Cockroach allergen specific igE antibody test kit

Abstract

The present application relates to the technical field of immune detection, and particularly relates to a cockroach allergen specific IgE antibody detection reagent. The detection reagent comprises a sensitizer composition solution, wherein the sensitizer composition comprises: sensitizers Bla g1, Bla g2, Bla g4, Bla g5, Bla g6, Bla g7, Bla g9 and Bla g11. Compared with other options (such as 2, 3, 5, etc. mixed therein), the detection reagent provided by the present application has the advantages of high detection efficiency, and has the same detection efficiency as natural extracts, better storage stability than natural extracts, smaller batch differences and better detection stability than natural extracts.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunodetection, in particular to a cockroach allergen specific IgE antibody detection reagent. BACKGROUND

[0002] Allergic reaction, also known as allergy or type I allergy, is a specific immune response of the body to the same antigen re-stimulation, causing tissue damage or physiological function disorder. The antigen substance causing allergic reaction is called allergen, also known as allergen or sensitizer. When the body inhales, ingests, contacts or injects a substance containing a sensitizing component, the body's B cells will produce specific IgE antibodies, and IgE will make the body in a sensitized state with mast cells or basophils. When the body is exposed to the allergen again, an allergic reaction will occur, and clinical symptoms will appear. Most patients with allergic diseases in clinical practice usually only receive symptomatic treatment, and the real cause of the allergy has not been found. Therefore, it is very important to provide an allergen detection technology or method to assist clinicians in diagnosing allergy or identifying allergens, so as to prevent and treat allergic conditions specifically.

[0003] Cockroaches are one of the causes of allergic reactions. The potential sources of related allergens in the environment include the cockroaches themselves, exuviae, secretions, eggshells and feces, and contact, inhalation and ingestion can all trigger allergic reactions, which can induce asthma, rhinitis, dermatitis and other allergic reactions. The common cockroach species in China include Periplaneta americana, Blattella germanica and Blattella asahinai, among which Blattella germanica is the most common. In 2009, a multi-center epidemiological survey of allergens in China covered 17 cities in North China, East China, South China and Southwest China, including 24 centers, a total of 6304 patients with asthma and / or rhinitis participated. The prevalence rate of Blattella germanica-induced allergy was 11.5%. The main diagnostic methods for allergic diseases are in vivo tests and in vitro tests. In vitro tests mainly include IgE detection based on serology. Specific IgE detection for Blattella germanica allergic patients can assess disease risk and provide medication guidance for the desensitization treatment process. According to the World Health Organization's Allergen Nomenclature Subcommittee, there are currently 11 Blattella germanica allergens, including Bla g 1-Bla g 9 and Bla g 11, Bla g 12. The contents and main locations of these 11 allergen proteins in Blattella germanica are different. Bla g 4 only exists in males, and the sensitization rates of different allergen proteins vary from 10% to 80%.

[0004] Currently, the specific IgE detection kits for Blattella germanica or cockroach allergens used in clinical practice all use natural extracts or biotinylated natural extracts. The natural extract of allergen has complex components and the content of the components is difficult to control, and it is difficult to control the quality and standardize the batch, which further leads to low accuracy and stability of the detection. SUMMARY

[0005] Therefore, the present application aims to provide a cockroach allergen-specific IgE antibody detection reagent with small batch-to-batch variation and good detection stability.

[0006] The present application provides a cockroach allergen-specific IgE antibody detection reagent, which comprises an allergen composition solution, wherein the allergen composition comprises: allergen Bla g 1, allergen Bla g2, allergen Bla g4, allergen Bla g5, allergen Bla g6, allergen Bla g7, allergen Bla g9 and allergen Bla g11.

[0007] The allergen composition solution of the present application comprises a diluent and:

[0008] biotin-labeled allergen Bla g 1, biotin-labeled allergen Bla g2,

[0009] biotin-labeled allergen Bla g4, biotin-labeled allergen Bla g5,

[0010] biotin-labeled allergen Bla g6, biotin-labeled allergen Bla g7, biotin-labeled allergen Bla g9 and biotin-labeled allergen Bla g11.

[0011] In the present application, the allergen composition is the allergen composition of Blattella germanica. That is, in the allergen composition of the present application, the allergen Bla g 1 is a recombinant Blattella germanica allergen Bla g 1, the allergen Bla g2 is a recombinant Blattella germanica allergen Bla g 2, the allergen Bla g4 is a recombinant Blattella germanica allergen Bla g 4, the allergen Bla g5 is a recombinant Blattella germanica allergen Bla g 5, the allergen Bla g6 is a recombinant Blattella germanica allergen Bla g 6, the allergen Bla g7 is a recombinant Blattella germanica allergen Bla g7, the allergen Bla g9 is a recombinant Blattella germanica allergen Bla g9, and the allergen Bla g11 is a recombinant Blattella germanica allergen Bla g11.

[0012] In the present application, the biotin is NHS-PEG 12 -Biotin.

[0013] The molar ratio of the allergen Bla g 1 to NHS-PEG 12 -Biotin is 1:(8-12); preferably, the molar ratio of the allergen Bla g1 to NHS-PEG 12 -Biotin is 1:10.

[0014] Allergen Bla g 2 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 2 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0015] Allergen Bla g 4 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 4 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0016] Allergen Bla g 5 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 5 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0017] Allergen Bla g 6 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 6 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0018] Allergen Bla g 7 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 7 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0019] Allergen Bla g 9 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 9 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0020] Allergen Bla g 11 with NHS-PEG 12 molar ratio 1 : (8-12) of Biotin; preferably, the allergen Bla g 11 with NHS-PEG 12 molar ratio 1 : 10 of Biotin.

[0021] In the present application, the mass ratio of the biotin-labeled allergen Bla g 1, the biotin-labeled allergen Bla g2, the biotin-labeled allergen Bla g4, the biotin-labeled allergen Bla g5, the biotin-labeled allergen Bla g6, the biotin-labeled allergen Bla g7, the biotin-labeled allergen Bla g9 and the biotin-labeled allergen Bla g11 is 1:(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.55-0.7):(0.8-1.2):(0.55-0.7):(0.55-0.7). Preferably, the mass ratio of the biotin-labeled allergen Bla g 1, the biotin-labeled allergen Bla g2, the biotin-labeled allergen Bla g4, the biotin-labeled allergen Bla g5, the biotin-labeled allergen Bla g6, the biotin-labeled allergen Bla g7, the biotin-labeled allergen Bla g9 and the biotin-labeled allergen Bla g11 is 1:1:1:1:0.625:1:0.625:0.625.

[0022] In the present application, the diluent of the allergen composition solution is 0.1M Tris-NaCl buffer solution containing 1wt%-3wt% bovine serum albumin, 2wt%-3wt% glycerol, 2wt%-3wt% sucrose, 0.1wt%-0.3wt% Proclin 300, 0.1wt%-0.3wt% Triton 100. The pH value of the 0.1M Tris-NaCl buffer solution is 7.4±0.05, and the concentration of NaCl is 0.15mol / L.

[0023] In the present application, the concentration of the allergen composition in the allergen composition solution is 0.2μg / mL-15μg / mL. As a preferred, the concentration of the allergen composition in the allergen composition solution is 5-6μg / mL. Preferably, the concentration of the allergen composition in the allergen composition solution is 5.5μg / mL.

[0024] In the present application, the allergen detection reagent of specific IgE antibody further comprises streptavidin-coated magnetic particle suspension, horseradish peroxidase-labeled anti-human IgE antibody and substrate solution.

[0025] In the present application, the volume ratio of the allergen composition solution, streptavidin-coated magnetic particle suspension, horseradish peroxidase-labeled anti-human IgE antibody in the allergen detection reagent of specific IgE antibody is 40:50:100.

[0026] The present application also provides a detection method of Blattella germanica specific IgE antibody, which comprises:

[0027] Step 1: mix the test substance, allergen composition solution and streptavidin-coated magnetic microparticle suspension, remove the supernatant after incubation;

[0028] Step 2: add horseradish peroxidase-labeled anti-human IgE antibody, remove the supernatant after incubation;

[0029] Step 3: add substrate solution, and determine the detection result according to the chemiluminescence intensity.

[0030] In the present application, the test substance includes a sample from an allergic patient or a sample from the environment.

[0031] In the present application, the method for determining the detection result according to the chemiluminescence intensity adopts a standard curve method.

[0032] The present application provides a reagent for detecting cockroach antibody, which comprises an allergen composition solution, wherein the allergen composition comprises: allergen Bla g1, allergen Bla g2, allergen Bla g4, allergen Bla g5, allergen Bla g6, allergen Bla g7, allergen Bla g9 and allergen Bla g11. Compared with other options (such as mixing 2, 3, 5, etc.), the reagent provided by the present application has the advantages of high detection efficiency, and has the same detection efficiency as natural extract, better storage stability than natural extract, smaller batch difference and better detection stability. DETAILED DESCRIPTION

[0033] The present application provides a cockroach allergen-specific IgE antibody detection reagent, and those skilled in the art can refer to the content herein to appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0034] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The recombinant allergen Bla g1, the recombinant allergen Bla g2, the recombinant allergen Bla g4, the recombinant allergen Bla g5, the recombinant allergen Bla g6, the recombinant allergen Bla g7, the recombinant allergen Bla g9 and the recombinant allergen Bla g11 can be purchased in the market, such as Bosung Bio, INDOOR Biotechnologies, etc.

[0035] The present application will be further described in conjunction with the following examples:

[0036] Example 1 An allergen-containing reagent for detecting Blattella germanica-specific IgE antibodies and use thereof

[0037] I. An allergen-containing reagent:

[0038] 0.8 μg / mL biotin-labeled recombinant Bla g 1 ;

[0039] 0.8 μg / mL biotin-labeled recombinant Bla g 2;

[0040] 0.8 μg / mL biotin-labeled recombinant Bla g 4;

[0041] 0.8 μg / mL biotin-labeled recombinant Bla g 5;

[0042] 0.5 μg / mL biotin-labeled recombinant Bla g 6;

[0043] 0.8 μg / mL L biotin-labeled recombinant Bla g 7;

[0044] 0.5 μg / mL biotin-labeled recombinant Bla g 9;

[0045] 0.5 μg / mL biotin-labeled recombinant Bla g 11.

[0046] The concentration of biotin-labeled recombinant allergens in the allergen-containing reagent is 5.5 μg / mL. The diluent is 2% bovine serum albumin, 2.5% glycerol, 2.5% sucrose, 0.2% Proclin 300, 0.2% Triton 100 in 0.1 M Tris-NaCl buffer (pH = 7.4 ± 0.05, 0.15 M NaCl).

[0047] II. Preparation method:

[0048] 1. Preparation of biotin-labeled recombinant sensitizing proteins:

[0049] The eight recombinant sensitizing proteins were labeled using the biotinylation reagent NHS-PEG12-Biotin from Thermo Scientific. The NHS-PEG12-Biotin reagent was prepared at 20 mM with dimethyl sulfoxide, and mixed with 2 mg / ml recombinant sensitizing proteins at the appropriate ratio. The mixture was shaken at room temperature for 2 hours. The reaction solution was dialyzed against 0.01 M PBS (pH = 7.2) for 3 times, 8 hours each time, to obtain the biotin-labeled recombinant sensitizing proteins.

[0050] The ratio of recombinant sensitizing proteins to biotinylation reagent was optimized, in terms of molar ratio,

[0051] The labeling ratio of recombinant Bla g 1 to the biotinylated reagent is 1:10;

[0052] The labeling ratio of recombinant Bla g 2 to the biotinylated reagent is 1:10;

[0053] The labeling ratio of recombinant Bla g 4 to the biotinylated reagent is 1:10;

[0054] The labeling ratio of recombinant Bla g 5 to the biotinylated reagent is 1:10;

[0055] The labeling ratio of recombinant Bla g 6 to the biotinylated reagent is 1:20;

[0056] The labeling ratio of recombinant Bla g 7 to the biotinylated reagent is 1:10;

[0057] The labeling ratio of recombinant Bla g 9 to the biotinylated reagent is 1:10;

[0058] The labeling ratio of recombinant Bla g 11 to the biotinylated reagent is 1:10.

[0059] 2. Preparation of a diluent containing an allergen reagent:

[0060] (1) Take Tris 12.11 g, NaCl 8.77 g in a flask, then add 600 mL purified water in the flask, fully stir to completely dissolve the real reagent;

[0061] (2) Adjust the pH to control the pH in the range of 7.35-7.45;

[0062] (3) Take 20 g of bovine serum albumin, 2 mL of Proclin 300, 2 mL of Triton 100, 25 mL of glycerol and 25 g of sucrose and add them into the above beaker;

[0063] (4) Finally, make up to 1000 mL, filter with a 0.2 μm filter to obtain.

[0064] 3. Preparation of an allergen reagent:

[0065] The biotin-labeled recombinant Bla g 1, biotin-labeled recombinant Bla g 2, biotin-labeled recombinant Bla g 4, biotin-labeled recombinant Bla g 5, biotin-labeled recombinant Bla g 6, biotin-labeled recombinant Bla g 7, biotin-labeled recombinant Bla g 9, and biotin-labeled recombinant Bla g 11 are prepared into 5.5 μg / mL by using the above diluent according to the mass ratio of 1:1:1:1:0.625:1:0.625:0.625, to obtain an allergen reagent.

[0066] III. Application of the allergen-containing reagent

[0067] The allergen-containing reagent is used for detecting Blattella germanica specific IgE antibody, and the application method is as follows:

[0068] (1) 40 μl of the allergen-containing reagent is mixed with 50 μl of streptavidin-coated magnetic microparticle suspension and 50 μl of sample of allergic patient, incubated at 37°C for 15 min, and the supernatant is removed by magnetic separation;

[0069] (2) 100 μl of horseradish peroxidase-labeled anti-human IgE antibody is added, mixed, incubated at 37°C for 15 min, and the supernatant is removed by magnetic separation;

[0070] (3) Substrate solution is added, and the chemiluminescence intensity is determined;

[0071] (4) The concentration of IgE in the sample to be detected can be obtained according to the calibration curve and algorithm.

[0072] Test Example 1 Comparison of labeling ratios of 8 kinds of recombinant sensitizing proteins labeled with biotin

[0073] The experimental method is described in Example 1, and the concentration values of the listed samples are the results determined by Phadia Immuno CAP Blattella germanica allergen-specific IgE detection reagent. The experimental results are shown in Table 1 below:

[0074] Table 1. Comparison of labeling ratios of 8 kinds of recombinant sensitizing proteins labeled with biotin

[0075]

[0076]

[0077]

[0078]

[0079] The results show that the reactivity differs when the labeling ratio of recombinant sensitizing proteins and biotinylated reagent is different, and the best detection effect is obtained when the ratio of recombinant Bla g 1, recombinant Bla g 2, recombinant Bla g 4, recombinant Bla g 5, recombinant Bla g 7, recombinant Bla g 9 and recombinant Bla g 11 is 1:10, and the best detection effect is obtained when the ratio of recombinant Bla g 6 is 1:20.

[0080] Test Example 2 Comparison of storage stability of the allergen-containing reagent of Example 1 and biotinylated natural extract

[0081] The experimental method is described in Example 1. The experimental results are shown in Table 2 below:

[0082] Table 2. Comparison of storage stability of anto-allergen reagent and biotinylated natural extract

[0083]

[0084]

[0085] The results show that the anto-allergen reagent has better storage stability than the conventional biotinylated natural extract.

[0086] Test Example 3 Comparison of detection efficiency of Example 1 anto-allergen reagent and biotinylated natural extract

[0087] The experimental method is as described in Example 1. The experimental results and coincidence rate statistics are shown in Tables 3.1 and 3.2 below:

[0088] Table 3.1. Comparison of detection efficiency of anto-allergen reagent and biotinylated natural extract

[0089]

[0090]

[0091] Table 3.2. Coincidence rate statistics

[0092]

[0093] The results show that the anto-allergen reagent has comparable detection efficiency to the conventional biotinylated natural extract.

[0094] Test Example 4 Comparison of Example 1 anto-allergen reagent and biotinylated natural extract between batches

[0095] The experimental method is as described in Example 1. The experimental results are shown in Table 4 below:

[0096] Table 4. Comparison of Example 1 anto-allergen reagent and biotinylated natural extract between batches

[0097]

[0098] The results show that the anto-allergen reagent has less difference between batches and better detection stability than the conventional biotinylated natural extract.

[0099] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A reagent for detecting cockroach allergen-specific IgE antibodies, characterized in that, The solution includes an allergen composition solution, said allergen composition solution comprising a diluent and: Biotin-labeled allergens Bla g 1, Bla g 2, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 9, and Bla g 11; wherein the mass ratio of biotin-labeled allergens Bla g 1, Bla g 2, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 9, and Bla g 11 is 0.8:0.8:0.8:0.8:0.5:0.8:0.5:0.5; The biotin is NHS-PEG. 12 -Biotin, Allergen Bla g 1 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g 2 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g 4 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g5 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g 6 and NHS-PEG 12 - The molar ratio of biotin is 1:20; the allergen Bla g 7 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g 9 and NHS-PEG 12 -The molar ratio of biotin is 1:10; the allergen Bla g 11 and NHS-PEG 12 -The molar ratio of biotin is 1:10; The diluent is a 0.1 M Tris-NaCl buffer containing 2 wt% bovine serum albumin, 2.5 wt% glycerol, 2.5 wt% sucrose, 0.2 wt% Proclin 300, and 0.2 wt% Triton 100.

2. The detection reagent according to claim 1, characterized in that, The allergen composition is an allergen composition of the German cockroach.

3. The detection reagent according to claim 1 or 2, characterized in that, The concentration of the allergen composition in the solution is 0.2 μg / mL to 15 μg / mL.

4. The detection reagent according to claim 1 or 2, characterized in that, It also includes streptavidin-coated magnetic microparticle suspensions, horseradish peroxidase-labeled anti-human IgE antibodies, and substrate solutions.

5. A non-diagnostic method for detecting German cockroach-specific IgE antibodies, characterized in that, include: Step 1: Mix the analyte, the allergen composition solution, and the streptavidin-coated magnetic microparticle suspension, incubate, and then remove the supernatant; Step 2: Add horseradish peroxidase-labeled anti-human IgE antibody, incubate, and then remove the supernatant; Step 3: Add the substrate solution and judge the detection result based on the chemiluminescence intensity.

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