Sensitivity and specificity antigen preparation for allergy detection and preparation method thereof

Abstract

The invention discloses an antigen preparation with sensitivity and specificity for allergy detection and a preparation method of the antigen preparation. According to the method, serum from volunteers allergic to fish is used, an allergy test is carried out based on a micro-fluidic chip, magnetic beads and a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and allergen identification is carried out by comparing a Uniprot database with a proteomics server ExPASy. Crucian is used as an aquatic product model, and 12 common and rare allergens in the species are extracted from the developed preparation. In addition, results of in-vitro allergy experiments and specific parvalbumin ELISA detection show that the preparation can effectively reduce false negative and false positive in allergy detection, and the antigen preparation developed by the research is expected to play an important role in diagnosis of allergic diseases, especially in the aspect of prevention and treatment of fish allergic diseases. The preparations provide new prospects for special-effect, safe and convenient allergic disease tests.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an antigen preparation for comprehensive detection of fish allergy and a preparation method thereof. Background Art

[0002] Allergic diseases have been listed by the World Health Organization (WHO) as one of the three major diseases to be prioritized for prevention and treatment in the 21st century. It is estimated using data from 89 countries that more than 250 million people are affected globally. Common food allergies are mainly caused by milk, eggs, nuts, peanuts, soybeans, wheat, sesame, fish, and shellfish. Among them, fish is one of the most common foods causing allergic reactions in children and adults globally, and only a few cases of fish allergy tend to subside with age. It is reported that the number of deaths due to fish allergy accounts for 9% of the deaths from allergic reactions.

[0003] Currently, in the treatment of fish allergy, there is a need to solve the problem of incomplete recognition of fish allergens and the lack of component discrimination diagnosis of fish allergens in the clinical environment. Based on the fact that the research focus on fish allergens has been limited to parvalbumin, more research is needed to understand the involvement of other fish allergens, as well as several other strategies for allergen-specific immunotherapy (AIT), including peptide vaccines, DNA vaccines, mixed allergens, and the use of nanobodies with the ability to treat multiple allergens. The clinical diagnosis of fish allergy requires the use of whole extracts, and there is an urgent need to develop more sensitive fish antigen preparations for the diagnosis of fish allergy patients.

[0004] "Antigen preparation" refers to any substance component that can produce an antigenic or immunogenic response. Detailed sensitization characteristics can improve the effectiveness of immunotherapy and can be used in precision medicine. Currently, the main cost factor in allergic disease management is unrecognized and untreated allergic diseases. To ensure the best diagnostic and treatment effects, it is particularly important to develop antigen preparations containing as many allergens as possible.

[0005] Indonesian scientists Wijaya et al. prepared a skin prick test reagent for myofibrillar protein and sarcoplasmic protein of Scylla Serrata. The results showed that separating crab protein into myofibrillar and sarcoplasmic proteins for skin prick test could improve the sensitivity and specificity of allergy diagnosis. Polish scientists Cukrowska et al. conducted a randomized placebo-controlled trial on children with atopic dermatitis (AD) and cow's milk protein (CMP) allergy using a probiotic preparation containing a mixture of Lactobacillus rhamnosus O900, Lactobacillus rhamnosus O908, and Lactobacillus casei O918 strains. The research results showed that the mixture of probiotic strains provided benefits for children with AD and CMP allergies. Egyptian scientists Mohammed et al. extracted allergens from pollen, animal emissions, and mites to prepare a preparation for sublingual immunotherapy and analyzed it by storing it at 2 - 104 °C for 9 months. The results showed that the concentration of all prepared allergens (indicated by the concentration of protein content) decreased exponentially over time. The above research provided guidance for the effectiveness of protein preparations.

[0006] Currently, the most commonly used method is to use enzyme-linked immunosorbent assay (ELISA) to determine the content of allergens in target foods. Australian researchers Ruethers et al. used three commercially available ELISA kits to detect 57 species of teleost fish, and the detection rates ranged from 26% to 61%. The fish species with the highest detection rates included carp, cod, and salmon, indicating that the ability of fish ELISA kits to detect allergens in fish is limited. Currently, the application of LC-MS / MS in food allergen analysis is still relatively new, and complex separation, purification, and degradation are required during the protein identification process of LC-MS. To avoid these, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) is used to analyze the initially intact protein. MALDI-TOF MS is an analytical technique that measures it based on its mass-to-charge ratio (m / z) and intensity. Summary of the Invention

[0007] The purpose of the present invention is to develop an antigen preparation for comprehensive detection of fish allergy, aiming to provide a new prospect for effective, safe, and convenient allergy disease testing, and is expected to play an important role in the diagnosis of allergic diseases, especially in the prevention and treatment of fish allergic diseases.

[0008] In view of the above invention purpose, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method capable of eluting and collecting as many allergens as possible from a single species, and the method includes the following steps:

[0010] Step S1. Couple activated tosyl magnetic beads with mouse monoclonal anti-human IgE antibody;

[0011] Step S2. Couple the coupled magnetic beads with IgE in the pooled sera of multiple allergic patients;

[0012] Step S3. Fabricate a microfluidic chip channel made of polydimethylsiloxane (PDMS) by soft lithography technology as the channel for capturing allergens.

[0013] Step S4. Use crucian carp as an aquatic product model, and extract sarcoplasmic proteins from fish muscle for capturing crucian carp allergens.

[0014] Step S5. Perform allergen immunomagnetic separation in the microfluidic chip channel, and collect the eluted allergens.

[0015] Preferably, step S1 includes the following steps:

[0016] S11 Functionalize the magnetic beads according to the manufacturer's instructions. Take 2.5 μL of tosyl magnetic beads with a diameter of 1.5 μm (20 μg / μL) and put them into a centrifuge tube. Add coupling solution (0.1 M sodium borate buffer solution, pH 9.5) on a magnetic stand to wash and remove the supernatant, repeating three times. Add 2.5 μL of mouse monoclonal anti-human IgE antibody (2.73 mg / mL), 8.75 μL of 0.1 M sodium borate buffer solution, and 5.20 μL of 3 M anhydrous ammonium sulfate to the washed magnetic beads for coupling;

[0017] S12 After coupling, place it in an incubator for overnight incubation (12 h, 25 °C, 850 rpm). After 12 h, add 20 μL of blocking solution (5% (m / m) BSA, 10 mM PBS, 0.05% Tween 20, pH 7.4) and continue to incubate (6 h, 25 °C, 850 rpm). After the incubation, collect the blocked anti-IgE-coated magnetic bead microspheres as functionalized magnetic beads. Wash the obtained functionalized magnetic beads three times with PBST (10 mM PBS with 0.05% Tween 20 (v / v), pH 7.4) for standby.

[0018] Preferably, in step S2, serum samples from patients with moderate or above allergy levels are selected, and pooled serum samples from multiple allergic patients are selected. Step S2 includes the following steps: Dilute the serum sample 10-fold with PBST (10 mM PBS with 0.05% Tween 20 (v / v), pH 7.4). Take 13 μL of the diluted serum sample and add it to the functionalized magnetic beads, and place it on an incubator for incubation (850 rpm, 25 °C, 5 min). After incubation, remove the supernatant, and then wash three times with PBST buffer (PBS containing 0.05% Tween-20, 0.01 M, pH 7.4). Collect the washed antibody-coated magnetic beads for subsequent experimental steps.

[0019] Preferably, step S3 includes the following steps: Wash the channel with PBST buffer, repeat twice, wash once with deionized water, and pass through BSA solution (5% BSA (v / v), 10 mM PBS, 0.05% Tween 20, pH 7.4) to block the channel for 1 h.

[0020] Preferably, step 4 includes the following steps: Take 6 g of stored crucian carp muscle, add 40 mL of buffer (40 mM Tris-HCl containing 20 mM EDTA), and then homogenize (10 times / s, 10 min). Take the homogenized homogenate and centrifuge (9300 g, 10 min). After centrifugation, collect the supernatant for subsequent capture of crucian carp allergens.

[0021] Preferably, step S5 includes the following steps: Wash the blocked channel twice with PBST buffer and once with deionized water. Add 27 μL of crucian carp myoplasm protein solution to the IgE-coated magnetic beads and vortex to mix evenly. Pass the mixed solution into the microfluidic channel for incubation (850 rpm, 25 °C, 1 h). Then place the chip on a magnet for immunomagnetic separation, and pass 10 μL of 5% (v / v) acetic acid solution into each single channel to elute the captured protein and collect the eluate.

[0022] The second aspect of the present invention provides an allergy detection antigen preparation prepared by the above method: The eluate after successful elution is used as the antigen preparation, which contains multiple different types of allergens of crucian carp.

[0023] The third aspect of the present invention provides a method for establishing a database of crucian carp allergens based on a microfluidic chip and MALDI-TOF MS. The method includes the following steps:

[0024] Step 1: Digest and enzymatically hydrolyze the obtained antigen preparation with trypsin;

[0025] Step 2: Detect using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS);

[0026] Step 3: Compare the retrieved proteins with the UniProt database, use a proteomics server to assign the measured ion peaks, identify the allergens captured by the MBs, and establish a crucian carp allergen library.

[0027] Preferably, step 1 includes the following steps: Take 1 μL of the eluate collected in the above step, measure its absorbance at a wavelength of 280 nm in a ultra-micro spectrophotometer, repeat the measurement five times and take the average value, and substitute it into the regression equation a = 0.275ρB + 0.166 to calculate the relative concentration of the protein in the eluate, where a is the measured absorbance and ρB is the protein concentration (mg / mL). Add trypsin (protein / trypsin = 30:1, w / w) to the eluate for incubation (600 rpm, 37 °C, 1 h). After the incubation, perform digest analysis by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0028] Preferably, step 2 includes the following steps: After calibrating the MALDI-TOF MS instrument, take 1.5 μL of the eluate on a MALDI target plate (MSP 96 target plate) and wait for it to air-dry and deposit naturally. Subsequently, cover it with the matrix for analyzing proteins. Cover 1 μL of sinapic acid (SA) (15 mg / mL, acetonitrile / water / trifluoroacetic acid (TFA) = 500:499:1, v / v) on the eluate and calibration, and cover 1 μL of α-cyano-4-hydroxycinnamic acid (10 mg / mL, acetonitrile / water / trifluoroacetic acid (TFA) = 500:499:1, v / v) on the trypsin digest. Obtain a mass spectrum in the range of m / z 2000 - 20000 Da under positive ion mode and linear conditions. Use the automatic sample collection mode to sum the spectra generated by 500 laser irradiations, with a laser frequency of 20 Hz, a laser intensity of 65%, a detector gain of 10.3×, and at least 6 targets are set for each sample to obtain 6 mass spectra.

[0029] Preferably, the step 3 includes the following steps: retrieving proteins using the UniProt database (https: / / www.uniprot.org), assigning measured ion peaks, and identifying allergens captured by MBs. Performing mass spectrometry analysis on the protein and trypsin-digested mass spectrometry map using Bruker's FlexAnalysis software and the FindMod tool (https: / / expasy.org / ) provided by the Swiss Institute of Bioinformatics (SIB) in the Proteomics Server ExPASy (Expert Protein Analysis System). Identifying peaks by matching the experimentally measured peptide masses with the masses of theoretical peptides calculated from specific SwissProt / TrEMBL entries.

[0030] The fourth aspect of the present invention provides a database of crucian carp allergens established based on microfluidic chip and MALDI-TOF MS technology.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The present invention develops an antigen preparation for comprehensive detection of fish allergies and provides a method capable of eluting and collecting as many allergens as possible in a single species.

[0033] 2. The antigen preparation developed in this study is expected to play an important role in the diagnosis of allergic diseases, especially in the prevention and treatment of fish allergic diseases.

[0034] 3. The present invention provides a method for establishing a database of crucian carp allergens based on microfluidic chip and MALDI-TOF MS, and establishes its allergen database, providing more comprehensive allergen information for fish allergy detection.

[0035] 4. The antigen preparation developed by the present invention provides a new prospect for specific, safe and convenient allergy disease testing. Description of the Drawings

[0036] Figure 1 The average mass spectrometry map (A) of 6 representative results obtained by performing allergy tests on serum samples of 5 fish-allergic patients. The average spectrum map (B) of 6 representative MALDI-TOF MS results obtained after enzymatic digestion of the allergen eluate obtained in the allergy test.

[0037] Figure 2 The parvalbumin standard curve obtained by determination using a specific parvalbumin ELISA kit (96T, Oumarsi Biotechnology Co., Ltd., Shanghai, China).

[0038] Figure 3 The representative MALDI-TOF MS spectra obtained from in vitro allergen-specific IgE tests using serum samples from patients with IgE-mediated food allergies. Sample 6 and Sample 7 are sera from patients with IgE-mediated fish allergy, and Sample 8 and Sample 9 are sera from patients with IgE-mediated mango and crab allergies. The pooled sample is the pooled serum of 4 normal individuals.

[0039] Figure 4 Representative MALDI-TOF mass spectra obtained from three independent experiments. Detailed implementation mode

[0040] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0041] Example 1: Preparation of crucian carp myosin solution

[0042] The extraction steps of myosin are as follows:

[0043] 1. Take 6 g of stored crucian carp muscle, add 40 mL of buffer (40 mM Tris-HCl containing 20 mM EDTA), and homogenize (10 times / s, 10 min).

[0044] 2. Centrifuge the homogenized homogenate (9300 g, 10 min). After centrifugation, collect the supernatant, which is the fish myosin solution and is used for subsequent experimental steps.

[0045] Example 2: Functionalization of magnetic beads

[0046] 1. Take 2.5 μL of tosyl magnetic beads with a diameter of 1.5 μm, add coupling solution (0.1 M sodium borate buffer, pH 9.5), wash and then remove the supernatant, and repeat the washing three times.

[0047] 2. Add 2.5 μL of mouse monoclonal anti-human IgE antibody, 10 μL of 0.1 M sodium borate buffer, and 5 μL of 3 M anhydrous ammonium sulfate to the washed magnetic beads for coupling, and incubate overnight at a constant temperature (12 h, 25 °C, 850 rpm).

[0048] 3. After 12 h, add 20 μL of blocking solution (5% (m / m) BSA, 10 mM PBS, 0.05% Tween 20, pH 7.4) and continue to incubate (6 h, 25 °C, 850 rpm).

[0049] 4. After the cultivation, collect the well-sealed anti-IgE-coated magnetic beads as functionalized magnetic beads. Wash the functionalized magnetic beads three times with PBST for standby.

[0050] Example 3: Coupling of IgE antibody in serum by functionalized magnetic beads

[0051] 1. Screen serum samples from different allergic patients and group and number them. The screened sera are as follows: Degree of fish allergy: moderate allergy (serum of patient 1), severe allergy (serum of patient 2), severe allergy (serum of patient 3), moderate allergy (serum of patient 4), mild allergy (serum of patient 5).

[0052] 2. Dilute the serum samples 10 times with PBST. Take 13 μL of the diluted serum sample and add it to the functionalized magnetic beads and incubate (850 rpm, 25 °C, 5 min).

[0053] 3. After the cultivation, remove the supernatant and wash three times with PBST buffer. Collect the magnetic beads conjugated with IgE antibody for subsequent experimental steps.

[0054] Example 4: Immunomagnetic separation of allergens in the microfluidic chip channel

[0055] 1. Pretreatment of the microfluidic chip: A microfluidic chip channel made of polydimethylsiloxane (PDMS) prepared by soft lithography technology. The channel is a straight channel with an internal length of 3 cm and a cross-section of 200 μm × 200 μm, and its bottom is combined with a glass substrate to form a seal. Pass PBST buffer through the channel for washing, repeat twice, and wash once with deionized water. Pass BSA solution to block the channel for 1 h.

[0056] 2. Immunomagnetic separation and capture of allergens in the chip: Wash the sealed channel twice with PBST buffer and once with deionized water. Add 27 μL of myosin solution to the IgE-coated magnetic beads and shake well to mix evenly. Pass the mixed solution into the microfluidic channel for incubation (850 rpm, 25 °C, 1 h). Then place the chip on a magnet for immunomagnetic separation, and pass 10 μL of 5% (v / v) acetic acid solution into each single channel to elute the captured protein, and collect the eluate for digestion.

[0057] Example 5: Preparation of antigen preparation

[0058] The captured allergens are eluted through 5% HAc in the microfluidic chip channel, and the eluate after successful elution is collected as the antigen preparation.

[0059] Example 6: MALDI-TOF MS mass spectrometry test of antigen preparation

[0060] 1. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments were performed using a Bruker Microflex LRF mass spectrometer. Cytochrome C and myoglobin were used, and 1 mL of deionized water was added and mixed evenly to prepare a calibration solution. The MALDI-TOF MS instrument was calibrated using the double-charged and single-charged ions therein.

[0061] 2. 1.5 μL of the eluate was taken on a MALDI target plate (MSP 96 target plate) and waited to air-dry and deposit naturally. Subsequently, it was covered with the matrix sinapic acid (SA) for analyzing proteins, and a mass spectrum in the range of m / z 2000 - 20000 Da was obtained under positive ion mode and linear conditions.

[0062] 3. The spectra generated by 500 laser irradiations were summed using the automatic sample collection mode. The laser frequency was 20 Hz, the laser intensity was 65%, the detector gain was 10.3×, and at least 6 target points were set for each sample to obtain 6 mass spectra. The average mass spectrum obtained is as Figure 1 (A) shown.

[0063] Example 7: Enzymatic digestion and identification of allergens

[0064] 1. 1 μL of the eluate collected in the above steps was taken, and its absorbance at a wavelength of 280 nm was measured in an ultra-micro spectrophotometer, and the measurement was repeated five times and the average value was taken.

[0065] 2. Substitute into the regression equation a = 0.275ρB + 0.166 to calculate the relative concentration of the protein in the eluate, where a is the measured absorbance and ρB is the protein concentration (mg / mL). Trypsin (protein / trypsin = 30:1, w / w) was added to the eluate for incubation (600 rpm, 37 °C, 1 h).

[0066] 3. After the incubation, the digest was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The average mass spectrum of the enzymatic fingerprint peptide segments obtained is as Figure 1 (B) shown.

[0067] 4. Use the UniProt database (https: / / www.uniprot.org) to retrieve proteins, assign measured ion peaks, and identify allergens captured by MBs. Perform mass spectrometry analysis on the protein and trypsin-digested mass spectrometry spectra using Bruker's FlexAnalysis software and the FindMod tool (https: / / expasy.org / ) provided by the Swiss Institute of Bioinformatics (SIB) on the Proteomics Server ExPASy (Expert Protein Analysis System). Identify peaks by matching the experimentally measured peptide masses with the masses of theoretical peptides calculated from specific Swiss Prot / TrEMBL entries.

[0068] Example 8: Establish a crucian carp allergen database

[0069] Potential allergens identified from the average mass spectrometry and peptide fingerprint spectra are used as almost all potential allergens representing the species, and an allergen library is established based on this. The specific allergen information is shown in Table 1. Among them, the peak resolution of all mass spectra used for averaging is greater than 10,000, and the mass accuracy is greater than 1‰. The coverage rate of the peptide segments in the established allergen library is calculated with reference to the entire amino acid sequence of the protein in the Uniprot database.

[0070] Table 1 Crucian carp allergen database and peptide segment coverage rate

[0071]

[0072]

[0073] Example 9: ELISA test for allergenic proteins

[0074] Send the collected antigen preparation to the testing unit for enzyme-linked immunosorbent assay. Determine the parvalbumin content in the antigen preparation according to the instructions of the specific parvalbumin ELISA kit (96T, Oumarsi Biotechnology Co., Ltd., Shanghai, China). The measured parvalbumin standard curve is as Figure 2 shown, where the detected parvalbumin concentration in the antigen preparation is 5.4 μg / mL, equivalent to an equivalent content of 27 mg / kg in muscle.

[0075] Example 10: In vitro allergy detection

[0076] The specific steps for in vitro allergy detection are as follows:

[0077] 1. Screen the serum samples of different volunteers and group them for numbering. The screened sera are as follows: Degree of fish allergy: moderate allergy (test serum 6), severe allergy (test serum 7).

[0078] Degree of non - fish allergy: mango allergy (test serum 8), crab allergy (test serum 9), normal person (test sera 10 - 13).

[0079] 2. Use the above sera for in vitro allergy testing. The eluates obtained from the allergy testing are subjected to MALDI - TOF MS detection and compared with the established database. The results obtained are as Figure 3 shown.

[0080] 3. From Figure 3 the results, it can be seen that the prepared antigen preparation can detect the serum allergy status of fish - allergic patients. Compared with the control group and the blank group, it shows the sensitivity and effectiveness of the antigen preparation, and can greatly reduce the false negative rate and false positive rate.

[0081] Example 11: Reproducibility test

[0082] The specific steps of the reproducibility test are as follows:

[0083] 1. According to Examples 1 - 6, use the same serum samples to conduct 3 repeated experiments and compare the representative results of 3 independent experiments. The results obtained are as Figure 4 shown.

[0084] 2. Compare the mass spectra obtained from three independent experimental runs. The similarity scores calculated are 0.9230 (Run 1 vs. Run 2), 0.9263 (Run 1 vs. Run 3), and 0.9053 (Run 2 vs. Run 3). The average value is 0.91 ± 0.01 (RSD 1.1%), indicating that the method has good reproducibility for identifying potential allergens in fish muscle.

[0085] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above - mentioned embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method capable of eluting and collecting as many allergens as possible from a single species, characterized in that: The method comprises the following steps: Step S1. using activated tosyl magnetic beads coupled with mouse monoclonal anti-human IgE antibody; Step S2. The coupled magnetic beads are then combined with IgE in the combined serum of multiple allergic patients; Step S3. Using soft lithography technology to prepare a microfluidic chip channel made of polydimethylsiloxane (PDMS) as a channel for capturing allergens. Step S4. Using crucian carp as a model of aquatic products, extracting sarcoplasmic protein from fish muscle for capturing crucian carp allergens. Step S5. Perform allergen immunomagnetic separation in the microfluidic chip channel and collect the eluted allergens.

2. A method according to claim 1 that can elute and collect as many allergens as possible from a single species, characterized in that: The step S1 comprises the following steps: S11 Functionalization of magnetic beads was performed according to the manufacturer's instructions. Pick 2.5 μL of 1.5 μm diameter tosyl magnetic beads (20 μg / μL) were placed in a centrifuge tube, and the coupling solution (0.1 M sodium borate buffer solution, pH 9.5) was added to the magnetic stand to elute and remove the supernatant, and repeated three times. 2.5 μL of mouse monoclonal anti-human IgE antibody (2.73 mg / mL), 8.75 μL of 0.1 M sodium borate buffer solution and 5.20 μL of 3 M anhydrous ammonium sulfate were added to the washed magnetic beads for coupling; After S12 coupling, place in an incubator for constant temperature overnight culture (12h, 25℃, 850rpm). After 12h, add 20μL of blocking solution (5% (m / m) BSA, 10mM PBS, 0.05% Tween 20, pH 7.4) and continue to culture (6h, 25℃, 850rpm). After the culture is completed, collect the blocked anti-IgE coated magnetic beads as functionalized magnetic beads. Wash the obtained functionalized magnetic beads three times with PBST (10mM PBS with 0.05% Tween 20 (v / v), pH 7.4) for standby use.

3. A method according to claim 1 capable of eluting and collecting as many allergens as possible from a single species, characterized in that: The step S2 uses serum samples from patients with moderate or above allergic degree, and the step S2 uses combined serum samples from multiple allergic patients. The step S2 includes the following steps: diluting the serum sample 10 times with PBST (10mM PBS with 0.05% Tween 20 (v / v), pH 7.4), taking 13μL of the diluted serum sample and adding it to the functionalized magnetic beads and placing it on a culture machine for culture (850rpm, 25℃, 5min). After the culture is completed, the supernatant is removed, and then washed three times with PBST buffer (PBS containing 0.05% Tween-20, 0.01M, pH 7.4). Collect the washed antibody-coated magnetic beads for subsequent experimental steps.

4. A method according to claim 1 capable of eluting and collecting as many allergens as possible from a single species, characterized in that: The step S3 comprises the following steps: passing PBST buffer into the channel for washing, repeating twice, washing once with deionized water, passing BSA solution (5% BSA (v / v), 10 mM PBS, 0.05% Tween 20, pH 7.4) into the channel to block the channel for 1 hour.

5. A method for eluting and collecting as many allergens as possible from a single species according to claim 1, characterized in that: Step 4 comprises the following steps: taking 6 g of stored crucian carp muscle, adding 40 mL of buffer (40 mM Trsi-HCl containing 20 mM EDTA) and then homogenizing (10 times / s, 10 min). Taking the homogenate and centrifuging (9300 g, 10 min). After the centrifugation, the supernatant is collected for subsequent capture of crucian carp allergens.

6. A method for eluting and collecting as many allergens as possible from a single species according to claim 1, characterized in that: The step S5 includes the following steps: the closed channel is washed twice with PBST buffer and once with deionized water. 27 μL of crucian carp myoplasmic protein solution is added to the IgE-coated magnetic beads and vortexed to mix evenly. The mixed solution is passed into the microfluidic channel for incubation (850 rpm, 25° C., 1 h). The chip is then placed on a magnet for immunomagnetic separation, and 10 μL of 5% (v / v) acetic acid solution is passed into each channel to elute the captured protein and collect the eluate.

7. An allergy detection antigen preparation obtained by the method of claim 2 capable of eluting and collecting as many allergens as possible from a single species, characterized in that: The eluate after successful elution will be collected as an antigen preparation, which contains multiple different types of crucian carp allergens.

8. A method for eluting and collecting as many allergens as possible from a single species as claimed in claim 1, wherein the method is based on a microfluidic chip and MALDI-TOF MS to establish a database of crucian carp allergens, characterized in that: The method comprises the following steps: Step 1, digesting the obtained antigen preparation with trypsin; Step 2, detecting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS); Step 3: Retrieve proteins by comparing with the UniProt database, assign measured ion peaks using a proteomics server, identify allergens captured by MBs and establish a crucian carp allergen library.

9. The method for establishing a crucian carp allergen database based on microfluidic chip and MALDI-TOF MS according to claim 8, characterized in that: The step 1 includes the following steps: taking 1 μL of the eluate collected in the above step, measuring its absorbance at a wavelength of 280 nm in an ultra-micro spectrophotometer, repeating the measurement five times to obtain an average value, substituting into the regression equation a=0.275ρB+0.166 to calculate the relative concentration of protein in the eluate, where a is the measured absorbance and ρB is the protein concentration (mg / mL). Adding trypsin (protein / trypsin=30:1, w / w) to the eluate for incubation (600 rpm, 37°C, 1h). After the incubation, the digestion product is analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

10. The method for establishing a crucian carp allergen database based on microfluidic chip and MALDI-TOF MS according to claim 8, characterized in that: The step 2 includes the following steps: after calibrating the MALDI-TOF MS instrument, take 1.5 μL of the eluent onto a MALDI target plate (MSP 96 target plate) and wait for it to air dry and deposit naturally. Then, the matrix for analyzing the protein is used for overlaying, and 1 μL of sinapinic acid (SA) (15 mg / mL, acetonitrile / water / trifluoroacetic acid (TFA) = 500:499:1, v / v) is overlaid on the eluent and calibration, and 1 μL of α-cyano-4-hydroxycinnamic acid (10 mg / mL, acetonitrile / water / trifluoroacetic acid (TFA) = 500:499:1, v / v) is overlaid on the trypsin digest. A mass spectrum with an m / z range of 2000-20000 Da is obtained under positive ion mode and linear conditions. The spectra generated by 500 laser irradiations were summed using the automatic sample acquisition mode, with a laser frequency of 20 Hz, a laser intensity of 65%, a detector gain of 10.3×, and at least 6 target points were set for each sample to obtain 6 mass spectra.

11. The method for establishing a crucian carp allergen database based on microfluidic chip and MALDI-TOF MS according to claim 8, characterized in that: Step 3 includes the following steps: Searching for proteins using the UniProt database (https: / / www.uniprot.org), assigning measured ion peaks, and identifying allergens captured by MBs. Mass spectrometry analysis of proteins and trypsin digest mass spectra was performed using Bruker's FlexAnalysis software and the FindMod tool (https: / / expasy.org / ) provided by the Swiss Institute of Bioinformatics (SIB) in the proteomics server ExPASy (Expert Protein Analysis System). Peak identification was performed by matching the experimentally measured peptide masses with the masses of theoretical peptides calculated from specific SwissProt / TrEMBL entries.

12. A method as claimed in claim 1 capable of eluting and collecting as many allergens as possible from a single species, wherein the database of crucian carp allergens is established based on microfluidic chips and MALDI-TOF MS technology.

Images