Preparation of natural alternaria antigen and allergen-specific ige antibody detection kit thereof

Abstract

This invention relates to a reagent for detecting Alternaria alternata allergen-specific IgE antibodies and a magnetic particle chemiluminescence detection kit. The invention involves activating an Alternaria alternata strain (accession number ATCC 66981) and culturing it on a solid culture medium. Freshly cultured Alternaria alternata spores and mycelia are collected, and Alternaria alternata protein is extracted and biotin-labeled to obtain biotin-labeled Alternaria alternata protein. This biotin-labeled protein is then added to a preservation solution to obtain the Alternaria alternata allergen-specific IgE antibody detection reagent. This invention uses a specific strain and a screened and optimized culture medium formula to extract Alternaria alternata protein, resulting in high protein yield and good reactivity. When used for magnetic particle chemiluminescence detection of Alternaria alternata allergen-specific IgE antibodies, it exhibits advantages such as high sensitivity and controllable batch-to-batch variation, which is beneficial for the widespread application of magnetic particle chemiluminescence methods for Alternaria alternata allergen-specific IgE antibodies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnostic reagents, and particularly relates to a natural Alternaria alternata antigen preparation and an allergen-specific IgE antibody detection kit. BACKGROUND

[0002] There are more than 100,000 species of fungi in nature, among which those that can be dispersed in the air can cause allergic diseases including allergic rhinitis and asthma. Alternaria alternata is a common allergen widely existing indoors and outdoors. Related investigation and research shows that 14% of children with asthma are allergic to fungi, and 46% of children allergic to fungi are allergic to Alternaria alternata; another large-sample clinical epidemiology study found that 19% of patients with respiratory diseases are allergic to at least one kind of mold, and 60% of these patients allergic to mold are patients allergic to Alternaria alternata; it can be seen that Alternaria alternata is closely related to respiratory diseases. However, the research and application of Alternaria alternata are not as extensive and in-depth as those of dust mites and pollen.

[0003] In existing research, there are few literatures about natural Alternaria alternata antigen proteins, which are mainly focused on the preparation and application of recombinant proteins. Although the preparation method of recombinant proteins has the advantages of low allergenicity and high purity, some allergenic proteins of Alternaria alternata cannot be completely determined at present, and all components of Alternaria alternata cannot be obtained. The application of recombinant Alternaria alternata proteins in in vitro allergen diagnosis usually lacks components, which can lead to low sensitivity of detection reagents.

[0004] Most of the existing Alternaria alternata allergy diagnostic reagents are based on immunoblotting, enzyme-linked immunoassay, and fluorescence immunoassay. The magnetic microparticle chemiluminescence detection technology is increasingly developed and improved, has higher detection sensitivity, and can realize full automation, full quantification, short time consumption, etc. The preparation of high-quality natural Alternaria alternata antigen proteins and their application in the diagnosis of the magnetic microparticle chemiluminescence platform will help to improve the accuracy of in vitro Alternaria alternata allergen diagnosis. SUMMARY

[0005] The technical problem solved by the present application is to overcome the shortcomings of the prior art and provide an Alternaria alternata allergen-specific IgE antibody detection reagent containing natural Alternaria alternata antigen proteins and a magnetic microparticle chemiluminescence detection kit.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0007] The application provides a preparation method of Alternaria alternata allergen specific IgE antibody detection reagent, which comprises the following steps: culturing an activated Alternaria alternata strain with a preservation number of ATCC 66981 on a solid culture medium, taking fresh Alternaria alternata spores and mycelia cultured, extracting Alternaria alternata protein and obtaining biotin-labeled Alternaria alternata protein through biotin labeling, and adding a preservative solution to obtain the Alternaria alternata allergen specific IgE antibody detection reagent.

[0008] The formula of the solid culture medium is as follows: 15-30 g of glucose, 2-5 g of yeast extract, 0.8-1.5 g of potassium phosphate dibasic, 0.3-0.8 g of magnesium sulfate heptahydrate, 0.005-0.015 g of ferrous sulfate heptahydrate, 0.3-0.8 g of potassium chloride and 15-25 g of agar, and water is added to 1 L.

[0009] Further, the formula of the solid culture medium is as follows: 18-22 g of glucose, 2-4 g of yeast extract, 0.8-1.2 g of potassium phosphate dibasic, 0.4-0.6 g of magnesium sulfate heptahydrate, 0.008-0.012 g of ferrous sulfate heptahydrate, 0.4-0.6 g of potassium chloride and 18-22 g of agar, and water is added to 1 L.

[0010] In the embodiment of the application, the culture condition is as follows: the temperature is 25-30 DEG C, and the culture period is 6-12 days.

[0011] In the embodiment of the application, the preservative solution is a phosphate buffer solution containing 1-2 wt% BSA, 1-3 wt% glycerol and 2-5 wt% mannitol, and the pH value of the preservative solution is 7.2-7.5.

[0012] According to some specific embodiments of the application, the Alternaria alternata spores and mycelia are frozen by liquid nitrogen, and then are immersed in an extraction solution for extraction, after the extraction, the supernatant is obtained by centrifugation, the supernatant is mixed with a Sulfo-NHS-biotin solution to react at room temperature, and then the Sulfo-NHS-biotin labeled Alternaria alternata protein is obtained by dialysis using a 0.05-0.2 M CB buffer solution with a pH value of 9.0-9.5, and the Sulfo-NHS-biotin labeled Alternaria alternata protein is added into the preservative solution to obtain the Alternaria alternata allergen specific IgE antibody detection reagent.

[0013] In the embodiment of the application, the molar ratio of the Alternaria alternata protein in the supernatant to the Sulfo-NHS-biotin in the Sulfo-NHS-biotin solution is 1:15-25, for example, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24 or 1:25.

[0014] Further preferably, the molar ratio of the Alternaria alternata allergen protein in the supernatant and the Sulfo-NHS-biotin in the Sulfo-NHS-biotin solution is 1:18-22.

[0015] In the embodiment of the present application, the formula of the extraction solution is: sodium chloride 4-8 g, sodium bicarbonate 1.5-3.5 g, phenol 3-5 g, and distilled water to 1 L, wherein preferably distilled water is used.

[0016] According to the specific embodiment of the present application, the formula of the extraction solution is: sodium chloride 5 g, sodium bicarbonate 2.75 g, phenol 4 g, and distilled water to 1 L.

[0017] In the embodiment of the present application, the concentration of the biotin-labeled Alternaria alternata allergen protein in the Alternaria alternata allergen-specific IgE antibody detection reagent is 5-30 μg / mL, for example, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL.

[0018] The present application also provides a magnetic microparticle chemiluminescence detection kit comprising the Alternaria alternata allergen-specific IgE antibody detection reagent prepared by the preparation method or the Alternaria alternata allergen-specific IgE antibody detection reagent.

[0019] In the embodiment of the present application, the magnetic microparticle chemiluminescence detection kit further comprises a streptavidin-coated magnetic microparticle suspension, an alkaline phosphatase-labeled mouse anti-human IgE antibody solution, a biotin-labeled anti-human IgE antibody solution, and a luminescent substrate solution.

[0020] In the embodiment of the present application, the concentration of the streptavidin-coated magnetic microparticle suspension is 0.4-1 mg / mL, for example, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL.

[0021] In the embodiment of the present application, the solvent of the streptavidin-coated magnetic microparticle suspension is a phosphate buffer solution with a pH value of 7.2-7.5 containing 1-2 wt% BSA, 1-3 wt% glycerol, and 2-5 wt% mannitol.

[0022] In the embodiments of the present application, the concentration of the alkaline phosphatase-labeled mouse anti-human IgE antibody and the biotin-labeled anti-human IgE antibody is independently 0.1-2.0 μg / mL.

[0023] In the embodiments of the present application, the anti-human IgE antibody is mixed with Sulfo-NHS-LC-biotin, and the alkaline phosphatase-labeled mouse anti-human IgE antibody is obtained by dialysis in a phosphate buffer containing 1-2 wt% BSA at pH 7.2-7.5.

[0024] In the embodiments of the present application, the luminescent substrate solution is AMPPD solution.

[0025] In the embodiments of the present application, the magnetic microparticle chemiluminescence detection kit further comprises a calibrator and a quality control.

[0026] According to some embodiments, the concentration of the calibrator is 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, 100 IU / mL.

[0027] According to some embodiments, the concentration of the quality control is 0.7 IU / ml, 17.5 IU / ml.

[0028] The present application also provides the use of the above-mentioned Alternaria alternata allergen-specific IgE antibody detection reagent or magnetic microparticle chemiluminescence detection kit in the quantitative detection of Alternaria alternata allergen-specific IgE.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The present application uses a specific strain to culture and extract Alternaria alternata protein in combination with a screened and optimized culture medium formula, and the obtained reagent is used for the magnetic microparticle chemiluminescence detection of Alternaria alternata allergen-specific IgE antibody, and has the advantages of high sensitivity and controllable batch difference. The production process of the Alternaria alternata allergen-specific IgE antibody detection reagent of the present application is simple, the yield of Alternaria alternata protein is high, the production cost is low, and the reaction activity is good, which is conducive to the popularization and application of the magnetic microparticle chemiluminescence method for Alternaria alternata allergen-specific IgE antibody. DETAILED DESCRIPTION

[0031] The present application will be further described below in combination with examples. However, the present application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different specific requirements, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features involved in each embodiment of the present application can be combined with each other as long as there is no conflict between them.

[0032] In the following examples, the implementation conditions not specified are standard conditions in the industry, and the reagents not specified are all commercially available products.

[0033] The detection instrument used in the following examples is the SMART 6500HOB fully automated chemiluminescence analyzer from Jiangsu Haooubo Biomedical Co., Ltd. (HOB).

[0034] Example 1

[0035] Preparation of a reagent for detecting Alternaria allergen-specific IgE antibodies.

[0036] 1.1 Preparation of solid culture medium: Weigh each component according to the formula of each culture medium in Table 1, add water to make up to 1L, dispense into petri dishes, autoclave and set aside for later use.

[0037] 1.2 Under aseptic conditions, open the ampoule containing the Alternaria alternata strain (from the American Type Culture Collection (ATCC): Alternaria alternata (Fries) Keissler (ATCC 66981)), and use a pipette to add 1 mL of sterile water to the ampoule to form a suspension. Let it stand overnight at room temperature (25°C) to ensure the activity of the fungus.

[0038] 1.3 The solid culture medium prepared above was used for fermentation culture. After sterilizing and cooling the inoculation loop over an alcohol lamp, the loop was dipped into the suspension in the ampoule and streaked on the solid culture medium. The inoculation loop should not be allowed to break the surface of the plate. Then the culture dish was placed upside down in an incubator at 25°C for 10 days. Take 0.5g of freshly cultured Alternaria spores and mycelium, freeze them in liquid nitrogen, and then transfer them to a pre-cooled (liquid nitrogen) Teflon container. Add pre-cooled steel balls, place the container in a separator, shake for 1 minute, transfer the powder to a 1.5mL centrifuge tube, add 1mL of extraction solution (formulation: 5g sodium chloride, 2.75g sodium bicarbonate, 4g phenol, and distilled water to 1L), invert the centrifuge tube and carefully mix the suspension (do not vortex), centrifuge for 5 minutes, and take the supernatant. The supernatant contains total Alternaria spore protein (the supernatant corresponding to medium 1 is labeled as antigen protein solution 1, the supernatant corresponding to medium 2 is labeled as antigen protein solution 2, and so on up to antigen protein solution 11), and store at -20℃.

[0039] Table 1

[0040]

[0041] The protein concentrations in the different antigen protein solutions were determined using the BCA method: The BCA assay kit was provided by Thermo Fisher Scientific. The BSA standard protein was diluted to 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, and 0.05 mg / mL according to the instructions. The BCA working solution was prepared by mixing reagent A and reagent B in a volume ratio of 50:1. Reagent A consisted of 1% BCA disodium salt, 0.4% sodium hydroxide, 0.16% sodium tartrate, 2% anhydrous sodium carbonate, and 0.95% sodium bicarbonate. Reagent B consisted of 4% copper sulfate. BCA kit sample loading: Dilute the sample with PBS according to the sample concentration. Add 20 μL of each sample and standard to a 96-well plate. Perform 3 replicates for each sample. Add 300 μL of working solution, mix for 10 s, and react at 37°C for half an hour. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration according to the standard curve and take the average value. The results are shown in Table 2.

[0042] Table 2 (Unit: mg / mL)

[0043]

[0044] 1.4 The above antigen protein solution was added to a 10 mg / mL Sulfo-NHS-biotin solution (solvent: DMF) at a molar ratio of 1:15 to Alternaria alternata protein. The reaction was carried out at room temperature for 1 h. After the reaction, the mixture was dialyzed against 0.1 M CB buffer at pH 9.5. The obtained Sulfo-NHS-biotin-labeled Alternaria alternata antigen protein was stored in an appropriate amount of phosphate buffer (pH = 7.4) containing 2 wt% BSA, 3 wt% glycerol and 5 wt% mannitol to prepare a 10 μg / mL biotin-labeled Alternaria alternata allergen solution, which is the Alternaria alternata allergen-specific IgE antibody detection reagent. (The antigen protein solution 1 is labeled as detection reagent 1, the antigen protein solution 2 is labeled as detection reagent 2, and so on up to detection reagent 11).

[0045] Example 2

[0046] A magnetic microparticle chemiluminescence detection kit was prepared for the detection of Alternaria allergen-specific IgE antibodies.

[0047] The magnetic microparticle chemiluminescence detection kit includes streptavidin-coated magnetic microparticle suspension, biotin-labeled Alternaria alternata allergen solution, alkaline phosphatase-labeled anti-human IgE antibody, biotin-labeled anti-human IgE antibody, calibrators, quality control materials, and substrates based on AMPPD luminescence. The specific preparation method is as follows:

[0048] Streptavidin-coated magnetic microparticle suspension: 10 mg / mL streptavidin magnetic beads (1 μm in diameter) were mixed and magnetically separated. The mixture was then resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol and 5 wt% mannitol. After washing three times and magnetic separation, the mixture was finally resuspended in phosphate buffer (pH 7.4) containing 2 wt% BSA, 3 wt% glycerol and 5 wt% mannitol to obtain a streptavidin-coated magnetic microparticle suspension with a concentration of 0.4 mg / mL.

[0049] Biotin-labeled Alternaria allergen solution: Alternaria allergen-specific IgE antibody detection reagent prepared in Example 1.

[0050] Alkaline phosphatase-labeled anti-human IgE antibody solution: The alkaline phosphatase (AP) to be activated was dialyzed, then a coupling agent was added for reaction, the free coupling agent was removed, and then the activated alkaline phosphatase was added to the activated anti-human IgE antibody solution for coupling reaction. The free AP was then removed. The concentration of alkaline phosphatase-labeled anti-human IgE antibody in the alkaline phosphatase-labeled anti-human IgE antibody solution was 1.5 μg / mL.

[0051] Biotin-labeled anti-human IgE antibody solution: Anti-human IgE antibody was added to dimethylformamide containing Sulfo-NHS-LC-biotin, and then dialyzed with phosphate buffer containing 2 wt% BSA at pH 7.4. The concentration of biotin-labeled anti-human IgE antibody in the biotin-labeled anti-human IgE antibody solution was 1.5 μg / mL.

[0052] Calibrators: IgE protein derived from WHO standards was diluted with 0.1M Tris-HCl buffer at pH 7.4 to prepare calibrators of 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, and 100 IU / mL.

[0053] Quality control samples: IgE protein derived from WHO standards was prepared into 0.7 IU / mL and 17.5 IU / mL quality control samples using 0.1M Tris-HCl buffer at pH 7.4.

[0054] The luminescent substrate solution is an alkaline phosphatase chemiluminescent substrate based on AMPPD luminescence, which is produced in-house. For details, please refer to the invention patent "An Enzymatic Chemiluminescent Substrate for Alkaline Phosphatase", authorized announcement number CN 104990912 B.

[0055] The above-mentioned kits were used to quantitatively detect the Alternaria-specific IgE content in clinical samples, including 10 positive samples (P) and 10 negative samples (N). The results were compared with those from Phadia's Immuno CAP assay, and the results are shown in Table 3 (Phadia unit: kUA / L, HOB unit: IU / mL, 1 kUA / L = 1 IU / mL). The detection method for the magnetic microparticle chemiluminescence detection kit was as follows: The calibrator, streptavidin-coated magnetic microparticle suspension, and biotin-labeled anti-human IgE antibody were mixed and incubated at 37°C for 15 min. The supernatant was removed by magnetic separation, and the mixture was washed three times. Alkaline phosphatase-labeled anti-human IgE antibody was added, mixed, and incubated at 37°C for 15 min. The supernatant was removed by magnetic separation, and the mixture was washed three times. The luminescent substrate solution was added, and the mixture was incubated at 37°C for 5 min. The chemiluminescence intensity was measured, and a graph was plotted with the calibrator concentration on the x-axis and the chemiluminescence intensity on the y-axis. The standard curve was calculated by mixing the test sample with streptavidin-coated magnetic microparticle suspension and biotin-labeled Alternaria alternata allergen solution, incubating at 37°C for 15 min, magnetically separating the supernatant, and washing three times. Alkaline phosphatase-labeled anti-human IgE antibody was added, mixed, and incubated at 37°C for 15 min. The supernatant was then magnetically separated and washed three times. A luminescent substrate solution was added, and the mixture was incubated at 37°C for 5 min. The chemiluminescence intensity was measured and substituted into the standard curve to calculate the concentration of Alternaria alternata allergen-specific IgE in the test sample. The cutoff value was 0.35 IU / mL, and a value greater than or equal to 0.35 IU / mL was considered positive.

[0056] Table 3

[0057]

[0058]

[0059] The positive and negative concordance rates between the reagent test results and Phadia are shown in Table 4:

[0060] Table 4

[0061]

[0062]

[0063] Tables 3 and 4 show that the detection reagents for Alternaria alterniflora protein extracted from different culture media have different effects. It can be seen that different culture media will affect the antigenic sites and components of the antigenic protein. Among them, detection reagent 1 has a lower background and the highest positive and negative concordance rate.

[0064] Example 3

[0065] Optimize the detection reagent for Alternaria allergen-specific IgE antibody.

[0066] The crude allergen extract 1 was added to a 10 mg / mL Sulfo-NHS-biotin solution (solvent: DMF) at molar ratios of Alternaria alternata protein to Sulfo-NHS-biotin of 1:15, 1:20, and 1:25, respectively. The reaction was carried out at room temperature for 1 hour. After the reaction, the mixture was dialyzed against 0.1 M CB buffer at pH 9.5. The obtained Sulfo-NHS-biotin-labeled Alternaria alternata antigen protein was stored in an appropriate amount of phosphate buffer (pH = 7.4) containing 2 wt% BSA, 3 wt% glycerol, and 5 wt% mannitol to prepare a 10 μg / mL biotin-labeled Alternaria alternata allergen solution, which is the Alternaria alternata allergen-specific IgE antibody detection reagent (the 1:15 molar ratio is labeled as detection reagent A, the 1:20 molar ratio as detection reagent B, and the 1:25 molar ratio as detection reagent C).

[0067] According to Example 2, the above-mentioned detection reagents A, B, and C were respectively combined with streptavidin-coated magnetic microparticle suspension, alkaline phosphatase-labeled anti-human IgE antibody, biotin-labeled anti-human IgE antibody, calibrators, quality control materials, and AMPPD-based luminescence substrates to form a magnetic microparticle chemiluminescence detection kit. The corresponding detection reagent A was labeled as Magnetic Microparticle Chemiluminescence Detection Kit A, the corresponding detection reagent B was labeled as Magnetic Microparticle Chemiluminescence Detection Kit B, and the corresponding detection reagent C was labeled as Magnetic Microparticle Chemiluminescence Detection Kit C.

[0068] The levels of Alternaria-specific IgE in clinical samples were quantitatively detected using the above-mentioned magnetic particle chemiluminescence detection kits A, B, and C, including 20 positive samples (P) and 10 negative samples (N). The results were compared with those obtained using Phadia's Immuno CAP assay, and the results are shown in Table 5. (Phadia unit: kUA / L, HOB unit: IU / mL, 1 kUA / L = 1 IU / mL).

[0069] Table 5

[0070]

[0071]

[0072] The positive and negative concordance rates between the magnetic particle chemiluminescence detection kit A, magnetic particle chemiluminescence detection kit B, and magnetic particle chemiluminescence detection kit C and Phadia are shown in Table 6.

[0073] Table 6

[0074]

[0075] Table 6 shows that by adjusting the ratio of conjugated biotinylation, the background value of the reagent can be reduced and the positive detection rate can be improved. The overall concordance rate is highest when the molar ratio of Alternaria alternata protein to Sulfo-NHS-biotin in crude allergen extract 1 is 1:20.

[0076] Example 4

[0077] The accuracy, limit of detection, linearity, repeatability, specificity, precision, and inter-batch difference of the chemiluminescence detection kit B for magnetic microparticles were tested, as detailed below:

[0078] Accuracy testing:

[0079] A high-concentration sample A (accuracy reference H) of known concentration is added to a low-concentration sample B (accuracy reference L) at a volume ratio not exceeding 1:19 to obtain the test sample. The concentration of the test sample should be within the reagent detection range. The recovery rate is calculated according to the formula:

[0080]

[0081] In the formula:

[0082] R – Recovery rate;

[0083] V—Volume of the accuracy reference H;

[0084] V0 — Volume of the accuracy reference material L;

[0085] C—Detection concentration of the mixed sample;

[0086] C0—Concentration of accuracy reference material L;

[0087] C S —The concentration of the accuracy reference material H.

[0088] The accuracy analysis data is shown in Table 7:

[0089] Table 7 (Unit: IU / mL)

[0090]

[0091] The results showed that the recovery rate was in the range of 85% to 115%, which met the requirements.

[0092] Limit of detection:

[0093] Five samples with concentrations close to the detection limit (detection limit references 1-5) were tested, with each sample tested five times. The test results were sorted by magnitude. The blank limit and detection limit can be considered reasonably set if the following conditions are met:

[0094] a) The number of test results below the blank limit should be less than or equal to 3;

[0095] b) No test results were found that were higher than the lower limit of the reference range.

[0096] Using the calibration point CAL1 (zero calibration point) as the sample, conduct at least 20 tests. Calculate the luminescence value Mean+2SD, and back-calculate the concentration using linear regression of the luminescence values ​​at the CAL1 and CAL2 calibration points from at least 5 results to obtain the blank limit.

[0097] The results of the detection limit analysis are shown in Tables 8 and 9:

[0098] Table 8 (Unit: IU / mL)

[0099]

[0100] Table 9 (Unit: IU / mL)

[0101]

[0102]

[0103] The detection limits showed that the detection limits were set reasonably, all not exceeding 0.1 IU / mL, which met the requirements.

[0104] Linear detection:

[0105] Dilute high-value samples (linear reference standards) that are close to the upper limit of the linear interval with low-value samples that are close to the lower limit of the linear interval. Dilute proportionally to at least 5 concentrations, where the low-value concentration sample must be close to 0.1 IU / mL. Repeat the test 3 times for each concentration, calculate the average value of each concentration point, and fit the average value of the measured concentrations to the theoretical concentration using the least squares method to form a linear relationship, and calculate the linear correlation coefficient r.

[0106] The experimental results are shown in Table 10:

[0107] Table 10 (Unit: IU / mL)

[0108]

[0109] The results show that the linear correlation coefficient r is greater than 0.9900, which meets the requirements.

[0110] Repeatability testing:

[0111] The samples were tested 10 times each at two concentration levels: 0.35 IU / mL to 0.7 IU / mL (repeatability reference 1) and 3.5 IU / mL to 17.5 IU / mL (repeatability reference 2). The mean M and standard deviation SD of the test results were calculated using the following formula to obtain the coefficient of variation CV:

[0112] CV = SD / M × 100%

[0113] In the formula:

[0114] CV — Coefficient of Variation;

[0115] M — the average value of 10 concentration measurements;

[0116] SD — the standard deviation of 10 concentration measurements.

[0117] The experimental results are shown in Table 11:

[0118] Table 11 (Unit: IU / mL)

[0119]

[0120] The results showed that the intra-batch CV of repeatability results was no higher than 10%, which met the requirements.

[0121] Specific detection:

[0122] Samples containing total IgE at a concentration of not less than 60 IU / mL and without specific IgE antibodies, and samples with IgA concentrations of not less than 700 μg / mL, IgM concentrations of 500 μg / mL, and IgG concentrations of 7000 μg / mL were tested. Each sample was tested once, and the results are shown in Table 12.

[0123] Table 12 (Unit: IU / mL)

[0124] Sample Concentration Sample with total IgE at 60 IU / mL and without specific IgE antibodies 0.04 700 μg / mL IgA 0.01 500 μg / mL IgM 0.01 7000 μg / mL IgG 0.02

[0125] The results showed that all measurements were no higher than 0.1 IU / mL, which met the requirements.

[0126] Inter-batch difference detection:

[0127] Three batches of magnetic microparticle chemiluminescence detection kits were prepared using three batches of crude allergen extract 1 according to magnetic microparticle chemiluminescence detection kit B. These three batches of magnetic microparticle chemiluminescence detection kits were used to detect the clinical samples in Example 3, and the batch-to-batch differences were calculated.

[0128] The experimental results are shown in Table 13:

[0129] Table 13 (Unit: IU / mL)

[0130] Sample No. Batch 1 Batch 2 Batch 3 M SD CV N1 0.15 0.17 0.17 0.16 0.01 8.9％ N2 0.24 0.21 0.25 0.23 0.02 8.9％ N3 0.27 0.32 0.29 0.29 0.03 8.5％ N4 0.17 0.14 0.16 0.16 0.02 9.7％ N5 0.24 0.21 0.22 0.22 0.02 7.4％ N6 <0.1 <0.1 <0.1 / / / N7 <0.1 <0.1 <0.1 / / / N8 0.11 <0.1 0.13 0.12 0.01 9.7％ N9 0.28 0.32 0.33 0.31 0.02 8.1％ N10 0.16 0.17 0.18 0.17 0.01 5.1％ P1 0.38 0.37 0.40 0.38 0.01 3.5％ P2 0.40 0.39 0.38 0.39 0.01 3.0％ P3 1.28 1.24 1.21 1.24 0.04 3.1％ P4 2.36 2.43 2.35 2.38 0.04 1.8％ P5 14.49 14.80 15.71 15.00 0.64 4.2％ P6 18.21 17.15 16.99 17.45 0.66 3.8％ P7 28.80 28.78 29.67 29.08 0.51 1.7％ P8 43.74 47.50 45.30 45.52 1.89 4.2％ P9 51.98 51.98 47.03 50.33 2.86 5.7％ P10 86.86 89.62 83.58 86.69 3.02 3.5％ P11 0.35 0.36 0.37 0.36 0.01 3.5％ P12 0.40 0.40 0.39 0.40 0.01 1.5％ P13 0.36 0.37 0.38 0.37 0.01 3.0％ P14 0.71 0.73 0.73 0.72 0.01 1.7％ P15 6.81 7.01 6.61 6.81 0.20 2.9％ P16 10.57 10.60 10.07 10.42 0.30 2.8％ P17 43.00 46.62 43.93 44.52 1.88 4.2％ P18 22.48 22.71 23.15 22.78 0.34 1.5％ P19 48.70 49.65 44.96 47.77 2.48 5.2％ P20 72.89 68.38 66.64 69.30 3.23 4.7％

[0131] In Table 13, P1 to P20 are positive samples, and N1 to N10 are negative samples.

[0132] The results showed that the batch-to-batch variation (CV) of the three batches of reagent kits was no greater than 10%, indicating that the batch-to-batch differences of the reagent kits were small and the performance was stable.

[0133] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing a reagent for detecting Alternaria allergen-specific IgE antibodies, characterized in that, Alternaria alternata strain with accession number ATCC 66981 was activated and cultured on solid medium. Freshly cultured Alternaria alternata spores and mycelia were collected, frozen in liquid nitrogen, and then extracted with extraction buffer. After extraction, the supernatant was collected by centrifugation. The supernatant was mixed with Sulfo-NHS-biotin solution and reacted at room temperature. After the reaction, the mixture was dialyzed with 0.05-0.2M CB buffer at pH 9.0-9.5 to obtain Sulfo-NHS-biotin-labeled Alternaria alternata protein. The Sulfo-NHS-biotin-labeled Alternaria alternata protein was added to the preservation solution to obtain the Alternaria alternata allergen-specific IgE antibody detection reagent. The solid culture medium comprises: 18-22g glucose, 2-4g yeast extract, 0.8-1.2g dipotassium hydrogen phosphate, 0.4-0.6g magnesium sulfate heptahydrate, 0.008-0.012g ferrous sulfate heptahydrate, 0.4-0.6g potassium chloride, 18-22g agar, and water to a final volume of 1L. The molar ratio of Alternaria alternata protein in the supernatant to Sulfo-NHS-biotin in the Sulfo-NHS-biotin solution is 1:18~22.

2. The preparation method of the Alternaria allergen-specific IgE antibody detection reagent according to claim 1, characterized in that, The cultivation conditions are: temperature 25~30℃, cultivation period 6~12 days.

3. The method for preparing the Alternaria allergen-specific IgE antibody detection reagent according to claim 1, characterized in that, The preservation solution is a phosphate buffer containing 1-2 wt% BSA, 1-3 wt% glycerol and 2-5 wt% mannitol, and the pH value of the preservation solution is 7.2-7.

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4. The method for preparing the Alternaria allergen-specific IgE antibody detection reagent according to claim 1, characterized in that, The extract is formulated as follows: 4-8g sodium chloride, 1.5-3.5g sodium bicarbonate, 3-5g phenol, and water to a final volume of 1L.

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