Quantitative detection reagent for peanut allergen specificity IgE (Immunoglobulin E)

Abstract

The invention relates to the field of biological detection, in particular to a quantitative detection reagent for peanut allergen specificity IgE (Immunoglobulin E). The invention provides a detection reagent. The detection reagent comprises a biotinylation-peanut allergen extract and / or a biotinylation-peanut allergen component Arah3; the biotinylation-peanut allergen extract is prepared from biotin and a peanut natural allergen extract, the biotinylation-peanut allergen component Arah3 is prepared from biotin and a peanut allergen component Arah3. The invention provides an allergen-containing reagent for detecting peanut allergen specificity IgE (Immunoglobulin E), the main component of the reagent is obtained by adding a peanut allergen component Arah3 into a natural extract and mixing, so that the abundance of allergen is increased, and the problem of missed diagnosis of natural extract detection is solved. The sensitivity of the allergen-containing reagent provided by the invention is higher than that of a natural extract.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a peanut allergen specific IgE quantitative detection reagent. Background Art

[0002] The diagnosis and treatment of allergic diseases requires the identification of allergens and targeted prevention and treatment of allergens. Therefore, identifying allergens is the core of allergic disease treatment. Only by identifying allergens can we effectively take measures to avoid contact with allergens and decide on immunotherapy plans. It also helps to predict the natural course of allergic diseases in children.

[0003] There are about 160 kinds of food that cause allergies, among which milk, eggs, peanuts, soybeans, wheat, fish, crustaceans and tree nuts are identified by the FDA as the eight major allergic foods. About 90% of food allergies are caused by these eight foods. Peanut allergy is an immediate allergic reaction mediated by immunoglobulin E (IgE). Only trace amounts of allergens can cause severe allergic reactions. The most common contact with peanut allergens is food ingestion (91%), followed by skin contact (8%) and inhalation (1%). Allergic reactions caused by oral ingestion of peanuts are more severe and can be life-threatening, while allergic reactions caused by skin or air contact are relatively mild. Its clinical symptoms often involve multiple systems, including the skin, cardiovascular, digestive and respiratory systems, mainly manifesting many uncomfortable symptoms, such as itching, angioedema, rhinoconjunctivitis, asthma and diarrhea, etc. Severe systemic reactions such as anaphylactic shock may occur. These symptoms may occur alone or with complications. Peanuts are an important cause of severe food allergies in both adults and children. Peanut allergy is different from allergies to other foods (such as milk, eggs, soy and wheat). Peanut allergy is generally more severe and persistent. Allergic symptoms rarely disappear completely, and only 20% of people with peanut allergy can tolerate it. Clinical diagnosis methods for peanut allergy include in vivo and in vitro tests. In vivo tests include food provocation tests, skin prick tests (SPT), specific patch tests (APT), etc. In vitro tests include food-specific IgE (sIgE) antibody and food-specific IgG (sIgG) antibody detection. Currently, food provocation tests are the gold standard for diagnosing peanut allergy, but they are time-consuming, labor-intensive, expensive, and dangerous, and have certain limitations in clinical application.

[0004] The diagnostic methods for allergic diseases mainly include in vivo tests and in vitro tests. In vitro tests are mainly based on serological IgE detection. Specific IgE detection of patients with peanut allergy can assess the risk of disease and provide medication guidance for the desensitization treatment process. The list of the World Health Organization / International Union of Immunological Societies Allergen Nomenclature Subcommittee lists 16 peanut allergens, of which Arah1, Arah2, Arah3, and Arah6 are the main allergenic proteins. The main peanut allergen Arah3 is an 11S seed storage protein. It is well known that the 11S protein has multiple isoforms in many species. It is known that there are 5 different genes encoding Ara h3 subtypes in peanuts. Due to the heterogeneity of this allergenic protein, purification from natural sources is considered difficult. The choice of ELISA type is initially determined by the purity and specificity of the antigen and antibody. Among them, the selection of antigens and the quality of antigens are very important for the accuracy of the test results. By increasing the abundance of antigens, the phenomenon of the risk of missed detection when using a single natural extract as an antigen can be improved.

[0005] At present, there are four registered numbers for peanut allergen-specific IgE detection kits in China, including: (1) the imported peanut f13 allergen-specific IgE antibody detection kit (fluorescent immunoassay), the allergen-containing reagent component used in this kit is the antigen-coated cap; (2) the domestic peanut f013 allergen-specific IgE antibody detection kit (fluorescent magnetic particle chemiluminescent immunoassay), the allergen-containing reagent component used in this kit is biotinylated peanut protein extract; (3) the domestic peanut allergen-specific IgE antibody detection kit (enzyme-linked immunosorbent capture assay), the allergen-containing reagent component used in this kit is peanut allergen-biotin; (4) the domestic peanut allergen-specific IgE antibody detection kit (enzyme immunoassay), the allergen-containing reagent component used in this kit is biotinylated allergen; the main components of the allergen-containing reagent components used in the above kits are all natural extracts of peanuts. Summary of the invention

[0006] In view of this, the present invention provides a peanut allergen-specific IgE quantitative detection reagent. The present invention provides an allergen-containing reagent for peanut allergen-specific IgE detection, the main component of which is obtained by mixing a natural extract with a peanut allergen component Arah3, so as to increase the abundance of allergens and address the problem of missed diagnosis in natural extract detection. The allergen-containing reagent provided by the present invention has higher sensitivity than natural extracts.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a detection reagent, comprising: a biotinylated-peanut allergen extract and / or a biotinylated-peanut allergen component Arah3;

[0009] The biotinylated-peanut allergen extract comprises: biotin and a natural peanut allergen extract;

[0010] The biotinylated peanut allergen component Arah3 comprises: biotin and peanut allergen component Arah3.

[0011] In some embodiments of the present invention, the above detection reagent further comprises: streptavidin-coated magnetic particles, anti-human IgE enzyme-labeled antibodies, calibrators, biotinylated anti-human IgE antibodies and buffer.

[0012] The present invention also provides a method for preparing the above-mentioned detection reagent, wherein the biotin is mixed with the peanut natural allergen extract and the peanut allergen component Arah3, respectively, reacted, and dialyzed to obtain the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3, respectively.

[0013] The present invention also provides a method for preparing the above detection reagent, comprising the following steps:

[0014] The buffer comprises: ~Buffer One or more of;

[0015] S1: mixing the biotin with the peanut natural allergen extract and the peanut allergen component Arah3, reacting and dialyzing, to obtain the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3, respectively;

[0016] S2: Separately mix the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3 with a buffer Mixing to obtain a solution of the biotinylated-peanut allergen extract and a solution of the biotinylated-peanut allergen component Arah3;

[0017] S3: Take the streptavidin-coated magnetic particles and the buffer I. After mixing, magnetic separation, and discarding the supernatant, a precipitate is obtained;

[0018] S4: Take the precipitate and the buffer solution Mixing to obtain a solution of the streptavidin-coated magnetic particles;

[0019] S5: Take the anti-human IgE enzyme-labeled antibody and the buffer Mixing to obtain a solution of the anti-human IgE enzyme-labeled antibody;

[0020] S6: Take the IgE antibody and the buffer Mixing to obtain the calibrant;

[0021] S7: Take the biotin-labeled mouse anti-human IgE antibody and the buffer The biotinylated anti-human IgE antibody was obtained by mixing.

[0022] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut natural allergen extract is 1:(5-40).

[0023] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut natural allergen extract is 1: (5-10), 1: (10-20) or 1: (20-40).

[0024] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut natural allergen extract is 1:5, 1:10, 1:20 or 1:40.

[0025] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut natural allergen extract is 1:20.

[0026] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut allergen component Arah3 is 1:(5-20).

[0027] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut allergen component Arah3 is 1:(5-10) or 1:(10-20).

[0028] In some embodiments of the present invention, in the above preparation method, the molar ratio of the biotin to the peanut allergen component Arah3 is 1:10.

[0029] In some embodiments of the present invention, in the above preparation method, the final concentration of the solution of the biotinylated-peanut allergen extract is 15-25 μg / mL; the final concentration of the solution of the biotinylated-peanut allergen component Arah3 is 15-25 μg / mL.

[0030] In some embodiments of the present invention, in the above preparation method, the final concentration of the solution of the biotinylated-peanut allergen extract is 20 μg / mL; the final concentration of the solution of the biotinylated-peanut allergen component Arah3 is 20 μg / mL.

[0031] In some embodiments of the present invention, in the above preparation method, the buffer Includes: 1% (v / v) bovine serum albumin, 1‰ (v / v) polyethylene glycol 6000, and glycine-NaOH buffer pH 7-9;

[0032] The buffer I includes: Tris-HCl buffer pH 7~8;

[0033] The buffer Includes: 1~3% (v / v) human serum albumin and Tris-HCl buffer pH 7~8;

[0034] The buffer Includes: 5% (v / v) horse serum and phosphate buffer pH 7;

[0035] The buffer Includes: 1% (v / v) bovine serum albumin, 1‰ (v / v) polyethylene glycol 6000, and phosphate buffer pH 7-8;

[0036] The buffer Includes: 5% (v / v) horse serum, 2% (v / v) glycerol, 10% (v / v) sucrose, and phosphate buffer pH 7.

[0037] In some embodiments of the present invention, in the above preparation method, the final concentration of the solution of the streptavidin-coated magnetic microparticles is 0.5~3 mg / mL; the final concentration of the solution of the anti-human IgE enzyme-labeled antibody is 1 mg / mL; the final concentration of the calibrator is 0~100 IU / mL; and the final concentration of the biotinylated-anti-human IgE antibody is 3 μg / mL.

[0038] The present invention also provides the use of the above detection reagent and / or the detection reagent prepared by the above preparation method in the preparation of a peanut allergen-specific IgE quantitative detection product.

[0039] The present invention also provides a detection product, comprising: the above detection reagent and / or the detection reagent obtained by the above preparation method and an acceptable auxiliary agent, carrier and / or device.

[0040] The present invention provides a detection reagent, comprising: a biotinylated-peanut allergen extract and / or a biotinylated-peanut allergen component Arah3;

[0041] The biotinylated-peanut allergen extract comprises: biotin and a natural peanut allergen extract;

[0042] The biotinylated peanut allergen component Arah3 comprises: biotin and peanut allergen component Arah3.

[0043] The present invention provides an allergen-containing reagent for peanut allergen-specific IgE detection. The main component of the reagent is obtained by mixing a natural extract with a peanut allergen component Arah3, thereby increasing the abundance of allergens and addressing the problem of missed diagnosis in natural extract detection. The allergen-containing reagent provided by the present invention has higher sensitivity than natural extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0045] Figure 1 Shows HPLC analysis of natural peanut extract;

[0046] Figure 2 Shown is the HPLC analysis of mixed allergens of natural peanut extract and peanut added components. DETAILED DESCRIPTION

[0047] The invention discloses a peanut allergen-specific IgE quantitative detection reagent.

[0048] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0050] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0051] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0052] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0053] The invention provides a peanut allergen-specific IgE quantitative detection kit, which comprises: magnetic particles coated with streptavidin, biotinylated-peanut allergen solution, anti-human IgE enzyme-labeled antibody, calibrator, and biotin-labeled anti-human IgE antibody.

[0054] Preparation of the streptavidin-coated magnetic microparticle suspension:

[0055] The streptavidin-coated magnetic microparticle stock solution is resuspended in 0.05M Tris-HCl buffer (pH=7~8), washed repeatedly for 4 times (5~10 min / time), and magnetically separated. Finally, the magnetic microparticles are resuspended in 0.05M Tris-Nacl buffer (pH=7~8) containing 1~3% human serum albumin (Albumin Human, HSA) to obtain a streptavidin-coated magnetic microparticle suspension with a concentration of 0.5~3mg / mL, preferably 1~2mg / mL.

[0056] Preparation of the biotinylated-peanut allergen solution:

[0057] (1) Extraction of natural peanut allergen extract: Select fresh and high-quality peanut kernels, rub to remove the red skin, and then crush them in a tissue crusher and pass through a 40-mesh sieve. Then mix the peanut powder with n-hexane at a ratio of 1:10 (g / mL), defatted under magnetic stirring at 4°C for 2 hours, let it stand for 1 hour, discard the supernatant, and repeat the defatting once. Place the defatted peanut powder in a fume hood overnight to allow the n-hexane to evaporate completely. Mix the defatted peanut powder with pre-cooled 0.01 mol / L PBS (pH 7.4, containing 0.01 mol / L β-mercaptoethanol and 0.05% EDTA·2Na) at a ratio of 1:10 (g / mL), and magnetically stir at 4°C overnight. The next day, centrifuge the extract at 4°C, 11,000×g for 30 minutes, collect the supernatant, extract the precipitate with the extract for another 4 hours, and combine the supernatants after centrifugation to obtain the peanut allergen.

[0058] The peanut allergen component Arah3 was obtained by extracting total RNA from peanuts, reverse transcribed into c DNA, and amplifying the Arah3 gene sequence by PCR. The sequence was cloned into the pET-28a vector to construct the Arah3-pET-28a expression vector, which was verified and sequenced. The successfully sequenced plasmid was transferred into the Rosetta (DE3) expression host bacteria to induce expression. The recombinant protein expressed in the form of inclusion bodies was dissolved and dialyzed for renaturation. The recombinant protein was purified by nickel column to obtain the peanut allergen component Arah3.

[0059] (2) The molar ratio of biotin to the peanut natural allergen extract is 1:5 to 1:40; the preferred labeling ratio is 1:20. The molar ratio of biotin to the peanut allergen component Arah3 is 1:5 to 1:20; the preferred labeling ratio is 1:10.

[0060] (3) Preparation method of biotinylated-peanut allergen: biotin reagent and peanut natural allergen extract are mixed in a ratio of 1:20, and biotin reagent and peanut allergen component Arah3 are mixed in a ratio of 1:10. After shaking reaction at 2~8℃ for 2 hours, the reaction solution is loaded into a dialysis bag with MWCO: 3.5kD, and placed in 100-1000 fold volume of 0.01M phosphate buffer saline (PBS) (pH=7~8), and dialyzed for 24 hours to obtain biotinylated-peanut natural allergen extract and biotinylated-peanut allergen component Arah3, respectively.

[0061] (4) The biotinylated peanut natural allergen extract and the biotinylated peanut allergen component Arah3 were prepared into an antigen solution using a glycine-NaOH buffer (pH 7-9) containing a protein protective agent (bovine serum albumin) and a surfactant (polyethylene glycol 6000).

[0062] (5) The concentration of the biotinylated peanut allergen extract solution is 15-25 μg / mL, preferably 20 μg / mL; the concentration of the biotinylated peanut allergen component Arah3 solution is 15-25 μg / mL, preferably 20 μg / mL.

[0063] Preparation of the horseradish peroxidase-labeled anti-human IgE antibody

[0064] The horseradish peroxidase labeled with anti-human IgE is prepared with a phosphate buffer (pH=7) containing 5% horse serum to a concentration of 1 mg / mL.

[0065] Preparation of the calibrator

[0066] (1) IgE antibody is dissolved in phosphate buffer (pH 7-8) containing a protein protectant (bovine serum albumin) and a surfactant (polyethylene glycol 6000).

[0067] (2) 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, 50 IU / mL, and 100 IU / mL.

[0068] The biotinylated anti-human IgE antibody and its solution preparation:

[0069] Take Sulfo-NHS-LC-Biotin and add dimethyl sulfoxide to prepare 20mmol of biotin, add 5.0mg of anti-human IgE antibody, and react at room temperature for 3 hours. After the reaction, the system is dialyzed in phosphate buffer at 4°C overnight to obtain biotin-labeled anti-human IgE antibody, and phosphate buffer (pH=7) containing 5% horse serum, 2% glycerol and 10% sucrose is used to prepare a 3μg / mL solution to obtain biotin-labeled anti-human IgE antibody.

[0070] The method for detecting the peanut allergen-specific IgE:

[0071] (1) Conduct parallel testing of the calibrator and the sample to be tested;

[0072] (2) Mix the gradient calibrant and the sample to be tested with the streptavidin-coated magnetic microparticle suspension, then add the biotinylated anti-human IgE antibody solution and the biotinylated peanut allergen extract solution respectively, mix well, incubate at 37°C for 15 minutes, and perform magnetic separation to discard the supernatant;

[0073] (3) Add anti-human IgE enzyme-labeled antibody solution, mix well, incubate at 37°C for 15 minutes, and magnetically separate and discard the supernatant;

[0074] (4) Add substrate and measure the chemiluminescence intensity.

[0075] The present invention provides a peanut allergen-specific IgE detection kit, comprising the peanut allergen-specific IgE quantitative detection reagent and an acceptable auxiliary agent or carrier. The kit comprises: magnetic particles coated with streptavidin, biotinylated-peanut allergen solution, anti-human IgE enzyme-labeled antibody, calibrator, biotinylated-anti-human IgE antibody; the biotinylated-peanut allergen comprises natural peanut extract and peanut allergen component Arah3.

[0076] In Examples 1 to 6 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0077] The present invention will be further described below in conjunction with embodiments:

[0078] Example 1

[0079] This embodiment provides a peanut allergen-specific IgE quantitative detection kit, which includes: streptavidin-coated magnetic particles, biotinylated-peanut allergen solution, anti-human IgE enzyme-labeled antibody, calibrator, and biotin-labeled anti-human IgE antibody.

[0080] 1) Preparation of the streptavidin-coated magnetic microparticle suspension:

[0081] Place the streptavidin-coated magnetic particles in 0.05M Tris-HCl buffer (pH=7~8) and shake and wash for 5~10 minutes, separate with a magnet, discard the supernatant, and repeat 3~4 times; add 0.05M Tris-Nacl buffer (pH=7~8) containing 1~3% human serum albumin (Albumin Human, HSA) and resuspend to obtain a 1.0 mg / mL streptavidin-coated magnetic particle suspension.

[0082] 2) Preparation of the biotinylated peanut allergen:

[0083] Preparation method of biotinylated-peanut allergen: The biotin reagent is mixed with peanut natural allergen and recombinant allergenic protein Arah3 in a specific ratio, and after shaking reaction at 2~8℃ for 1 hour, the reaction solution is loaded into a dialysis bag with MWCO: 3.5kD, and placed in a 100-1000 fold volume of 0.01M phosphate buffer saline (phosphate buffer saline, PBS) (pH=7~8), and dialyzed for 24 hours to obtain biotinylated-peanut natural allergen extract and biotinylated-peanut allergen component Arah3, respectively.

[0084] 3) Preparation of the anti-human IgE enzyme-labeled antibody and its solution:

[0085] The anti-human IgE enzyme-labeled antibody is a mouse anti-human IgE antibody labeled with horseradish peroxidase, which is dissolved in a phosphate buffer (pH 7-9) containing a protein protective agent (bovine serum albumin) and a surfactant (polyethylene glycol 6000) to prepare a 1.0 mg / mL anti-human IgE enzyme-labeled antibody solution;

[0086] 4) Preparation of the horseradish peroxidase-labeled mouse anti-human IgE antibody solution:

[0087] The HRP-labeled mouse anti-human IgE antibody was prepared into a 1.0 μg / mL solution using a protein protective agent and a 0.05 MTris-NaCl buffer (pH=7~8) of a surfactant to obtain a horseradish peroxidase-labeled mouse anti-human IgE antibody solution.

[0088] 5) The specific IgE antibody calibration kit (magnetic particle chemiluminescence method) comprises:

[0089] Biotin-labeled anti-human IgE antibody, magnetic particle suspension, horseradish peroxidase-labeled mouse anti-human IgE, and calibrator; preferably, the concentration of biotin-labeled anti-human IgE antibody is 1.0 μg / mL; the calibrator is IgE protein, and preferably, the concentration of the calibrator is 7 concentration points, including 0.0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, 50 IU / mL, and 100 IU / mL.

[0090] Example 2

[0091] This embodiment provides a detection method using the kit prepared in Example 1, which is compatible with the Antu Bio fully automatic chemiluminescence instrument, has a high degree of automation, is simple and efficient. The method comprises the following steps:

[0092] 1) Add 20 μL of calibrator or sample to be tested into the reaction cup;

[0093] 2) Then add 20 μL of streptavidin-coated magnetic microparticle suspension and 20 μL of biotinylated anti-human IgE antibody solution or biotinylated peanut allergen solution to the reaction cup and incubate at 37±0.5℃ for 15 min;

[0094] 3) After the incubation, magnetic separation, removal of supernatant, and washing with washing solution three times;

[0095] 4) Then add 60 μL of horseradish peroxidase-labeled mouse anti-human IgE antibody solution and incubate at 37±0.5℃ for 15 min;

[0096] 5) After the incubation, magnetic separation, removal of supernatant, and washing with washing solution three times;

[0097] 6) Then add 50 μL of substrate A solution (containing luminol reagent) and 50 μL of substrate B solution (containing peroxide reagent), shake and mix for 18 seconds before testing;

[0098] 7) Select an appropriate curve fitting method. This kit recommends using a five-parameter standard curve. Use the concentration of the IgE calibrator as the horizontal axis and the corresponding chemiluminescent signal value as the vertical axis to establish a calibration curve. Calculate the concentration of peanut allergen-specific IgE in the sample to be tested based on the chemiluminescent signal value of the sample to be tested.

[0099] According to the above steps, the optimal ratio of biotin reagent and antigen is first selected. The results are shown in Table 1 and Table 2:

[0100] Table 1

[0101]

[0102] Table 2

[0103]

[0104] The results showed that the optimal mixing ratio of biotin reagent and peanut natural allergen extract was 1:20; the optimal mixing ratio of biotin reagent and peanut allergen component Arah3 was 1:10.

[0105] Example 3

[0106] Natural peanut extract and peanut recombinant allergenic component Arah3 were labeled with biotin reagent and tested in parallel in 25 samples. The results are shown in Table 3:

[0107] Table 3

[0108]

[0109] The results showed that when natural peanut extract was used alone to test samples, there would be a phenomenon of low sample reactivity or missed detection. However, when the peanut allergen component Arah3 was used alone to test samples, the reactivity of these low-reactive and missed samples increased, which could supplement the reactivity of natural peanut extract.

[0110] Example 4

[0111] The specific steps for comparing the allergens of natural peanut extract and peanut added components were as follows:

[0112] 1) The mobile phase begins to flow. The pump pushes the eluent or solvent through the system at a specified flow rate.

[0113] 2) Injection. Once the sample enters the mobile phase flow path, it will flow from the injection point to the head of the chromatographic column along with the mobile phase.

[0114] 3) Compound separation. The compounds are physically separated in the stationary phase of the chromatographic column. After elution from the chromatographic column, the separated sample components come to the detector.

[0115] 4) Analyte detection. Detect the target analyte using an electrical signal generated based on specific properties.

[0116] 5) Generate a chromatogram. CDS converts the detected analyte signal into a chromatogram represented by analyte signal and time.

[0117] like Figure 1 and Figure 2 The results showed that the main allergenic proteins in natural peanut extracts were present, and the abundance of Arah3 increased after adding components and mixing.

[0118] Example 5

[0119] The allergen reactivity of natural peanut extract and mixed allergens of peanut added components were compared using the Antu Bio-Automatic Chemiluminescence Analyzer (the experimental procedures have been described in Example 2).

[0120] Table 4

[0121]

[0122] The results showed that compared with natural peanut extracts, the mixed allergen reactivity of peanut added components was higher.

[0123] Example 6

[0124] The allergen compliance rate of natural peanut extract and mixed peanut added components was analyzed using the Antu Bio fully automatic chemiluminescence instrument (the experimental steps have been described in Example 2).

[0125] Table 5

[0126]

[0127] The results showed that after adding the peanut allergen component Arah3 in a specific proportion and mixing, the sensitivity was improved compared with conventional reagents.

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection reagent, characterized in that: include: Biotinylated-peanut allergen extract and / or biotinylated-peanut allergen fraction Arah3; The biotinylated-peanut allergen extract comprises: biotin and a natural peanut allergen extract; The biotinylated peanut allergen component Arah3 comprises: biotin and peanut allergen component Arah3.

2. The detection reagent according to claim 1, characterized in that Also includes: Streptavidin-coated magnetic particles, anti-human IgE enzyme-labeled antibody, calibrator, biotinylated anti-human IgE antibody and buffer.

3. The method for preparing the detection reagent according to claim 1, characterized in that: The biotin is mixed with the peanut natural allergen extract and the peanut allergen component Arah3, reacted, and dialyzed to obtain the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3, respectively.

4. The method for preparing the detection reagent according to claim 2, characterized in that: The steps include: The buffer comprises: ~Buffer One or more of; S1: mixing the biotin with the peanut natural allergen extract and the peanut allergen component Arah3, reacting and dialyzing, to obtain the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3, respectively; S2: Separately mix the biotinylated-peanut allergen extract and the biotinylated-peanut allergen component Arah3 with a buffer Mixing to obtain a solution of the biotinylated-peanut allergen extract and a solution of the biotinylated-peanut allergen component Arah3; S3: Take the streptavidin-coated magnetic particles and the buffer I. After mixing, magnetic separation, and discarding the supernatant, a precipitate is obtained; S4: Take the precipitate and the buffer solution Mixing to obtain a solution of the streptavidin-coated magnetic particles; S5: Take the anti-human IgE enzyme-labeled antibody and the buffer Mixing to obtain a solution of the anti-human IgE enzyme-labeled antibody; S6: Take the IgE antibody and the buffer Mixing to obtain the calibrant; S7: Take the biotin-labeled mouse anti-human IgE antibody and the buffer The biotinylated anti-human IgE antibody was obtained by mixing.

5. The preparation method according to claim 3 or 4, characterized in that: The molar ratio of the biotin to the peanut natural allergen extract is 1:(5-40); The molar ratio of the biotin to the peanut allergen component Arah3 is 1:(5-20).

6. The preparation method according to claim 4 or 5, characterized in that: The final concentration of the solution of the biotinylated peanut allergen extract is 15-25 μg / mL; the final concentration of the solution of the biotinylated peanut allergen component Arah3 is 15-25 μg / mL.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The buffer Includes: 1% (v / v) bovine serum albumin, 1‰ (v / v) polyethylene glycol 6000, and glycine-NaOH buffer pH 7-9; The buffer I includes: Tris-HCl buffer pH 7~8; The buffer Includes: 1~3% (v / v) human serum albumin and Tris-HCl buffer pH 7~8; The buffer Includes: 5% (v / v) horse serum and phosphate buffer pH 7; The buffer Includes: 1% (v / v) bovine serum albumin, 1‰ (v / v) polyethylene glycol 6000 and phosphate buffer pH 7~8; The buffer Includes: 5% (v / v) horse serum, 2% (v / v) glycerol, 10% (v / v) sucrose and phosphate buffer pH 7.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The final concentration of the solution of the streptavidin-coated magnetic microparticles is 0.5-3 mg / mL; the final concentration of the solution of the anti-human IgE enzyme-labeled antibody is 1 mg / mL; the final concentration of the calibrator is 0-100 IU / mL; and the final concentration of the biotinylated anti-human IgE antibody is 3 μg / mL.

9. Use of the detection reagent according to claim 1 or 2 and / or the detection reagent prepared by the preparation method according to any one of claims 3 to 8 in preparing a peanut allergen-specific IgE quantitative detection product.

10. The detection product is characterized in that, include: The detection reagent according to claim 1 or 2 and / or the detection reagent obtained by the preparation method according to any one of claims 3 to 8, and an acceptable auxiliary agent, carrier and / or device.

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