Beef allergen specific IgE antibody detection kit and application thereof

Abstract

The invention relates to the technical field of immunological detection, in particular to a beef allergen specific IgE antibody detection kit and application thereof. The beef extract is pretreated by adopting a streptavidin-biotin system, the beef allergen extract is labeled with biotin in different proportions, and the background can be reduced and the specificity can be improved by a method for preparing a biotinylation-beef allergen extract solution.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and in particular to a beef allergen-specific IgE antibody detection kit and application thereof. Background Art

[0002] Allergic reaction belongs to type I hypersensitivity reaction, which refers to the reaction that occurs when an immune body is stimulated by the same substance again: allergens enter the body, stimulate B cells to proliferate and differentiate into plasma cells, and produce specific IgE (Specific Immununoglobulin E, sIgE) antibodies. This antibody binds to the surface of mast cells and basophils, and the body is in a sensitized state. The bound cells are called sensitized target cells, and the sensitized state can last for several weeks. If there is no contact with allergens for a long time, the sensitized state will gradually disappear. When the allergen enters the body again, it binds to two or more adjacent IgE antibodies on the surface of sensitized target cells, causing cross-linking of membrane cell FCR, triggering sensitized target cell degranulation, and releasing and synthesizing biologically active mediators such as histamine, thereby inducing allergies in the body.

[0003] The prevalence of beef allergy ranges from 1% to 10% worldwide, with the most common reports in European countries. Among children with atopic dermatitis (AD), the incidence of beef allergy ranges from 3.28% to 6.52%, and among the general public it is 0.3%. And with the improvement of people's lives, the demand for beef is also increasing, and beef allergies have also generally increased in recent years.

[0004] Currently, there are three domestic products of in vitro diagnostic reagents for quantitative detection of beef allergen-specific IgE antibodies in human serum or plasma. The reaction principle is based on the immunological detection principle of antigen-antibody reaction, including: Hunan Haooubo Biotechnology Co., Ltd.'s beef (F27) allergen-specific IgE antibody detection kit (magnetic particle chemiluminescence method, Hunan Medical Device Registration No. 20212402094), Jiangsu Haooubo Biotechnology Co., Ltd.'s beef allergen-specific IgE antibody detection kit (enzyme-linked immunosorbent capture method, Su Medical Device Registration No. 20202401703) and Changsha Haike Biotechnology Co., Ltd.'s beef F027 allergen-specific IgE antibody detection kit (fluorescent magnetic particle chemiluminescence immunoassay, Hunan Medical Device Registration No. 20202401492).

[0005] The existing technology based on manual operation of enzyme-free test is cumbersome, time-consuming, and has low throughput. In addition, the presence of non-specific binding IgG, IgA, and IgM in human serum can interfere with the test and may lead to false positive results.

[0006] The existing technology based on colloidal gold is also a manual experiment with complex operation and methodological limitations. The sensitivity of colloidal gold is different from that of chemiluminescence. Summary of the invention

[0007] In view of this, the present invention provides a beef allergen-specific IgE antibody detection kit and its application. The beef extract is pretreated by a streptavidin-biotin system, and the beef allergen extract is labeled with biotin in different proportions. By preparing a biotinylated beef allergen extract solution, the background can be reduced and the specificity can be improved.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a biotin-labeled beef allergen solution, the preparation method of which comprises:

[0010] Step (a1): subjecting the beef allergen extract to column adsorption for 5 to 60 min, centrifuging, treating the obtained supernatant at 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C for 2 to 12 h, and dialyzing to obtain beef allergen;

[0011] Step (a2): biotin and the beef allergen are mixed at a molar ratio of 1:(3-30), shaken at 2-8° C. for 0.5-4 h, and dialyzed at a molecular weight cutoff of 3.5 kD for 24 h to obtain a biotin-labeled beef allergen solution.

[0012] In some specific embodiments of the present invention, the method for preparing the beef allergen extract in the method for preparing the biotin-labeled beef allergen solution comprises: homogenizing the beef, centrifuging, and obtaining the beef allergen extract.

[0013] In some specific embodiments of the present invention, the method for preparing the beef allergen extract in the method for preparing the biotin-labeled beef allergen solution further comprises the step of freeze-drying the beef allergen extract after centrifugation;

[0014] The freeze-drying process includes pre-freezing, sublimation drying, and analytical drying of a beef allergen extract having a solid matter concentration of 15% to obtain a freeze-dried powder.

[0015] In some specific embodiments of the present invention, the column adsorption treatment in the preparation method of the biotin-labeled beef allergen solution comprises the following steps:

[0016] (c1), mix the chromatography medium and K2HPO4, centrifuge and remove the supernatant;

[0017] (c2) mixing the beef allergen extract and the chromatographic medium in a mass ratio of 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50 for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, and centrifuging to obtain a purified material.

[0018] In some specific embodiments of the present invention, the chromatography medium in the method for preparing the biotin-labeled beef allergen solution comprises cobalt ion affinity chromatography medium beads and / or nickel ion affinity chromatography medium beads.

[0019] In some specific embodiments of the present invention, the biotin-labeled beef allergen solution further comprises a phosphate buffer solution with a pH of 7 to 8 containing a preservative, a surfactant and a protein protectant.

[0020] In some specific embodiments of the present invention, the biotin-labeled beef allergen solution further comprises a blocking agent containing antibodies, wherein the antibodies comprise one or more of IgG, IgM, IgA, Anti-IgG, Anti-IgM or Anti-IgA.

[0021] The present invention also provides the use of the biotin-labeled beef allergen solution in preparing a beef allergen detection kit.

[0022] The present invention also provides a kit for detecting beef allergen-specific IgE, comprising the above-mentioned biotin-labeled beef allergen solution.

[0023] In some specific embodiments of the present invention, the kit further comprises magnetic particles coated with streptavidin and anti-human IgE antibodies labeled with horseradish peroxidase.

[0024] The present invention also provides a method for detecting beef allergen-specific IgE, which is based on the above-mentioned kit.

[0025] In some specific embodiments of the present invention, the above method comprises the following steps:

[0026] Step (d1): mixing the sample to be tested, the magnetic particles coated with streptavidin, and the biotin-labeled beef allergen solution, incubating, removing the supernatant, and washing;

[0027] Step (d2): mixing with the anti-human IgE antibody labeled with horseradish peroxidase, incubating, removing the supernatant, and washing;

[0028] Step (d3): adding substrate, shaking and mixing, and then measuring the chemiluminescence intensity;

[0029] Step (d4): Calculate the concentration of beef allergen-specific IgE in the sample to be tested according to the standard curve.

[0030] In some specific embodiments of the present invention, the concentration of the streptavidin-coated magnetic particles in the above method is 0.1-3 mg / mL.

[0031] In some specific embodiments of the present invention, the concentration of beef allergen in the biotin-labeled beef allergen solution of the above method is 0.1-40.0 μg / mL.

[0032] In some specific embodiments of the present invention, the concentration of the antibody in the above method is 10-100 μg / mL.

[0033] In some specific embodiments of the present invention, the substrate in the above method comprises luminol and / or peroxide.

[0034] In some specific embodiments of the present invention, the incubation temperature in the above method is 15-40°C.

[0035] In some specific embodiments of the present invention, the incubation time of the above method is 5 to 40 min.

[0036] The detection kit of the present invention and its application have the following effects:

[0037] In order to solve the problems of inaccurate quantification, cumbersome manual operation and poor specificity of current technology, we optimized it and selected the magnetic particle luminescence platform: using streptavidin to prepare magnetic particles and horseradish peroxidase to label anti-human IgE antibodies with chemiluminescent immunoassay, which is fully automatic to ensure the accuracy of the test results and the test throughput.

[0038] When preparing the kit, the background of the reagent can be reduced by pre-treating the conventional beef extract, and the specificity of the test result can be improved by adding a blocking agent to the formula. The kit of the present application can detect beef allergen-specific IgE antibodies in a small amount of serum / plasma samples quickly and simply, with high sensitivity, high specificity, full automation, and full quantitativeness. Specifically:

[0039] 1) After pre-treatment of beef allergens, the allergenic proteins can be effectively purified and the background can be reduced;

[0040] 2) By adding blockers, the false positive signal value caused by interference from IgG, IgM, and IgA in the sample can be effectively reduced, thereby improving the detection specificity;

[0041] 3) Compared with manual experiments such as colloidal gold and immunoblotting, it has the advantages of short reaction time, simple operation and high throughput;

[0042] 4) The kit of the present invention is suitable for the magnetic microparticle chemiluminescence instrument platform, with fully automated detection, short detection time and low detection cost. DETAILED DESCRIPTION

[0043] The present invention discloses a beef allergen-specific IgE antibody detection kit and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The invention provides a beef allergen-specific IgE detection kit, which comprises a magnetic particle suspension, a beef allergen, and an anti-human IgE antibody labeled with horseradish peroxidase; wherein the beef allergen is modified with biotin, the anti-human IgE antibody is modified with horseradish peroxidase, and the magnetic particle suspension is coated with streptavidin.

[0045] The concentration of the streptavidin-coated magnetic microparticle stock solution is 0.1-3 mg / mL, preferably 0.5-1.8 mg / mL.

[0046] The beef allergen is pretreated, and the pretreatment means include separation and purification of allergenic proteins, physical treatment, and chemical treatment. Separation and purification of allergenic proteins include fractionation and adsorption column treatment; physical treatment includes temperature treatment at 37-100°C; chemical treatment includes sodium azide-peroxide treatment. Preferably, it is column adsorption treatment, and the treatment time is 5-60 min. After treatment, centrifugation is performed to obtain the supernatant, and then heat treatment is performed at 37-100°C, and the time is preferably 2-12 h.

[0047] Beef allergens are modified with biotin. The biotin labeling steps are as follows:

[0048] (1) The beef natural allergen extract is a natural extract freeze-dried powder, which is dissolved in a purified aqueous solution or phosphate buffer, treated with column adsorption for 5 to 60 minutes, centrifuged to obtain the supernatant, and then heat-treated at 37 to 100°C for preferably 2 to 12 hours, and dialyzed in phosphate buffer;

[0049] (2) Dissolve biotin in dimethyl sulfoxide (DMSO) at a concentration of 10-40 mmol / L;

[0050] (3) The dissolved biotin is reacted with beef allergen at a molar ratio of 1:3-1:30, preferably 1:5-1:20, and the reaction is shaken at 2-8°C for 0.5-4 h, preferably 1-3 h. After the reaction, dialyze the reaction solution into a dialysis bag with a MWCO: 3.5 kD, place it in a 100-1000 fold volume of phosphate buffer (pH=7-8), and dialyze it for 24 hours to obtain a biotinylated beef allergen extract.

[0051] The biotinylated beef allergen extract is prepared with a phosphate buffer (pH=7-8) (the buffer may also be a Bis-Tris buffer or a Tris buffer) containing a preservative (including at least one of ProClin300, Bronidox, and ampicillin sodium), a surfactant (including any one of dodecyldimethylaminoethyl lactone, sodium taurocholate, TX-100, CHAPS, and CTAB), and a protein protectant (including at least two of aminopyrine ADP, BB2, glycerol, sugars, and EDTA). The concentration of the beef allergen is 0.1-40.0 μg / mL, preferably 5-20 μg / mL. A blocking agent is added, and the blocking agent may be at least one of IgG, IgM, IgA, Anti-IgG, Anti-IgM, and Anti-IgA from different sources, and each added amount is 10-100 μg / mL.

[0052] The present invention also provides a method for detecting beef allergen-specific IgE, using the above-mentioned kit, comprising the following steps:

[0053] (1) Mixing the sample to be tested, a suspension of streptavidin-coated magnetic particles, and a biotin-labeled beef allergen solution, and incubating them;

[0054] (2) After the incubation, magnetic separation, removal of supernatant, and washing with washing solution three times;

[0055] (3) Add horseradish peroxidase-labeled anti-human IgE antibody at a mass ratio of 1:1000-1:8000, mix and incubate, repeat step 2 for magnetic separation and remove the supernatant;

[0056] (4) Add substrate solution, shake and mix, and then measure the chemiluminescence intensity;

[0057] (5) Calculate the concentration of IgE in the sample to be tested based on the standard curve.

[0058] The incubation temperature in step (1) and step (3) is 15-40°C, preferably 37°C; the incubation time in step (1) and step (3) is 5-40 min, preferably 15-20 min.

[0059] The present invention also provides application of the kit in detecting beef allergen-specific IgE.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0066] The present invention will be further described below in conjunction with embodiments:

[0067] Example 1

[0068] 1. Preparation of beef allergen

[0069] Raw material extraction - The raw material is beef protein crude extract freeze-dried powder. The preparation method is: beef slices, soaked in ultrapure water or phosphate buffer, ground, centrifuged, and protein solution is obtained. The concentration of the protein solution is 3~6 mg / mL, and freeze-dried. The crude extract itself has problems such as poor specificity, high background, and poor stability, so beef allergens need to be pretreated.

[0070] Pretreatment methods include separation and purification of allergenic proteins, physical treatment, and chemical treatment. Among them, separation and purification of allergenic proteins include fractionation and adsorption column treatment; physical treatment includes heat treatment at a temperature of 37~100℃; chemical treatment includes sodium azide-peroxide treatment.

[0071] Fractionation treatment method:

[0072] Sephacryl molecular sieve was used to separate proteins according to their molecular weight, and 20~75 KD protein markers were enriched for assessment.

[0073] Adsorption column treatment method:

[0074] 1. Mix the fine beads of cobalt or nickel with K2HPO4 for 2 minutes and centrifuge to remove the supernatant;

[0075] 2. The mass ratio of beef protein solution to beads is 1:10~1:50. Mix thoroughly for 10 min and centrifuge to obtain purified material.

[0076] Heat treatment method:

[0077] Place in an incubator or water bath for treatment, the temperature is preferably 35°C ~ 70°C, specifically 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C; the time is preferably 2 ~ 12 h, specifically 2 h, 4 h, 6 h, 8 h, 10 h, 12 h.

[0078] Sodium Azide-Peroxide Treatment Method:

[0079] The mass fraction of sodium azide-peroxide treatment is preferably 10%.

[0080] Tables 1 to 4 show the effects of different treatments and condition parameters on background:

[0081] Table 1: Effects of different pretreatments on beef allergen background

[0082]

[0083] Table 2: Ratio of beef protein solution to beads in adsorption column treatment

[0084]

[0085] When the mass ratio of beef protein solution to beads was between 1:50 and 1:10, the background (N1 and N2) was significantly reduced, and the background reduction effect was more obvious when the column ratio increased.

[0086] Table 3: Temperatures during heat treatment

[0087]

[0088] After heat treatment, the background (N1) decreased. N2 was the false positive sample without treatment, and its reactivity showed a downward trend after heat treatment. Except for a few positive samples whose signal values ​​decreased after treatment at 70°C, the reactivity of the samples increased slightly after heat treatment.

[0089] Table 4: Heat treatment duration

[0090]

[0091] N1 is the background sample, and the background decreases with the increase of heat treatment time. P1 and P2 are missed samples when not treated, and the reactivity tends to increase with the increase of heat treatment time.

[0092] In summary, it was found that the effect of column adsorption treatment with a treatment time of 5 to 60 min, centrifugation to obtain the supernatant after treatment, and then heat treatment at 37 to 100°C for 2 to 12 h was unexpectedly good.

[0093] 2. Kit Preparation

[0094] A beef allergen-specific IgE detection kit is prepared based on the pre-treated beef allergen, the kit comprising: a biotin-labeled beef f27 allergen (antigen content 0.010-4.0 μg / test), a magnetic particle suspension and an enzyme conjugate;

[0095] The preparation method of the biotin-labeled beef f27 allergen is as follows:

[0096] (1) The beef allergen extract was treated by column adsorption for 10 min, centrifuged to obtain the supernatant, and then heat treated at 50°C for 6 h, and then dialyzed against phosphate buffer.

[0097] (2) Dissolve biotin in dimethyl sulfoxide (DMSO) to a concentration of 20 mmol / L;

[0098] (3) The dissolved biotin is reacted with beef allergen at a molar ratio of 1:3-1:30, preferably 1:5-1:20, and the reaction is shaken at 2-8°C for 2 h. After the reaction, dialyze the reaction solution into a dialysis bag with a MWCO: 3.5 kD, place it in a 100-1000 fold volume of phosphate buffer (pH=7-8), and dialyze it for 24 hours to obtain a biotinylated beef allergen extract.

[0099] Table 5: Effects of different pretreatments on kit performance

[0100]

[0101] Table 6: Biotin and beef allergen reactions (protein:biotin)

[0102]

[0103] P1 and P2 are boundary samples. Signal values ​​above 450,000 can be detected as positive. These two samples can be detected when the labeling ratio is above 1:5. As the labeling ratio increases, the reactivity increases and the background increases.

[0104] Example 2

[0105] Based on the kit described in Example 1 (beef allergen was treated with a column and then heat treated), IgG (Sigma-Aldrich, IgG, I8765-5MG), IgM (Sigma-Aldrich, IgM, I8260-1MG), IgA (Sigma-Aldrich, IgA, I4036-1MG), Anti-IgG (Sigma-Aldrich, Anti-human-IgG, I5260-1ML), Anti-IgM (Sigma-Aldrich, Anti-human-IgM, MABX1911-10KC), and Anti-IgA (Sigma-Aldrich, Anti-human-IgA, I0884-1MG) from different sources were added to the beef allergen to evaluate the effects of different blockers on the performance of the kit.

[0106] Table 7: Effects of different blocking agents on kit performance

[0107]

[0108] As shown in the results of Table 7: after adding the blocker, the signal value of the false positive sample was significantly reduced, the specificity was improved, and the detection of the positive sample was not affected, and the sensitivity was not affected.

[0109] Example 3

[0110] The negative samples were tested 20 times using the kit described in Example 2 (blockers were Anti-IgG and Anti-IgM), and the average value plus 2 times the standard deviation was taken as the blank limit (LoB) of the kit. The obtained LoB was less than 0.1 IU / mL.

[0111] Table 8: Kit LoB

[0112]

[0113] 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 biotin-labeled beef allergen solution, characterized in that: The preparation method comprises: Step (A1): subjecting the beef allergen extract to column adsorption treatment, centrifuging, treating the obtained supernatant at 35-100°C for 2-12 h, and dialyzing to obtain beef allergen; Step (A2): biotin and the beef allergen are mixed at a molar ratio of 1:(3-30), shaken, and dialyzed to obtain a biotin-labeled beef allergen solution.

2. The biotin-labeled beef allergen solution according to claim 1, characterized in that: It also includes a buffer solution with a pH of 7 to 8 containing preservatives, surfactants and protein protectants.

3. The biotin-labeled beef allergen solution according to claim 1 or 2, characterized in that: It also includes a blocking agent containing an antibody, wherein the antibody includes at least one of IgG, IgM, IgA, Anti-IgG, Anti-IgM, and Anti-IgA.

4. Use of the biotin-labeled beef allergen solution according to any one of claims 1 to 3 in the preparation of a beef allergen detection kit.

5. A kit for detecting beef allergen-specific IgE, characterized in that: The invention comprises the biotin-labeled beef allergen solution as claimed in any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that Also included are magnetic microparticles coated with streptavidin and anti-human IgE antibodies labeled with horseradish peroxidase.

7. A method for detecting beef allergen-specific IgE for non-diagnostic purposes, characterized in that: Detection based on the kit according to claim 5 or 6.

8. The method according to claim 7, characterized in that The following steps are involved: Step (B1): mixing the sample to be tested, the magnetic particles coated with streptavidin, and the biotin-labeled beef allergen solution, incubating, removing the supernatant, and washing; Step (B2): mixing with the anti-human IgE antibody labeled with horseradish peroxidase, incubating, removing the supernatant, and washing; Step (B3): adding substrate, shaking and mixing, and then measuring the chemiluminescence intensity; Step (B4): Calculate the concentration of beef allergen-specific IgE in the sample to be tested according to the standard curve.

9. The method according to claim 8, characterized in that include: The concentration of the streptavidin-coated magnetic particles is 0.1-3 mg / mL; and / or The concentration of beef allergen in the biotin-labeled beef allergen solution is 0.1-40.0 μg / mL; and / or The concentration of the antibody is 10-100 μg / mL; and / or The substrate includes luminol and / or peroxide.

10. The method according to claim 8 or 9, characterized in that include: The incubation temperature is 15-40°C; and / or The incubation time is 5 to 40 min.

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