepared according to the following steps: (1) Preparation of compound antibacterial agent: After washing and drying crab shells, crush them in a pulverizer; put the crushed crab shells into acetic acid solution, heat to 80-100℃, and stir in a mixer for 1-2 hours; then filter, rinse with water, and then add the rinsed material to sodium hydroxide solution, heat to 30-60℃, stir for 2 hours, add to acetic acid solution, heat to 30-60℃, and stir in a mixer; after filtration, dry to obtain a solid, then add ethanol and methanol, heat and clean, and then put into The chitosan solid was obtained by drying in a dryer. The chitosan solid was dissolved in a 1% (w / w) acetic acid solution and stirred at 30°C for 20 min to obtain a chitosan solution. The chitosan solution was heated to 40°C, and isopropanol, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone, and nano zinc oxide were added to the solution in sequence. The mixture was stirred at 2500-3000 r / min for 30-40 min to ensure complete dispersion. After cooling to room temperature, sodium citrate was added and stirred for 10-30 min to obtain a composite antibacterial agent. (2) Add surfactant and salt to buffer solution, stir in a mixer for 1-2 hours to obtain first mixture, adjust the pH of buffer solution to 6-6.9 with boric acid or acetic acid, add compound antibacterial agent to first mixture, stir for 1-2 hours to obtain second mixture, finally add sodium citrate, stir for 1-2 hours, adjust the pH of second mixture to 6.5 with boric acid or acetic acid to obtain washing buffer.

5. The method for preparing an allergen detection kit as described in claim 1, characterized in that, Antigens and quality control samples were coated onto nitrocellulose membranes using a spotting machine. The nitrocellulose membranes were then fixed onto PVC plates and cut into single-use reaction strips. Separately, enzyme conjugates, chromogenic solutions, blocking solutions, washing buffers, sample diluents, and stop solutions were prepared and placed together in a container for later use.

6. The application of the kit described in claim 1 in allergen detection.

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