A method for preparing a shrimp allergen mixture

Abstract

The present invention provides a method for preparing a shrimp allergen mixture. The preparation method comprises the following steps: (1) using acetone to defat, air-dry and crush to obtain defatted shrimp powder; (2) preparing a shrimp allergen extract by mixing the defatted shrimp powder and the extract in a mass volume ratio of 1g:(10mL-20mL); (3) dialyzing and microfiltration of the shrimp allergen extract to obtain a shrimp allergen purified solution; (4) mixing the Arctic sweet shrimp allergen purified solution, the small white shrimp allergen purified solution and the white shrimp allergen purified solution prepared in steps (1) to (3) in a volume ratio of 1:(6-8):(1.5-3) to obtain a shrimp allergen mixture. The shrimp allergen mixture of the present invention has more comprehensive sensitizing components, higher biological activity and positive detection rate, and clean background, and is a high-precision allergy raw material more suitable for people with shrimp allergies in my country.

Description

Technical Field

[0001] The invention belongs to the field of raw material preparation for antibody detection, and particularly relates to a method for preparing a shrimp allergen mixture. Background Art

[0002] Allergic diseases have become another major chronic disease threatening human health after cardiovascular and cerebrovascular diseases, tumors, chronic respiratory diseases, diabetes and metabolic diseases, and mental and psychological diseases. An official report released by the World Health Organization pointed out that 22% to 25% of the world's population has suffered or is suffering from allergic diseases, and the prevalence rate continues to rise. A survey of allergic diseases in my country shows that 2 / 3 of the 30 million asthma patients in my country suffer from allergic asthma, the average prevalence of allergic rhinitis in adults in 18 major cities is 17.6%, and the prevalence of asthma in children reached 7.57% in 2019, almost doubling every ten years.

[0003] The in vitro detection of allergen-specific antibodies and the subsequent identification of allergens associated with allergic diseases is currently the safest, most reliable and most definitive diagnostic method. However, the in vitro diagnostic market for allergic diseases is currently dominated by international manufacturers, whose products occupy the high-end market and are very expensive. Domestic allergic disease diagnosis companies have developed rapidly in recent years, their market share has continued to expand, and they have gradually moved towards the high-end. Although domestic manufacturers are developing rapidly, key raw materials for allergic disease diagnosis have always relied on imports.

[0004] Among foods that are prone to cause food allergies, shrimp and its products are a very important category. The relevant research department of the Food and Agriculture Organization of the United Nations has listed it in the eight categories of allergenic foods. To study shrimp allergens, it is necessary to first obtain high-purity allergen components. However, there are many varieties of shrimp, and the allergic phenomena of the population also have regional characteristics. The differences between imported shrimp allergen raw materials and the living and eating habits of the Chinese people bring risks to the prevention and treatment of diseases. The applicability of imported shrimp allergens currently used in large quantities is difficult to guarantee, and false negative results are prone to occur, resulting in missed detection. The high price of imported shrimp allergen raw materials increases costs, and the stability of supply is affected by the international situation and there is a huge risk. However, since the allergenic components of shrimp allergens are relatively complex, the shrimp allergens prepared by traditional preparation methods are crude extracts, and there is a risk of missed diagnosis due to some allergenic components not being included in the extract.

[0005] The main allergenic proteins in shrimp include tropomyosin (TM), arginine kinase (AK), myosin light chain (MLC), sarcoplasmic calcium binding protein (SCP), pyruvate kinase (PK) and hemocyanin.

[0006] Therefore, it is necessary to develop a method for preparing shrimp allergen raw materials that can ensure the richness of different allergenic components and maintain high allergenicity. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method capable of extracting more and more complete allergen proteins from shrimp. The shrimp allergen sensitizing components obtained by the method are complete and have high allergenicity, and can be directly used in an in vitro detection kit.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for preparing a shrimp allergen mixture, the preparation method comprising the following steps:

[0010] (1) using acetone to defat, air-dry, and grind to obtain defatted shrimp powder;

[0011] (2) preparing a shrimp allergen extract by mixing the defatted shrimp powder and the extract in a mass volume ratio of 1 g: (10 mL to 20 mL);

[0012] (3) the shrimp allergen extract is dialyzed and microfiltered to obtain a shrimp allergen purified solution;

[0013] (4) Using sweet prawns, white shrimps and whiteleg shrimps as raw materials, the sweet prawns allergen purified liquid, the white shrimps allergen purified liquid and the whiteleg shrimps allergen purified liquid prepared according to steps (1) to (3) are mixed in a volume ratio of 1: (6-8): (1.5-3) to obtain the shrimp allergen mixture.

[0014] Preferably, in step (4), the volume ratio of the purified liquid of Artic sweet shrimp allergen, the purified liquid of white shrimp allergen and the purified liquid of whiteleg shrimp allergen is 1:7:2.

[0015] Preferably, in step (2), the extract is water or a buffer solution with a pH value of 7.0 to 8.5.

[0016] Further preferably, the buffer is a PBS buffer with a concentration of 0.01 to 0.02 M, a CBS buffer with a concentration of 0.01 to 0.02 M, a Tris-HCl buffer with a concentration of 0.04 to 0.06 M, or a Tris-HCl buffer with a concentration of 0.04 to 0.06 M containing KCl and DTT.

[0017] Still more preferably, the extract is a Tris-HCl buffer solution with a concentration of 0.04-0.06 M containing 0.1-0.3 M KCl and 0.4-0.8 mM DTT, and the pH value of the extract is 8.0-8.5.

[0018] Preferably, in step (2), the extract is prepared by mixing the defatted shrimp powder and the extract, standing the mixture at 2-6° C. for 1.5-2.5 h, then centrifuging the mixture at a speed of 8000-10000 r / min for 15-25 min, and taking the supernatant, which is the extract.

[0019] Preferably, in step (3), a dialysis bag with a molecular weight cutoff of 3000-4000Da and a PBS low-salt buffer solution are used as the dialysis fluid, and the microfiltration uses a 0.25-0.45 μm filter membrane.

[0020] Preferably, in step (1), the shrimp muscle tissue clear liquid is mixed with 2-6°C cold acetone at a ratio of 1 g: (8 mL-12 mL) for overnight extraction, during which the cold acetone is replaced 3-4 times, and then centrifuged at a speed of 3000-5000 r / min for 10-20 min, the precipitate is taken, and the precipitate is washed with 2-6°C cold acetone until the effluent is colorless, and the precipitate is air-dried and crushed to obtain the defatted shrimp powder.

[0021] A multi-process organically coordinated refining strategy is adopted to extract shrimps of different species, and then through comprehensive analysis and optimized compounding, a shrimp allergy mixture with complete components and higher positive compliance rate is obtained, thereby improving the accuracy of allergy diagnosis, greatly reducing missed diagnosis and misdiagnosis caused by missing allergenic components and cross-reactions, and enabling diagnostic results to better guide clinical practice, identify relevant allergens, and carry out accurate immunotherapy.

[0022] The present invention also provides a shrimp allergen mixture, which is prepared by the above preparation method.

[0023] The present invention also provides application of the shrimp allergen mixture in a shrimp allergy detection reagent or a detection kit.

[0024] Preferably, the detection reagent or detection kit adopts magnetic particle chemiluminescence technology.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The present invention adopts the form of freeze-dried products, which is convenient for storage and reduces the problem of low test results caused by dilution of the sample to be tested.

[0027] The shrimp allergen mixture prepared by the preparation method of the present invention has more comprehensive sensitizing components, higher biological activity and positive detection rate, and a clean background. The serum test results of shrimp allergic patients in my country are more accurate and have a lower risk of missed detection. The preparation method of the present invention is a high-precision allergy raw material that is more suitable for people with shrimp allergies in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the standard curve obtained based on the measured values ​​of BSA standards;

[0029] Figure 2 is the SDS-PAGE electrophoresis diagram of the allergen solution prepared in Examples 1 to 6;

[0030] Figure 3 The SDS-PAGE electrophoresis diagram of the purified liquid of the Arctic sweet shrimp allergen, the purified liquid of the white shrimp allergen and the purified liquid of the white shrimp allergen;

[0031] Figure 4 This is a diagram showing the results of a clinical human positive serum immunoblotting test;

[0032] Figure 5 This is a diagram showing the results of a clinical human negative serum immunoblotting experiment. DETAILED DESCRIPTION

[0033] 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.

[0034] In the following examples, the implementation conditions not specified are conventional conditions in the industry, and the reagents can be purchased from the market.

[0035] In the following embodiments, the white shrimp is also known as the vannamei shrimp, white-limbed shrimp, white shrimp, and vannamei shrimp, Penaeidae, Litopenaeus genus; the Arctic sweet shrimp is also known as the cold water shrimp, and the imported Arctic sweet shrimp used in this article; the small white shrimp is also known as the spiny tail white shrimp, Longarm shrimp family, white shrimp genus, produced in the waters near China; the mud shrimp is also known as the knife-headed new shrimp, Penaeidae, New shrimp genus, which is artificially cultivated

[0036] The temperature of cold acetone used in the following examples was about -20°C.

[0037] In the following examples, DTT is the abbreviation of dithiothreitol.

[0038] In the following examples, 0.01 M PBS buffer (pH 7.4): 8.0 g NaCl, 2.9 g Na2HPO4, 0.20 g KCl, and 0.20 g KH2PO4 were weighed and dissolved in 800 mL deionized water, and the pH value was adjusted to 7.4 with NaOH and HCl, and the volume was made up to 1 L with ultrapure water, and the solution was filtered through a 0.22 μm filter membrane and stored at room temperature;

[0039] 0.01M CBS buffer (pH 8.0): weigh 0.59g NaHCO3 and 0.23g Na2CO3 in 800mL deionized water, fully dissolve, adjust the pH to 8.0 with NaOH and HCl, dilute to 1L with ultrapure water, filter through 0.22μm membrane and store at room temperature;

[0040] 0.05M Tris-HCl (pH 8.2): Weigh 6.06g Tris in 800mL deionized water, dissolve, adjust pH to 8.2 with NaOH and HCl, make up to 1L with ultrapure water, filter through 0.22μm membrane and store at room temperature.

[0041] 0.05M Tris-HCl buffer (containing 0.2M KCl, 0.5mM DTT, pH 8.2): weigh 6.06g Tris, 14.90g KCl, 0.078g DTT in 800mL deionized water, dissolve, adjust pH to 8.2 with NaOH and HCl, dilute to 1L with ultrapure water, filter through 0.22μm membrane and store at room temperature;

[0042] 0.01M PBS buffer (containing 0.2M KCl, 0.5mM DTT, pH 7.4): weigh 8.0g NaCl, 2.9g Na2HPO4, 0.20g KCl, 0.20g KH2PO4, 14.90g KCl, 0.078g DTT in 800mL deionized water, dissolve and adjust the pH to 7.4 with NaOH and HCl, make up to 1L with ultrapure water, filter through 0.22μm membrane and store at room temperature.

[0043] Example 1

[0044] This embodiment provides a method for extracting allergens from Penaeus vannamei, and the steps are as follows:

[0045] (1) Degreasing: Wash the muscle tissue of Penaeus vannamei, grind it into a paste, add cold acetone and extract overnight. The ratio of cold acetone to muscle tissue is 1:10 (W / V). The cold acetone is replaced 3-4 times during the overnight extraction. After the extraction, centrifuge (4°C, 4000r / min, 15min) to obtain the precipitate. Wash the precipitate with cold acetone until the effluent is colorless. After the precipitate is air-dried at room temperature, it is crushed to obtain defatted shrimp meat powder.

[0046] (2) Extraction: Take 0.5 g of the above defatted shrimp meat powder in a 15 mL centrifuge tube, add 7.5 mL of ultrapure water, extract at 4°C for 2 h, and then centrifuge (4°C, 9000 r / min, 20 min) to collect the supernatant.

[0047] (3) Filtration and dialysis: The supernatant is filtered through a 0.22 μm sterile filter membrane to obtain a crude allergen protein solution. The crude allergen protein solution is placed in a 3500 Da dialysis bag and dialyzed in a 0.01 M PBS buffer (pH 7.4). The crude allergen protein solution is dialyzed three times, with the solution being replaced every 8 h, 12 h, and 24 h. After dialysis, the filtrate is filtered again using a 0.22 μm filter membrane. The filtrate is the purified solution of Penaeus vannamei allergens.

[0048] Example 2

[0049] This example provides a method for extracting allergens from Penaeus vannamei. The steps are basically the same as those in Example 1, except that the ultrapure water used in the extraction solution in step (2) is replaced with 0.01 M PBS buffer (pH 7.4).

[0050] Example 3

[0051] This example provides a method for extracting allergens from Penaeus vannamei. The steps are basically the same as those in Example 1, except that the ultrapure water used as the extract in step (2) is replaced with 0.01 M CBS buffer (pH 8.0).

[0052] Example 4

[0053] This example provides a method for extracting allergens from Penaeus vannamei. The steps are basically the same as those in Example 1, except that the ultrapure water used as the extracting solution in step (2) is replaced with 0.05 M Tris-HCl (pH 8.2).

[0054] Example 5

[0055] This example provides a method for extracting allergens from Penaeus vannamei. The steps are basically the same as those in Example 1, except that the ultrapure water used as the extracting solution in step (2) is replaced with 0.05M Tris-HCl buffer (containing 0.2M KCl, 0.5mM DTT, pH 8.2).

[0056] Example 6

[0057] This example provides a method for extracting allergens from Penaeus vannamei. The steps are basically the same as those in Example 1, except that the ultrapure water used in the extraction solution in step (2) is replaced with 0.01M PBS buffer (containing 0.2M KCl, 0.5mM DTT, pH 7.4).

[0058] The allergen solutions prepared in Examples 1 to 6 were subjected to SDS-PAGE identification. The electrophoresis conditions were: 12% separation gel, 5% concentration gel, constant voltage 140V. The SDS-PAGE electrophoresis diagram is shown in Figure 2 , wherein lane 1 is a marker used to indicate the molecular weight and position; lanes 2 to 7 are the purified allergen solutions prepared in Examples 1 to 6, respectively. Figure 1 It was shown that compared with Examples 2 to 4 and Example 6 (lanes 2 to 5, lane 7), the protein bands of the allergen solution prepared in Example 5 (lane 6) were the most abundant, indicating that 0.05M Tris-HCl buffer (containing 0.2M KCl, 0.5mM DTT, pH 8.2) had the best extraction effect and extracted the most types of proteins.

[0059] The protein concentration of the allergen solution prepared in Examples 1 to 6 was determined. The concentration was detected by the BCA method according to the instructions of the Bradford kit, which will not be described in detail here. The standard curve (the horizontal axis is the protein concentration, the vertical axis is the OD value) and the conversion formula were obtained based on the measured values ​​of the BSA standard. Figure 1 , substitute the OD value of the protein to be tested into the formula to calculate the protein concentration. The results are shown in Table 1.

[0060] Table 1

[0061] project Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Protein concentration (mg / mL) 3.96 5.18 6.23 4.64 7.81 6.42

[0062] Example 7

[0063] This embodiment provides a method for extracting Arctic sweet shrimp allergens, and the steps are as follows:

[0064] (1) Degreasing: Clean the muscle tissue of the Arctic sweet shrimp, grind it into a paste, add cold acetone and extract overnight. The ratio of cold acetone to muscle tissue is 1:10 (W / V). The cold acetone is replaced 3-4 times during the overnight extraction. After the extraction, centrifuge (4°C, 4000r / min, 15min) to obtain the precipitate. Wash the precipitate with cold acetone until the effluent is colorless. After the precipitate is air-dried, it is crushed to obtain defatted shrimp meat powder.

[0065] (2) Extraction: Take 0.5 g of the above defatted shrimp meat powder in a 15 mL centrifuge tube, add 7.5 mL of 0.05 M Tris-HCl buffer (containing 0.2 M KCl, 0.5 mM DTT, pH 8.2), extract at 4°C for 2 h, and then centrifuge (4°C, 9000 r / min, 20 min) to collect the supernatant.

[0066] (3) Filtration and dialysis: The supernatant is filtered through a 0.22 μm sterile filter membrane to obtain a crude allergen protein solution. The crude allergen protein solution is placed in a 3500 Da dialysis bag and dialyzed against a PBS low-salt buffer solution. The crude allergen protein solution is dialyzed three times, with the solution being replaced every 8 h, 12 h, and 24 h. After dialysis, the solution is filtered again using a 0.22 μm filter membrane. The filtrate is the purified solution of the Arctic sweet shrimp allergen.

[0067] This example also compares ultrapure water, 0.01M PBS buffer (pH 7.4), 0.01M CBS buffer (pH 8.0), 0.05M Tris-HCl (pH 8.2), and 0.01M PBS buffer (containing 0.2M KCl, 0.5mM DTT, pH 7.4) as the extracting solution in the extraction step. The types and concentrations of Arctic sweet shrimp proteins in the prepared allergen purified solutions are inferior to those of the allergen purified solution prepared using 0.05M Tris-HCl buffer (containing 0.2M KCl, 0.5mM DTT, pH 8.2) as the extracting solution.

[0068] Example 8

[0069] This embodiment provides a method for extracting shrimp allergens, and the steps are as follows:

[0070] (1) Degreasing: Clean the muscle tissue of small white shrimp, grind it into a paste, add cold acetone and extract overnight. The ratio of cold acetone to muscle tissue is 1:10 (W / V). Change the cold acetone 3-4 times during the overnight extraction. After the extraction, centrifuge (4°C, 4000r / min, 15min) to obtain the precipitate. Wash the precipitate with cold acetone until the effluent is colorless. After the precipitate is air-dried, grind it to obtain defatted shrimp meat powder.

[0071] (2) Extraction: Take 0.5 g of the above defatted shrimp meat powder in a 15 mL centrifuge tube, add 7.5 mL of 0.05 M Tris-HCl buffer (containing 0.2 M KCl, 0.5 mM DTT, pH 8.2), extract at 4°C for 2 h, and then centrifuge (4°C, 9000 r / min, 20 min) to collect the supernatant.

[0072] (3) Filtration and dialysis: The supernatant is filtered through a 0.22 μm sterile filter membrane to obtain a crude allergen protein solution. The crude allergen protein solution is placed in a 3500 Da dialysis bag and dialyzed against a PBS low-salt buffer solution. The crude allergen protein solution is dialyzed three times, with the solution being replaced every 8 h, 12 h, and 24 h. After dialysis, the solution is filtered again using a 0.22 μm filter membrane. The filtrate is the purified shrimp allergen solution.

[0073] This example also compares ultrapure water, 0.01M PBS buffer (pH 7.4), 0.01M CBS buffer (pH 8.0), 0.05M Tris-HCl (pH 8.2), and 0.01M PBS buffer (containing 0.2M KCl, 0.5mM DTT, pH 7.4) as the extracting solution in the extraction step. The types and concentrations of shrimp proteins in the prepared allergen purified solutions are not as good as the allergen purified solution prepared using 0.05M Tris-HCl buffer (containing 0.2M KCl, 0.5mM DTT, pH 8.2) as the extracting solution.

[0074] The purified solution of the Arctic sweet shrimp allergen prepared in Example 7, the purified solution of the small white shrimp allergen prepared in Example 8, and the purified solution of the white shrimp allergen prepared in Example 5 were subjected to SDS-PAGE to detect the shrimp allergenic components. The electrophoresis conditions were the same as above. The electrophoresis diagram is shown in Figure 3 , M: Marker, Lane 1: Arctic sweet shrimp, Lane 2: Whiteleg shrimp, Lane 3: Small white shrimp.

[0075] The purified liquid of Penaeus vannamei allergen prepared in Example 5, the purified liquid of sweet shrimp allergen prepared in Example 7 and the purified liquid of small white shrimp allergen prepared in Example 8 were used to detect the allergen reactivity by immunoblotting. The steps of the immunoblotting experiment are as follows: the SDS-PAGE electrophoresis gel is taken out and placed in a transfer membrane group, and the protein on the gel is transferred to a PVDF membrane by a transfer membrane instrument (20V, 4min 30s). Add 30mL of PBST buffer wash solution to the incubation box, place on a shaker to wash for 10 minutes, and discard the waste liquid; add 30mL of blocking solution, incubate on a shaker for 2 hours for blocking, and wash 3 times with PBST buffer; dilute human serum 5 times with PBST buffer, add diluted primary antibody (clinical human serum: positive serum or negative serum), incubate on a shaker at room temperature for 1 hour, wash with PBST buffer, add 30mL of diluted secondary antibody (sheep anti-human IgE-HRP), incubate on a shaker at room temperature for 1 hour, and wash 3 times with PBST buffer; prepare color development solution (Thermo Fisher SuperSignal chemiluminescent substrate) in a light-proof 1:1 (v / v) ratio, evenly infiltrate the entire PVDF membrane, incubate in the dark for 2 minutes, use a gel imager for image acquisition, expose for 120 seconds, and collect images for analysis. Among them, the results of clinical human positive serum immunoblotting experiments are shown in Figure 4Different shrimp species react differently to different positive sera. Clinical human negative sera results are shown in Figure 5 The shrimp allergen raw material prepared by the above method has a clean background and is non-reactive with negative serum.

[0076] Example 9

[0077] This embodiment provides a shrimp allergen mixture. The Arctic sweet shrimp allergen purified solution prepared in Example 7, the small white shrimp allergen purified solution prepared in Example 8, and the white shrimp allergen purified solution prepared in Example 5 were mixed at a mass ratio of 1:2:2, 1:5:1, and 1:7:2, respectively, to prepare three shrimp allergen mixtures.

[0078] Comparative Example 1

[0079] This comparative example provides a shrimp allergen mixture. The white shrimp allergen purified liquid prepared in Example 5, the shrimp allergen purified liquid, and the small white shrimp allergen purified liquid prepared in Example 8 were mixed at a mass ratio of 1:2:2, 1:5:1, and 1:7:2, respectively, to prepare three shrimp allergen mixtures.

[0080] The preparation method of the shrimp allergen solution is the same as that of Example 5, except that the raw material whiteleg shrimp is replaced with shrimp.

[0081] The performance of different shrimp allergens and shrimp allergen mixtures was detected by using nanomagnetic particle chemiluminescence, and the steps are as follows:

[0082] (1) Add 40 μL of serum to be tested and 50 μL of streptavidin nanoparticles labeled with shrimp allergens into the test tube, mix well, and incubate at 37°C for 15 minutes;

[0083] (2) Under the action of an external magnetic field, the magnetic particles settle, the supernatant is removed, and a cleaning solution is added. After the magnetic field is removed, the magnetic particles are shaken to resuspend them. This cleaning step is repeated three times to remove unbound human serum antibodies.

[0084] (3) Add 100 μl of alkaline phosphatase-labeled mouse anti-human IgE secondary antibody solution, mix well, and incubate at 37°C for 15 min.

[0085] (4) Under the action of the external magnetic field, the magnetic particles settle down, the supernatant is removed, 500ul of cleaning solution is added, and after removing the magnetic field, the magnetic particles are shaken to resuspend; this cleaning step is repeated 3 times to remove unbound enzyme-labeled antibodies and other impurities.

[0086] (5) Add 150ul of enzymatic chemiluminescent substrate, remove the magnetic field, mix thoroughly, incubate at 37°C for 5 minutes, and measure the luminescence value. Calculate the content of shrimp allergen-specific IgE antibodies in the sample to be tested according to the luminescence intensity and the standard curve, in units of IU / ml;

[0087] Magnetic separation reagent: magnetic particle suspension labeled with allergens, the concentration of magnetic particles is 0.1-1.0 mg / mL, and the concentration of allergen or allergen mixture is 10 μg / mL. The labeled allergens are the white shrimp allergen solution prepared in Example 5, the Arctic sweet shrimp allergen solution prepared in Example 7, the small white shrimp allergen solution prepared in Example 8, and the shrimp allergen mixture prepared in Example 9 and Comparative Example 1.

[0088] Enzyme-labeled reagent: A solution of mouse anti-human IgE antibody labeled with alkaline phosphatase at a concentration of 0.5 to 2 μg / mL.

[0089] Luminescent substrate: AMPPD luminescent substrate.

[0090] The sera to be tested included 10 clinical human negative sera and 19 clinical human positive sera. The test results were compared with the Phadia test results to see if they were consistent. Phadia was used as the gold standard for in vitro allergen detection. The detection method was referred to the instruction manual. The specific IgE detection values ​​(IU / mL) calculated based on the relative luminescence intensity values ​​were shown in Tables 2 and 3.

[0091] Table 2

[0092]

[0093]

[0094] In Table 2, the specific IgE detection value of the nanomagnetic particle chemiluminescence detection and Phadia detection is judged as negative if it is less than 0.35 IU / mL, and it is positive if it is greater than or equal to 0.35 IU / mL.

[0095] Table 2 shows that there are 10 serum samples with negative phadia detection. After the three types of shrimp, such as sweet shrimp, white shrimp and white shrimp, were tested separately and mixed in different proportions, the negative coincidence rate of chemiluminescence platform detection and phadia detection was 100%; there are 19 serum samples with positive phadia detection. When tested with sweet shrimp allergen alone, 9 samples were missed and the positive coincidence rate was only 52.6%. However, from the results, the background value of this variety of allergens was very low, but the positive value was higher than that of other varieties of allergens. The ratio can be optimized for use later; when tested with white shrimp allergen alone, the positive coincidence rate was 94.7, and there was still a phenomenon of missed detection; when tested with white shrimp allergen alone, 8 samples were missed and the positive coincidence rate was 57.9%, which was complementary to the test results of white shrimp. When the three shrimp allergens were compounded together, the performance was consistent with the gold standard of phadia, and neither missed detection nor false positives could occur. When the Arctic sweet shrimp, small white shrimp and whiteleg shrimp were mixed in the ratio of 1:2:2 and 1:5:1, no false positives occurred, but missed detections still occurred. When the Arctic sweet shrimp, small white shrimp and whiteleg shrimp were mixed in the ratio of 1:7:2, the positive and negative coincidence rates were both 100% compared with the phadia gold standard.

[0096] Table 3

[0097]

[0098]

[0099] In Table 3, the specific IgE detection value of the nanomagnetic particle chemiluminescence detection and Phadia detection was judged to be negative if it was less than 0.35 IU / mL, and it was positive if it was greater than or equal to 0.35 IU / mL.

[0100] Table 3 shows that when the Arctic sweet shrimp was replaced by the mud shrimp, the mud shrimp was tested alone on the chemiluminescence platform, and 2 false positives and 8 missed detections occurred, and both the positive and negative coincidence rates were low. After the three shrimps were mixed together in different proportions, the situation improved, but there were still missed detections, which was inconsistent with the Phadia test results.

[0101] 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 shrimp allergen mixture, characterized in that: The method for preparing the shrimp allergen mixture comprises the following steps: (1) Degreasing with acetone, air drying, and crushing to obtain defatted shrimp powder; (2) preparing a shrimp allergen extract by mixing the defatted shrimp powder and the extract in a mass volume ratio of 1 g: (10 mL to 20 mL); (3) The shrimp allergen extract is dialyzed and microfiltered to obtain a shrimp allergen purified solution; (4) Using sweet prawns, white shrimps and whiteleg shrimps as raw materials, the sweet prawns allergen purified solution, white shrimps allergen purified solution and whiteleg shrimps allergen purified solution prepared according to steps (1) to (3) are mixed in a volume ratio of 1: (6-8): (1.5-3) to obtain the shrimp allergen mixture. The extract is a 0.05M Tris-HCl buffer solution containing 0.2M KCl and 0.5mM DTT, and the pH value of the extract is 8.

2. In step (3), a dialysis bag with a molecular weight cutoff of 3000-4000 Da and a 0.01 M PBS buffer with a pH value of 7.4 are used as the dialysis fluid, and the microfiltration uses a 0.25-0.45 μm filter membrane.

2. The shrimp allergen mixture according to claim 1, characterized in that: In step (4), the volume ratio of the purified liquid of the sweet shrimp allergen, the purified liquid of the small white shrimp allergen and the purified liquid of the white shrimp allergen is 1:7:

2.

3. The shrimp allergen mixture according to claim 1, characterized in that: In step (2), the extract is prepared by mixing the defatted shrimp powder and the extract, standing the mixture at 2-6°C for 1.5-2.5 hours, and then centrifuging the mixture at a speed of 8000-10000 r / min for 15-25 minutes to obtain the supernatant, which is the shrimp allergen extract.

4. The shrimp allergen mixture according to claim 1, characterized in that: In step (1), the muscle tissue of the shrimp is mixed with 2-6°C cold acetone at a ratio of 1 g: (8 mL-12 mL) for overnight extraction, during which the cold acetone is replaced 3-4 times, and then centrifuged at a speed of 3000-5000 r / min for 10-20 min, the precipitate is taken, and the precipitate is washed with 2-6°C cold acetone until the effluent is colorless, and the precipitate is air-dried and crushed to obtain the defatted shrimp powder.

5. Use of the shrimp allergen mixture according to any one of claims 1 to 4 in the preparation of a shrimp allergy detection reagent or detection kit.

Images