Preparation of natural alternaria antigen and detection kit for allergen-specific IgE (Immunoglobulin E) antibody of natural alternaria antigen

Abstract

The invention relates to an alternaria allergen specific IgE antibody detection reagent and a magnetic particle chemiluminescence detection kit, which are prepared by the following steps: activating an alternaria strain with the preservation number of ATCC 66981, culturing on a solid culture medium, taking freshly-cultured alternaria spores and mycelia, and adding the magnetic particle chemiluminescence detection kit, and extracting alternaria protein, performing biotin labeling to obtain biotin-labeled alternaria protein, and adding the biotin-labeled alternaria protein into a preserving fluid to obtain the detection reagent for the allergen-specific IgE antibody of the alternaria. A specific strain is matched with a screened and optimized culture medium formula to culture and extract the alternaria protein, the yield of the alternaria protein is high, the reaction activity is good, and the method has the advantages of high sensitivity, controllable batch difference and the like when being used for magnetic particle chemiluminiscence detection of the alternaria allergen specific IgE antibody. The popularization and the application of the magnetic particle chemiluminescence method of the alternaria allergen specific IgE antibody are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of in vitro diagnostic reagents, and specifically relates to a natural Alternaria antigen preparation and an allergen-specific IgE antibody detection kit. Background Art

[0002] There are about 100,000 species of fungi in nature, most of which are those that can float in the air and cause allergic diseases including allergic rhinitis and asthma. Alternaria is a common allergen that is widely present indoors and outdoors. Relevant surveys and studies show that 14% of children with asthma are allergic to fungi, and children allergic to Alternaria account for 46% of children with fungal allergies; another large-sample clinical epidemiological study found that 19% of patients with respiratory diseases are allergic to at least one mold, and 60% of these patients who are allergic to mold are allergic to Alternaria; it can be seen that Alternaria is closely related to respiratory diseases. However, the research and application of Alternaria are not as extensive and in-depth as those of allergens such as dust mites and pollen.

[0003] In existing research, there are few literatures on natural Alternaria antigenic proteins, which mainly focus 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 of the allergenic proteins of Alternaria cannot be fully determined at this stage, and it is impossible to obtain all the allergenic protein components of Alternaria. When recombinant Alternaria proteins are used for in vitro allergen diagnosis, the lack of components usually leads to low sensitivity of detection reagents.

[0004] Most of the existing diagnostic reagents for Alternaria allergy are based on immunoblotting, enzyme-linked immunosorbent assay, and fluorescent immunoassay, while magnetic particle chemiluminescence detection technology is developing and improving, has higher detection sensitivity, and can achieve the advantages of full automation, full quantification, and short time consumption. The preparation of high-quality natural Alternaria antigen protein and its use in the diagnosis of magnetic particle chemiluminescence platform will help improve the accuracy of in vitro diagnosis of Alternaria allergens. Summary of the invention

[0005] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide an Alternaria allergen-specific IgE antibody detection reagent containing natural Alternaria antigen protein and a magnetic particle chemiluminescence detection kit.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing an Alternaria allergen-specific IgE antibody detection reagent, comprising the steps of activating an Alternaria strain with a deposit number of ATCC 66981 and culturing the same on a solid culture medium, taking freshly cultured Alternaria spores and mycelium, extracting Alternaria protein, and labeling the protein with biotin to obtain biotin-labeled Alternaria protein, and adding the biotin-labeled Alternaria protein to a preservation solution to obtain the Alternaria allergen-specific IgE antibody detection reagent.

[0008] The solid culture medium has a formula of: 15-30 g glucose, 2-5 g yeast extract, 0.8-1.5 g dipotassium hydrogen phosphate, 0.3-0.8 g magnesium sulfate heptahydrate, 0.005-0.015 g ferrous sulfate heptahydrate, 0.3-0.8 g potassium chloride, 15-25 g agar, and the volume is fixed to 1 L with water.

[0009] Furthermore, the formula of the solid culture medium is: 18-22 g of glucose, 2-4 g of yeast extract, 0.8-1.2 g of dipotassium hydrogen phosphate, 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, 18-22 g of agar, and the volume is made up to 1 L with water.

[0010] In the embodiment of the present invention, the culture conditions are: temperature 25-30° C., and culture period 6-12 days.

[0011] In the embodiment of the present invention, the storage 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 storage solution is 7.2-7.5.

[0012] According to some specific embodiments of the present invention, the spores and mycelium of Alternaria are frozen with liquid nitrogen, and extracted with an extract. After the extraction, the supernatant is centrifuged and mixed with a Sulfo-NHS-biotin solution, and then reacted at room temperature. After the reaction, dialysis is performed using a 0.05-0.2M CB buffer solution with a pH value of 9.0-9.5 to obtain Sulfo-NHS-biotin-labeled Alternaria protein, and the Sulfo-NHS-biotin-labeled Alternaria protein is added to a preservation solution to obtain the Alternaria allergen-specific IgE antibody detection reagent.

[0013] In an embodiment of the present invention, the molar ratio of the Alternaria 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, 1:25.

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

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

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

[0017] In an embodiment of the present invention, the concentration of biotin-labeled Alternaria protein in the Alternaria 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, and 30 μg / mL.

[0018] The present invention also provides a magnetic particle chemiluminescence detection kit, which comprises the Alternaria allergen-specific IgE antibody detection reagent prepared by the above preparation method or the Alternaria allergen-specific IgE antibody detection reagent.

[0019] In an embodiment of the present invention, the magnetic particle chemiluminescent detection kit further comprises a streptavidin-coated magnetic particle 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 an embodiment of the present invention, 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 an embodiment of the present invention, the solvent of the streptavidin-coated magnetic microparticle suspension is a phosphate buffer solution with a pH value of 7.2 to 7.5, containing 1 to 2 wt% BSA, 1 to 3 wt% glycerol and 2 to 5 wt% mannitol.

[0022] In the embodiment of the present invention, the concentrations of the alkaline phosphatase-labeled mouse anti-human IgE antibody and the biotin-labeled anti-human IgE antibody are independently 0.1-2.0 μg / mL.

[0023] In the embodiment of the present invention, the anti-human IgE antibody is mixed with Sulfo-NHS-LC-biotin and dialyzed with a phosphate buffer containing 1-2 wt % BSA and a pH value of 7.2-7.5 to obtain the alkaline phosphatase-labeled mouse anti-human IgE antibody.

[0024] In an embodiment of the present invention, the luminescent substrate solution is an AMPPD solution.

[0025] In an embodiment of the present invention, the magnetic microparticle chemiluminescence detection kit further includes a calibrator and a quality control product.

[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 product is 0.7 IU / ml, 17.5 IU / ml.

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

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

[0030] The present invention adopts a specific strain in combination with a screened and optimized culture medium formula to culture and extract Alternaria protein, and preserves it in a preservation solution after biotin labeling. When the obtained reagent is used for magnetic particle chemiluminescence detection of Alternaria allergen-specific IgE antibody, it has the advantages of high sensitivity and controllable batch difference. The Alternaria allergen-specific IgE antibody detection reagent of the present invention has a simple production process, a high yield of Alternaria protein, a low production cost and good reaction activity, which is conducive to the promotion and application of the magnetic particle chemiluminescence method of Alternaria allergen-specific IgE antibody. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

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

[0033] The detection instrument used in the following examples is a SMART 6500HOB fully automatic chemiluminescence analyzer from Jiangsu Hao Ou Bo Bio-pharmaceutical Co., Ltd. (HOB for short).

[0034] Example 1

[0035] Preparation of Alternaria allergen-specific IgE antibody detection reagent.

[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 culture dishes, and sterilize under high pressure for later use.

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

[0038] 1.3 Use the solid culture medium prepared above to carry out fermentation culture. After sterilizing and cooling the inoculation loop on an alcohol lamp, dip a loop of the suspension in the ampoule bottle to draw a line on the solid culture medium. Do not let the inoculation loop scratch the surface of the plate. Then put the culture dish upside down in a 25℃ incubator. The culture period is 10 days. Take 0.5 g of freshly cultured Alternaria spores and mycelium, freeze them in liquid nitrogen and transfer them to a precooled (liquid nitrogen) Teflon container, add precooled steel balls, put the container into a separator, shake for 1 minute, transfer the powder to a 1.5 mL centrifuge tube, add 1 mL of extract (formula: 5 g sodium chloride, 2.75 g sodium bicarbonate, 4 g phenol, add distilled water to 1 L), invert the centrifuge tube to carefully mix the suspension (do not vortex), centrifuge for 5 minutes, take the supernatant, the supernatant contains the total protein of Alternaria (the supernatant corresponding to culture medium 1 is marked as antigen protein solution 1, the supernatant corresponding to culture medium 2 is marked as antigen protein solution 2, and so on to antigen protein solution 11), and store at -20°C.

[0039] Table 1

[0040]

[0041] The BCA method was used to determine the protein concentration in the above different antigen protein solutions: the BCA detection kit was provided by Thermo Fisher Scientific, and 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. Prepare the BCA working solution: mix the volume reagent A:B=50:1, and mix well. Reagent A: 1% BCA disodium salt, 0.4% sodium hydroxide, 0.16% sodium tartrate, 2% anhydrous sodium carbonate, 0.95% sodium bicarbonate, reagent B: 4% copper sulfate. BCA kit sample addition: dilute the sample with PBS according to the sample concentration, add 20μL of each sample and standard to a 96-well plate, make 3 replicates of the sample, add 300μL of the working solution, mix for 10s, react at 37℃ for half an hour, then measure the absorbance at 562nm with a microplate reader, calculate the protein concentration according to the standard curve, take the average value, and 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 between Alternaria protein and Sulfo-NHS-biotin, and the reaction was carried out at room temperature for 1 hour. After the reaction, the solution was dialyzed using a 0.1 M CB buffer solution with a pH value of 9.5. The obtained Sulfo-NHS-biotin-labeled Alternaria antigen protein was stored in a phosphate buffer solution (pH = 7.4) containing an appropriate amount of 2 wt% BSA, 3 wt% glycerol and 5 wt% mannitol to prepare a 10 μg / mL biotin-labeled Alternaria allergen solution, i.e., an Alternaria allergen-specific IgE antibody detection reagent (the label corresponding to antigen protein solution 1 is detection reagent 1, the label corresponding to antigen protein solution 2 is detection reagent 2, and so on to detection reagent 11).

[0045] Example 2

[0046] A magnetic particle chemiluminescence detection kit for detecting specific IgE antibodies to Alternaria allergen was prepared.

[0047] The magnetic particle chemiluminescent detection kit includes a streptavidin-coated magnetic particle suspension, a biotin-labeled Alternaria allergen solution, an alkaline phosphatase-labeled anti-human IgE antibody, a biotin-labeled anti-human IgE antibody, a calibrator, a quality control product, and an AMPPD-based luminescent substrate. The specific preparation method is as follows:

[0048] Streptavidin-coated magnetic microparticle suspension: 10 mg / mL of streptavidin magnetic beads (particle size of 1 μm) were mixed and magnetically separated, resuspended in phosphate buffer (pH = 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol, washed 3 times, magnetically separated, and finally resuspended in phosphate buffer (pH 7.4) containing 2wt% BSA, 3wt% glycerol and 5wt% mannitol to obtain a streptavidin-coated magnetic microparticle suspension with a concentration of 0.4 mg / mL.

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

[0050] Alkaline phosphatase labeled anti-human IgE antibody solution: dialyze the alkaline phosphatase (AP) to be activated, then add a coupling agent to react, separate and remove the free coupling agent, then add the activated alkaline phosphatase to the activated anti-human IgE antibody solution for coupling reaction, and then separate and remove the free AP. The concentration of the alkaline phosphatase labeled anti-human IgE antibody in the alkaline phosphatase labeled anti-human IgE antibody solution is 1.5 μg / mL.

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

[0052] Calibrator: Take the IgE protein derived from the WHO standard and dilute it into 0 IU / mL, 0.35 IU / mL, 0.7 IU / mL, 3.5 IU / mL, 17.5 IU / mL, and 100 IU / mL calibrators using 0.1 M Tris-HCl buffer, pH 7.4.

[0053] Quality control: Take the IgE protein derived from the WHO standard and prepare it into 0.7 IU / mL and 17.5 IU / mL quality control products with 0.1 M Tris-HCl buffer, pH 7.4.

[0054] The luminescent substrate solution is an independently produced alkaline phosphatase enzymatic luminescent substrate based on AMPPD luminescence. For details, please refer to the invention patent "An enzymatic chemiluminescent substrate of alkaline phosphatase", authorization announcement number CN 104990912 B.

[0055] The above kits were used to quantitatively detect the content of Alternaria-specific IgE in clinical samples, including 10 positive samples (P) and 10 negative samples (N), and compared with the results of Immuno CAP test by Phadia. The results are shown in Table 3 (Phadia unit: kUA / L, HOB unit: IU / mL, 1kUA / L=1IU / mL). The detection method of the magnetic particle chemiluminescence detection kit is as follows: the calibrator, streptavidin-coated magnetic particle suspension, and biotin-labeled anti-human IgE antibody are mixed, incubated at 37°C for 15 minutes, magnetically separated to remove the supernatant, and washed three times; alkaline phosphatase-labeled anti-human IgE antibody is added, mixed, incubated at 37°C for 15 minutes, magnetically separated to remove the supernatant, and washed three times; luminescent substrate solution is added, incubated at 37°C for 5 minutes, and the chemiluminescence intensity is measured, and the concentration of the calibrator is used as the horizontal axis and the chemiluminescence intensity is used as the vertical axis to draw a graph. The sample to be tested was mixed with a magnetic particle suspension coated with streptavidin and a biotin-labeled Alternaria allergen solution, incubated at 37°C for 15 min, magnetically separated to remove the supernatant, and washed three times; an alkaline phosphatase-labeled anti-human IgE antibody was added, mixed, incubated at 37°C for 15 min, magnetically separated to remove the supernatant, and washed three times; a luminescent substrate solution was added, incubated at 37°C for 5 min, the chemiluminescence intensity was determined, and the concentration of the Alternaria allergen-specific IgE in the sample to be tested was calculated, and the cut off value was 0.35 IU / mL, and a value greater than or equal to 0.35 IU / mL was judged as positive.

[0056] Table 3

[0057]

[0058]

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

[0060] Table 4

[0061]

[0062]

[0063] Tables 3 and 4 show that the effects of the Alternaria protein detection reagents after culture and extraction with different culture media are different. It can be seen that different culture media will affect the antigenic sites and components of the antigen protein. Among them, the detection reagent 1 has a lower background and the highest positive and negative coincidence rate.

[0064] Example 3

[0065] Optimize the detection reagent of IgE antibody specific to Alternaria allergen.

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

[0067] According to Example 2, the above-mentioned detection reagents A, B and C are respectively mixed with a streptavidin-coated magnetic microparticle suspension, an alkaline phosphatase-labeled anti-human IgE antibody, a biotin-labeled anti-human IgE antibody, a calibrator, a quality control product and an AMPPD-based luminescent substrate to form a magnetic microparticle chemiluminescent detection kit, the corresponding detection reagent A is labeled as a magnetic microparticle chemiluminescent detection kit A, the corresponding detection reagent B is labeled as a magnetic microparticle chemiluminescent detection kit B, and the corresponding detection reagent C is labeled as a magnetic microparticle chemiluminescent detection kit C.

[0068] The above-mentioned magnetic microparticle chemiluminescence detection kit A, magnetic microparticle chemiluminescence detection kit B, and magnetic microparticle chemiluminescence detection kit C were used to quantitatively detect the content of Alternaria specific IgE in clinical samples, including 20 positive samples (P) and 10 negative samples (N), and compared with the Immuno CAP test results of Phadia, the results are shown in Table 5. (Phadia unit: kUA / L, HOB unit: IU / mL, 1kUA / L=1IU / mL).

[0069] Table 5

[0070]

[0071]

[0072] The positive and negative coincidence rates of magnetic microparticle chemiluminescence detection kit A, magnetic microparticle chemiluminescence detection kit B, magnetic microparticle 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 coupled biotinylation, the reagent background value can be reduced and the positive detection rate can be increased. When the molar ratio of Alternaria protein in the allergen crude extract 1 to Sulfo-NHS-biotin is 1:20, the total compliance rate is the highest.

[0076] Example 4

[0077] The accuracy, detection limit, linearity, repeatability, specificity, precision and batch difference of the magnetic microparticle chemiluminescence detection kit B were tested as follows:

[0078] Accuracy test:

[0079] A high-value sample A (accuracy reference H) with a known concentration is added to a low-concentration sample B (accuracy reference L) at a volume ratio of no more than 1:19 to obtain a sample to be tested. The concentration of the sample to be tested should be within the detection range of the reagent, and its recovery rate is calculated according to the formula:

[0080]

[0081] Where:

[0082] R——recovery rate;

[0083] V——volume of accuracy reference H;

[0084] V 0 ——volume of accuracy reference material L;

[0085] C——detection concentration of mixed sample;

[0086] C 0 ——Concentration of accuracy reference substance L;

[0087] C S ——Concentration of accuracy reference substance 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] Detection limit test:

[0093] Five samples with low concentrations close to the detection limit (detection limit reference samples 1 to 5) were tested, each sample was tested 5 times, and the test results were sorted by size. If the following conditions were met, the settings of the blank limit and the detection limit can be considered to be basically reasonable:

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

[0095] b) There are no test results higher than the lower limit of the reference interval.

[0096] Take calibration point CAL1 (zero calibration point) as the sample and test at least 20 times. Calculate the luminescence value Mean+2SD, and use the luminescence values ​​of calibration points CAL1 and CAL2 with no less than 5 results to make a straight line fit to back-calculate its concentration and obtain the blank limit.

[0097] The detection limit analysis results are shown in Table 8 and Table 9:

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

[0099]

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

[0101]

[0102]

[0103] The detection limit results showed that the detection limit was set reasonably, no higher than 0.1IU / mL, which met the requirements.

[0104] Linear detection:

[0105] Use low-value samples close to the lower limit of the linear interval to dilute high-value samples close to the upper limit of the linear interval (linear reference), and dilute them proportionally to at least 5 concentrations, among which the low-value concentration samples must be close to 0.1IU / mL. Repeat the test 3 times for each concentration, calculate the average value of each concentration point, and use the least squares method to fit the average value of the measured concentration to the theoretical concentration, 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 test:

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

[0112] CV=SD / M×100%

[0113] Where:

[0114] CV – coefficient of variation;

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

[0116] SD - standard deviation of the concentration results of 10 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] Specificity test:

[0122] Samples containing total IgE with a concentration of not less than 60 IU / mL and no specific IgE antibodies, and IgA samples with a concentration of not less than 700 μg / mL, IgM samples with a concentration of 500 μg / mL, and IgG samples with a concentration of 7000 μg / mL were tested. Each sample was tested once. The results are shown in Table 12:

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

[0124] sample concentration Samples with 60 IU / mL of total IgE and no specific IgE antibodies 0.04 700 μg / mL IgA 0.01 500 μg / mL IgM 0.01 7000μg / mL IgG 0.02

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

[0126] Batch-to-batch difference detection:

[0127] Three batches of allergen crude extract 1 were used to prepare three batches of magnetic microparticle chemiluminescence detection kits according to magnetic microparticle chemiluminescence detection kit B. The three batches of magnetic microparticle chemiluminescence detection kits were used to detect the clinical samples in Example 3, and the 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 inter-batch CV of the three batches of kits was no more than 10%, indicating that the kits had small inter-batch differences and stable performance.

[0133] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an Alternaria allergen-specific IgE antibody detection reagent, characterized in that: The Alternaria strain with the accession number of ATCC 66981 is activated and cultured on a solid culture medium, the freshly cultured Alternaria spores and mycelium are taken, Alternaria protein is extracted and labeled with biotin to obtain biotin-labeled Alternaria protein, and the biotin-labeled Alternaria protein is added to the preservation solution to obtain the Alternaria allergen-specific IgE antibody detection reagent, The solid culture medium has a formula of: 15-30 g glucose, 2-5 g yeast extract, 0.8-1.5 g dipotassium hydrogen phosphate, 0.3-0.8 g magnesium sulfate heptahydrate, 0.005-0.015 g ferrous sulfate heptahydrate, 0.3-0.8 g potassium chloride, 15-25 g agar, and the volume is fixed to 1 L with water.

2. The method for preparing the Alternaria allergen-specific IgE antibody detection reagent according to claim 1, characterized in that: The culture conditions are: temperature 25-30° C., and culture 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 storage 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 storage solution is 7.2-7.

5.

4. The method for preparing the Alternaria allergen-specific IgE antibody detection reagent according to claim 1, characterized in that: The spores and mycelium of Alternaria are frozen with liquid nitrogen, and extracted by adding an extract. After the extraction, the supernatant is centrifuged and mixed with a Sulfo-NHS-biotin solution, and then reacted at room temperature. After the reaction, dialysis is performed using a 0.05-0.2M CB buffer solution with a pH value of 9.0-9.5 to obtain Sulfo-NHS-biotin-labeled Alternaria protein, and the Sulfo-NHS-biotin-labeled Alternaria protein is added to a preservation solution to obtain the Alternaria allergen-specific IgE antibody detection reagent.

5. The method for preparing the Alternaria allergen-specific IgE antibody detection reagent according to claim 4, characterized in that: The molar ratio of the Alternaria protein in the supernatant to the Sulfo-NHS-biotin in the Sulfo-NHS-biotin solution is 1:15-25.

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

7. A detection reagent for specific IgE antibody to Alternaria allergen prepared by the method according to any one of claims 1 to 6, characterized in that: The concentration of biotin-labeled Alternaria protein in the Alternaria allergen-specific IgE antibody detection reagent is 5-30 μg / mL.

8. A magnetic microparticle chemiluminescence detection kit, characterized in that: It comprises the Alternaria allergen-specific IgE antibody detection reagent prepared by the preparation method according to any one of claims 1 to 6 or the Alternaria allergen-specific IgE antibody detection reagent according to claim 7.

9. The magnetic microparticle chemiluminescence detection kit according to claim 8, characterized in that: The magnetic particle chemiluminescence detection kit also includes a magnetic particle suspension coated with streptavidin, a mouse anti-human IgE antibody solution labeled with alkaline phosphatase, an anti-human IgE antibody solution labeled with biotin and a luminescent substrate solution.

10. The magnetic microparticle chemiluminescence detection kit according to claim 9, characterized in that: The concentration of the streptavidin-coated magnetic microparticle suspension is 0.4-1 mg / mL; and / or, the solvent of the streptavidin-coated magnetic microparticle suspension is a phosphate buffer having a pH value of 7.2 to 7.5 and containing 1 to 2 wt% BSA, 1 to 3 wt% glycerol and 2 to 5 wt% mannitol; and / or, the concentrations of the alkaline phosphatase-labeled mouse anti-human IgE antibody and the biotin-labeled anti-human IgE antibody are independently 0.1 to 2.0 μg / mL; and / or, mixing the anti-human IgE antibody with Sulfo-NHS-LC-biotin, and dialyzing with a phosphate buffer containing 1 to 2 wt % BSA and having a pH value of 7.2 to 7.5 to obtain the alkaline phosphatase-labeled mouse anti-human IgE antibody; And / or, the luminescent substrate solution is an AMPPD solution; And / or, the magnetic microparticle chemiluminescence detection kit also includes calibration products and quality control products.

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