Method for detecting zearalenone, aflatoxin and vomitoxin based on xMAP technology

Abstract

The invention belongs to the technical field of mould detection, and discloses a zearalenone, aflatoxin and vomitoxin detection method based on an xMAP technology, and the specific steps are as follows: S1, establishment of an antigen coating of the zearalenone xMAP detection method: coating a purified antigen with magnetic beads by using # imgabs0 # Antibody Coupling Kit; by utilizing the multi-labeling characteristic of the fluorescent encoding microspheres, multiple target molecules can be simultaneously identified in a single reaction, so that the sample collection quantity and processing steps are greatly reduced, and the tedious process of repeated experiments in a traditional method is avoided; by optimizing key parameters such as antigen coating conditions, working concentration of detection antibodies and signal amplification antibodies and the like, the sensitivity is remarkably improved on the premise of ensuring detection specificity, and meanwhile, a competitive immune test mode is combined with a high-throughput analysis instrument, so that single-time detection time is greatly shortened, the use amount of samples is extremely small, complex pretreatment is not needed, and the detection sensitivity is greatly improved. The operation process is standardized, simple and fast, and special equipment maintenance is not needed.

Description

Technical Field

[0001] The present invention belongs to the technical field of mold detection, and specifically provides a method for detecting zearalenone, aflatoxin and vomitoxin based on xMAP technology. Background Art

[0002] Mycotoxins are commonly found in feed and feed raw materials. They are a series of toxic secondary metabolites with a wide range of chemical structures produced by molds during their growth and reproduction. They have a wide range of toxic effects on humans and animals, causing mycotoxin contamination of animal feed, feed raw materials and human food, and can cause various symptoms such as liver toxicity and cancer in humans and animals.

[0003] Mycotoxins are commonly found in feed and feed ingredients, and the harm they cause has received global attention. Mycotoxins can enter the human food chain through contaminated grains, feed, and animal foods (milk, meat, eggs) provided by animals fed these feeds, ultimately endangering human health. Aflatoxin (AFB1), zearalenone (ZEM), and deoxynivalenol (DON) are three of the most widely studied mycotoxins and the most common toxins in contamination. Mycotoxins can pose varying degrees of threat to animal and human health at varying doses. Therefore, the detection of mycotoxins and toxin-producing molds is crucial. Currently, commonly used mycotoxin detection methods include thin-layer chromatography, liquid chromatography, and immunochemical analysis. These methods are mainly based on the physical and chemical properties of mycotoxins and cannot detect high-risk samples that have been contaminated by toxin-producing fungi but have not yet produced toxins. Therefore, the establishment of mycotoxin detection methods is crucial to preventing mycotoxin contamination and reducing toxin poisoning incidents. Methods for quickly and accurately detecting mycotoxin content are the prerequisite for effectively preventing and controlling mold contamination in feed. Summary of the Invention

[0004] The object of the present invention is to provide a method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting zearalenone, aflatoxins, and vomitoxin based on xMAP technology, the specific steps of which are as follows:

[0006] S1. Establishment of the xMAP Detection Method for Zearalenone

[0007] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen with magnetic beads and perform the specific operations according to the instructions.

[0008] S1.1. Validation of protein-coupled microspheres

[0009] Select the microspheres coupled to the zearalenone antigen, oscillate and sonicate for about 20 seconds to resuspend the microspheres, then dilute the coupled microspheres with detection buffer to a final concentration of 50 microspheres / μL. At the same time, prepare a 4μg / mL IgG detection anti-antibody solution labeled with phycoerythrin with detection buffer, and dilute the detection antibody solution to 0.0625μg / mL at a dilution ratio of 1:2; then, prepare two columns of 50μL of microsphere solution and relative antibody in each well on a 96-well plate, add 50μL of reaction buffer to the well containing the microsphere solution as a blank control, and then add 50μL of diluted IgG detection anti-antibody solution to each well, and use Gently pipette the solution in the wells several times to mix the solution; cover the 96-well plate, place it on a shaker, and incubate at room temperature for 30 minutes; after the incubation is complete, place the 96-well plate on the Luminex magnetic separation plate, quickly and forcefully flip the plate, and pour the solution in the wells into the biowaste bin. Next, use a pipette to aspirate 100 μL of reaction buffer into each reaction well, gently aspirate and release it several times, and then discard the liquid in the well using the same method. Repeat this washing step twice; finally, use a pipette to aspirate 100 μL of reaction buffer, gently aspirate and release it several times to resuspend the microspheres, and use the Luminex instrument to read 50-75 μL of the reaction results according to the system manual;

[0010] S1.2. Zearalenone xMAP test (competitive immunoassay)

[0011] Select the appropriate microsphere group that has been coupled to the antigen, vortex ultrasonic vibration for 20 seconds to resuspend the microspheres, then use PBS-TBN buffer to dilute the coupled microsphere group to a final concentration of 100 microspheres / species / μL of detection microsphere mixture, and use PBS-TBN buffer to dilute the competitive molecule to [IC70] or [IC80]; then add 25μL of PBS-TBN buffer to the background well, add 25μL of standard or sample to the corresponding well, add 25μL of diluted competitive molecule to each well, add 25μL of diluted corresponding antibody to each well, use a multichannel pipette to gently pipette several times to mix the reaction liquid, add 25μL of reaction microsphere mixture to each well, and use a multichannel pipette to gently pipette several times to mix the reaction liquid; cover the plate and use 8 00rpm shaking, incubate at room temperature for 60 minutes; after incubation, add 25ul diluted biotinylated anti-mouse IgG secondary antibody and incubate at room temperature for 60 minutes; use PBS-TBN buffer to dilute the SAPE reporter molecule to 4μg / mL, add 25μL diluted SAPE to each well, and then use a multichannel pipette to gently pipette several times to mix the reaction liquid, then cover the plate, shake at 800rpm, incubate at room temperature for 30 minutes, add 100μL of washing buffer (PBSTBN) to each well, do not stir the microspheres, and then repeat the multichannel pipette to gently pipette several times to mix the reaction liquid, wash twice in total, and finally add a final volume of 100μL reaction buffer to each well, and analyze 50-75μL of reaction solution using the Luminex instrument according to the operating instructions;

[0012] S1.3. Determination of the optimal coating concentration of zearalenone antigen

[0013] The antigen was diluted to 1, 3, and 5 μg / 1x10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000;

[0014] S1.4. Optimization of the amount of detection antibody

[0015] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0016] S1.5. Determination of SA-PE Antibody Working Concentration

[0017] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0018] S1.6 Specificity Test

[0019] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0020] S1.7 Sensitivity test

[0021] The standard zearalenone antigen (original concentration 6.8 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control.

[0022] S1.8. Determination of the optimal amount of coating protein

[0023] The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescent coded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of the three antigen amounts all reached above 10,000, indicating that the three coupled antibody amounts were all suitable. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 1ug;

[0024] S1.9. Determination of the optimal amount of antibody added

[0025] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0026] S1.10. Determination of the optimal SA-PE antibody working concentration

[0027] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0028] S1.11 Specificity test

[0029] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method. The results showed that, except for zearalenone, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0030] S1.12 Sensitivity test

[0031] The sensitivity test of diluted serum was performed using the established xMAP method;

[0032] S2. Establishment of xMAP detection method for aflatoxin B1

[0033] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen with magnetic beads. The specific operation method is the same as step S1.

[0034] S2.1. Validation of protein-coupled microspheres

[0035] Select microspheres coupled with aflatoxin B1 antigen, and the specific steps are the same as step S1.1;

[0036] S2.2. Aflatoxin B1 xMAP test (competitive immunoassay)

[0037] Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2;

[0038] S2.3. Determination of the optimal coating concentration of aflatoxin B1 antigen

[0039] Antigen was diluted to 1, 3, and 5 μg / 1 x 10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000;

[0040] S2.4. Optimization of the amount of detection antibody

[0041] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0042] S2.5. Determination of SA-PE Antibody Working Concentration

[0043] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0044] S2.6 Specificity Test

[0045] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0046] S2.7 Sensitivity test

[0047] The standard aflatoxin B1 antigen (original concentration 4.6 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The diluted serum was subjected to sensitivity testing using the established xMAP method, and PBS was used as a blank control.

[0048] S2.8. Determination of the optimal amount of coating protein

[0049] The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescence-encoded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of 3μg and 5μg antigen amounts reached above 10,000, indicating that all three coupled antibody amounts are suitable. Considering various factors, the optimal coupled antibody amount for 1×106 fluorescence-encoded microspheres is 3ug.

[0050] S2.9. Determination of the optimal amount of antibody added

[0051] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0052] S2.10. Determination of the optimal SA-PE antibody working concentration

[0053] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0054] S2.11 Specificity Test

[0055] The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for aflatoxin B1, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0056] S2.12 Sensitivity test

[0057] The sensitivity test of diluted serum was performed using the established xMAP method;

[0058] S3. Establishment of the xMAP Detection Method for Deoxynivalenol

[0059] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen onto magnetic beads. The specific operation is the same as step S1.

[0060] S3.1. Validation of Protein-Coupled Microspheres

[0061] Select microspheres coupled with vomitoxin antigen, and the specific steps are the same as step S1.1;

[0062] S3.2. DON xMAP test (competitive immunoassay)

[0063] Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2;

[0064] S3.3 Determination of the optimal coating concentration of vomitoxin antigen

[0065] Dilute the antigen and select 1, 3, and 5 μg / 1x 106 magnetic beads of recombinant antigen to coat the magnetic beads for protein coating verification. The effective coating concentration is when the MFI value reaches more than 10,000.

[0066] S3.4. Optimization of the amount of detection antibody

[0067] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0068] S3.5 Determination of SA-PE Antibody Working Concentration

[0069] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0070] S3.6 Specificity test

[0071] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0072] S3.7. Sensitivity test

[0073] The standard vomitoxin antigen (original concentration 8.2 mg / ml) was diluted 10-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control.

[0074] S3.8. Determination of the optimal amount of coating protein

[0075] The results showed that the MFI value increased with the increase of the amount of antibody. However, when the secondary antibody reached saturation, the MFI value of only 5 μg antigen reached more than 10,000, indicating that the amount of the three coupled antibodies can be used. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 5ug;

[0076] S3.9. Determination of the optimal amount of antibody added

[0077] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0078] S3.10. Determination of the Optimal SA-PE Antibody Working Concentration

[0079] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0080] S3.11 Specificity test

[0081] The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for vomitoxin, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0082] S3.12 Sensitivity test

[0083] The established xMAP method was used to perform sensitivity testing on diluted serum.

[0084] Preferably, the specific operation method of antigen coating in S1 is as follows: first, select magnetic beads and record the code of the laboratory magnetic beads, then vortex and sonicate for 20 seconds each to suspend the magnetic beads, then transfer 5.0x 106 magnetic beads to a USA Scientific microcentrifuge low binding rate test tube, and centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads; after removing the supernatant, add 100μl double distilled water, vortex and sonicate for 20 seconds each to suspend, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, and use 80μL activation buffer (100mM pH 6.2 sodium dihydrogen phosphate) by vortexing and sonicating for 20 seconds each to suspend; then, 10 μl of 50 mg / mL sulfosuccinimide (Sulfo-NHS) and 10 μl of 50 mg / mL dicarbodiimide (EDC) were added to the suspended magnetic beads, and the reaction volume was 100 μl; protected from light, incubated at room temperature with gentle shaking for 20 minutes, and centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads; the supernatant was removed and 250 μl of coupling buffer (50 mM, pH 7.0) was used. The beads were suspended by vortexing and sonicating for 20 seconds each with 5.0 MES buffer, and then centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads. After that, the beads were suspended with MES buffer of the same concentration and washed by centrifugation twice. After vortexing and sonicating for 20 seconds each with 100 μl of MES buffer of the same concentration, 1, 3, and 5 μg of antigen were added to the suspended magnetic beads to a total volume of 500 μl. After vortexing, the beads were incubated at room temperature for 2 hours with shaking and protected from light. After incubation, the beads were centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads. The supernatant was removed. First, vortex and ultrasonically suspend with 500 μL of PBS-TBN buffer for 20 seconds each, then centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, vortex and ultrasonically suspend with 1000 μL of PBS-TBN buffer for 20 seconds each, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, finally remove the supernatant, vortex and ultrasonically suspend with 1000 μL of PBS, 10% BSA, 0.05% sodium azide, pH7.4 mixed buffer for 20 seconds each, and store the coupled microspheres in a refrigerator at 2-8°C away from light.

[0085] Preferably, when using PBS-TBN buffer to dilute the coupled microsphere group into a detection microsphere mixture with a final concentration of 100 microspheres / species / μL as described in S1.2, it should be noted that each reaction requires 25 μL of the detection microsphere mixture.

[0086] Preferably, when the reaction background is high as described in S1.2, a magnetic plate can be used to carefully remove the supernatant by manual flipping or pipetting on a magnetic plate washer, taking care not to disturb the microspheres.

[0087] Preferably, as described in S1.12, the standard zearalenone antigen (original concentration is 6.8 mg / ml) is diluted 100-fold, followed by a 2-fold serial dilution, and the sensitivity of the diluted serum is tested using the established xMAP method. The results show that the minimum detection line of this method is 0.0664 ug / ml.

[0088] Preferably, as described in S2.12, the standard aflatoxin B1 antigen (original concentration is 4.6 mg / ml) is diluted 100-fold, followed by a 2-fold serial dilution, and the sensitivity test of the diluted serum is performed using the established xMAP method. The results show that the minimum detection line of this method is 0.18 ug / ml.

[0089] Preferably, as described in S3.12, the standard vomitoxin antigen (original concentration is 8.2 mg / ml) is diluted 10-fold, followed by a 2-fold serial dilution, and the diluted serum is subjected to sensitivity testing using the established xMAP method. The results showed that the minimum detection limit of this method is 102.5 ug / ml.

[0090] Preferably, the detection antibody is diluted into five concentration groups as described in S1.9, S2.9 and S3.9, namely 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml and 8ug / ml.

[0091] Preferably, the SA-PE antibody is diluted into four concentration groups as described in S1.10, S2.10 and S3.10, namely 1 ug / ml, 2 ug / ml, 4 ug / ml and 8 ug / ml.

[0092] The beneficial effects of the present invention are as follows:

[0093] By utilizing the multiple labeling characteristics of fluorescently encoded microspheres, multiple target molecules can be identified simultaneously in a single reaction, greatly reducing the amount of sample collection and processing steps, and avoiding the tedious process of repeated experiments required by traditional methods. By optimizing key parameters such as antigen coating conditions, working concentrations of detection antibodies and signal amplification antibodies, the sensitivity is significantly improved while ensuring detection specificity. At the same time, the competitive immunoassay mode is combined with high-throughput analytical instruments to greatly shorten the time of a single detection, use very little sample and do not require complex pre-treatment. The operation process is standardized, simple and fast, and no special equipment maintenance is required. The overall cost is significantly lower than traditional methods, providing an efficient and economical solution for food safety testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Schematic diagram of the verification results of the zearalenone antigen coupling efficiency of the present invention;

[0095] Figure 2 This is a schematic diagram of determining the optimal antibody concentration for detecting zearalenone of the present invention;

[0096] Figure 3 This is a schematic diagram showing the structure of determining the optimal SA-PE antibody working concentration for zearalenone of the present invention;

[0097] Figure 4 Schematic diagram of the zearalenone xMAP detection specificity test of the present invention;

[0098] Figure 5 Schematic diagram of the verification results of the aflatoxin B1 antigen coupling efficiency of the present invention;

[0099] Figure 6 This is a schematic diagram of determining the optimal antibody concentration for detecting aflatoxin B1 according to the present invention;

[0100] Figure 7 This is a schematic diagram for determining the optimal SA-PE antibody working concentration for aflatoxin B1 of the present invention;

[0101] Figure 8 Schematic diagram of the aflatoxin B1 xMAP detection specificity test of the present invention;

[0102] Figure 9 Schematic diagram of the verification results of the coupling efficiency of vomitoxin antigen of the present invention;

[0103] Figure 10 This is a schematic diagram for determining the optimal antibody concentration for detecting vomitoxin according to the present invention;

[0104] Figure 11 This is a schematic diagram for determining the optimal SA-PE antibody working concentration for vomitoxin of the present invention;

[0105] Figure 12 Schematic diagram of the specificity test for detecting vomitoxin xMAP of the present invention. DETAILED DESCRIPTION

[0106] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0107] like Figures 1 to 12 As shown, the embodiment of the present invention provides a method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology, and the specific steps are as follows:

[0108] S1. Establishment of the xMAP Detection Method for Zearalenone

[0109] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen with magnetic beads and perform the specific operations according to the instructions.

[0110] S1.1. Validation of protein-coupled microspheres

[0111] Select the microspheres coupled to the zearalenone antigen, oscillate and sonicate for about 20 seconds to resuspend the microspheres, then dilute the coupled microspheres with detection buffer to a final concentration of 50 microspheres / μL. At the same time, prepare a 4μg / mL IgG detection anti-antibody solution labeled with phycoerythrin with detection buffer, and dilute the detection antibody solution to 0.0625μg / mL at a dilution ratio of 1:2; then, prepare two columns of 50μL of microsphere solution and relative antibody in each well on a 96-well plate, add 50μL of reaction buffer to the well containing the microsphere solution as a blank control, and then add 50μL of diluted IgG detection anti-antibody solution to each well, and use Gently pipette the solution in the wells several times to mix the solution; cover the 96-well plate, place it on a shaker, and incubate at room temperature for 30 minutes; after the incubation is complete, place the 96-well plate on the Luminex magnetic separation plate, quickly and forcefully flip the plate, and pour the solution in the wells into the biowaste bin. Next, use a pipette to aspirate 100 μL of reaction buffer into each reaction well, gently aspirate and release it several times, and then discard the liquid in the well using the same method. Repeat this washing step twice; finally, use a pipette to aspirate 100 μL of reaction buffer, gently aspirate and release it several times to resuspend the microspheres, and use the Luminex instrument to read 50-75 μL of the reaction results according to the system manual;

[0112] S1.2. Zearalenone xMAP test (competitive immunoassay)

[0113] Select the appropriate microsphere group that has been coupled to the antigen, vortex ultrasonic vibration for 20 seconds to resuspend the microspheres, then use PBS-TBN buffer to dilute the coupled microsphere group to a final concentration of 100 microspheres / species / μL of detection microsphere mixture, and use PBS-TBN buffer to dilute the competitive molecule to [IC70] or [IC80]; then add 25μL of PBS-TBN buffer to the background well, add 25μL of standard or sample to the corresponding well, add 25μL of diluted competitive molecule to each well, add 25μL of diluted corresponding antibody to each well, use a multichannel pipette to gently pipette several times to mix the reaction liquid, add 25μL of reaction microsphere mixture to each well, and use a multichannel pipette to gently pipette several times to mix the reaction liquid; cover the plate and use 8 00rpm shaking, incubate at room temperature for 60 minutes; after incubation, add 25ul diluted biotinylated anti-mouse IgG secondary antibody and incubate at room temperature for 60 minutes; use PBS-TBN buffer to dilute the SAPE reporter molecule to 4μg / mL, add 25μL diluted SAPE to each well, and then use a multichannel pipette to gently pipette several times to mix the reaction liquid, then cover the plate, shake at 800rpm, incubate at room temperature for 30 minutes, add 100μL of washing buffer (PBSTBN) to each well, do not stir the microspheres, and then repeat the multichannel pipette to gently pipette several times to mix the reaction liquid, wash twice in total, and finally add a final volume of 100μL reaction buffer to each well, and analyze 50-75μL of reaction solution using the Luminex instrument according to the operating instructions;

[0114] S1.3. Determination of the optimal coating concentration of zearalenone antigen

[0115] The antigen was diluted to 1, 3, and 5 μg / 1x10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000;

[0116] S1.4. Optimization of the amount of detection antibody

[0117] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0118] S1.5. Determination of SA-PE Antibody Working Concentration

[0119] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0120] S1.6 Specificity Test

[0121] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0122] S1.7 Sensitivity test

[0123] The standard zearalenone antigen (original concentration 6.8 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control.

[0124] S1.8. Determination of the optimal amount of coating protein

[0125] The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescent coded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of the three antigen amounts all reached above 10,000, indicating that the three coupled antibody amounts were all suitable. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 1ug;

[0126] S1.9. Determination of the optimal amount of antibody added

[0127] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0128] S1.10. Determination of the optimal SA-PE antibody working concentration

[0129] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0130] S1.11 Specificity test

[0131] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method. The results showed that, except for zearalenone, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0132] S1.12 Sensitivity test

[0133] The sensitivity test of diluted serum was performed using the established xMAP method;

[0134] S2. Establishment of xMAP detection method for aflatoxin B1

[0135] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen onto magnetic beads. The specific operation is the same as step S1.

[0136] S2.1. Validation of protein-coupled microspheres

[0137] Select microspheres coupled with aflatoxin B1 antigen, and the specific steps are the same as step S1.1;

[0138] S2.2. Aflatoxin B1 xMAP test (competitive immunoassay)

[0139] Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2;

[0140] S2.3. Determination of the optimal coating concentration of aflatoxin B1 antigen

[0141] Antigen was diluted to 1, 3, and 5 μg / 1 x 10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000;

[0142] S2.4. Optimization of the amount of detection antibody

[0143] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0144] S2.5. Determination of SA-PE Antibody Working Concentration

[0145] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0146] S2.6 Specificity Test

[0147] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0148] S2.7 Sensitivity test

[0149] The standard aflatoxin B1 antigen (original concentration 4.6 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The diluted serum was subjected to sensitivity testing using the established xMAP method, and PBS was used as a blank control.

[0150] S2.8. Determination of the optimal amount of coating protein

[0151] The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescence-encoded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of 3μg and 5μg antigen amounts reached above 10,000, indicating that all three coupled antibody amounts are suitable. Considering various factors, the optimal coupled antibody amount for 1×106 fluorescence-encoded microspheres is 3ug.

[0152] S2.9. Determination of the optimal amount of antibody added

[0153] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0154] S2.10. Determination of the optimal SA-PE antibody working concentration

[0155] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0156] S2.11 Specificity Test

[0157] The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for aflatoxin B1, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0158] S2.12 Sensitivity test

[0159] The sensitivity test of diluted serum was performed using the established xMAP method;

[0160] S3. Establishment of the xMAP Detection Method for Deoxynivalenol

[0161] Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen with magnetic beads. The specific operation method is the same as step S1.

[0162] S3.1. Validation of Protein-Coupled Microspheres

[0163] Select microspheres coupled with vomitoxin antigen, and the specific steps are the same as step S1.1;

[0164] S3.2. DON xMAP test (competitive immunoassay)

[0165] Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2;

[0166] S3.3 Determination of the optimal coating concentration of vomitoxin antigen

[0167] Dilute the antigen and select 1, 3, and 5 μg / 1x 106 magnetic beads of recombinant antigen to coat the magnetic beads for protein coating verification. The effective coating concentration is when the MFI value reaches more than 10,000.

[0168] S3.4. Optimization of the amount of detection antibody

[0169] The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point.

[0170] S3.5 Determination of SA-PE Antibody Working Concentration

[0171] The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test.

[0172] S3.6 Specificity test

[0173] The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method;

[0174] S3.7 Sensitivity test

[0175] The standard vomitoxin antigen (original concentration 8.2 mg / ml) was diluted 10-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control.

[0176] S3.8. Determination of the optimal amount of coating protein

[0177] The results showed that the MFI value increased with the increase of the amount of antibody. However, when the secondary antibody reached saturation, the MFI value of only 5 μg antigen reached more than 10,000, indicating that the amount of the three coupled antibodies can be used. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 5ug;

[0178] S3.9. Determination of the optimal amount of antibody added

[0179] The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml;

[0180] S3.10. Determination of the Optimal SA-PE Antibody Working Concentration

[0181] The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml.

[0182] S3.11 Specificity test

[0183] The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for vomitoxin, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity.

[0184] S3.12 Sensitivity test

[0185] The established xMAP method was used to perform sensitivity testing on diluted serum.

[0186] First, the purified antigen is coated onto magnetic beads through an antigen coating step, followed by verification of the protein-coupled microspheres to ensure successful coupling. Next, an xMAP assay is established for each toxin. The optimal experimental conditions are determined by optimizing key parameters such as the antigen coating concentration and the working concentrations of the detection antibody and signal amplification antibody. During the method development process, specificity and sensitivity tests are also conducted. The entire method development process is standardized, simple and rapid.

[0187] Among them, the specific operation method of antigen coating in S1 is as follows: first, select magnetic beads and record the code of laboratory magnetic beads, then vortex and sonicate for 20 seconds each to suspend the magnetic beads, then transfer 5.0x 106 magnetic beads to a USA Scientific microcentrifuge low binding rate test tube and centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads; after removing the supernatant, add 100μl double distilled water, vortex and sonicate for 20 seconds each to suspend, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, and use 80μl activation buffer (100mM pH 6.2 sodium dihydrogen phosphate) by vortexing and sonicating for 20 seconds each to suspend; then, 10 μl of 50 mg / mL sulfosuccinimide (Sulfo-NHS) and 10 μl of 50 mg / mL dicarbodiimide (EDC) were added to the suspended magnetic beads, and the reaction volume was 100 μl; protected from light, incubated at room temperature with gentle shaking for 20 minutes, and centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads; the supernatant was removed and 250 μl of coupling buffer (50 mM, pH 7.0) was used. The beads were suspended by vortexing and sonicating for 20 seconds each with 5.0 MES buffer, and then centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads. After that, the beads were suspended with MES buffer of the same concentration and washed by centrifugation twice. After vortexing and sonicating for 20 seconds each with 100 μl of MES buffer of the same concentration, 1, 3, and 5 μg of antigen were added to the suspended magnetic beads to a total volume of 500 μl. After vortexing, the beads were incubated at room temperature for 2 hours with shaking and protected from light. After incubation, the beads were centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads. The supernatant was removed. First, vortex and ultrasonically suspend with 500 μL of PBS-TBN buffer for 20 seconds each, then centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, vortex and ultrasonically suspend with 1000 μL of PBS-TBN buffer for 20 seconds each, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, finally remove the supernatant, vortex and ultrasonically suspend with 1000 μL of PBS, 10% BSA, 0.05% sodium azide, pH7.4 mixed buffer for 20 seconds each, and store the coupled microspheres in a refrigerator at 2-8°C away from light.

[0188] By strictly following the steps in the instructions, it is ensured that the coupled microspheres can be successfully produced.

[0189] In S1.2, when using PBS-TBN buffer to dilute the coupled microsphere group into a detection microsphere mixture with a final concentration of 100 microspheres / species / μL, it should be noted that each reaction requires 25 μL of the detection microsphere mixture.

[0190] Each reaction requires 25 μL of detection microsphere mixture, allowing experimenters to quickly calculate the total volume required when preparing experimental materials, simplifying experimental preparation.

[0191] Among them, when the reaction background in S1.2 is high, you can choose to use a magnetic plate and carefully remove the supernatant by manual flipping or pipetting on a magnetic plate washer, taking care not to stir the microspheres.

[0192] When the reaction background is high, manual intervention can be performed to ensure that the experiment can proceed smoothly.

[0193] Among them, in S1.12, the standard zearalenone antigen (original concentration was 6.8 mg / ml) was diluted 100 times, and then a 2-fold serial dilution was performed. The sensitivity test of the diluted serum was performed using the established xMAP method. The results showed that the minimum detection line of this method was 0.0664ug / ml.

[0194] The sensitivity test table of the Zearalenone xMAP test is shown below

[0195]

[0196] When the standard antigen is 0.0664ug / ml, the MFI value is 1675, so the lowest detection line of this method is 0.0664ug / ml.

[0197] Among them, in S2.12, the standard aflatoxin B1 antigen (original concentration was 4.6 mg / ml) was diluted 100 times, and then a 2-fold serial dilution was performed. The sensitivity test of the diluted serum was performed using the established xMAP method. The results showed that the minimum detection line of this method was 0.18 ug / ml.

[0198] The sensitivity test table of aflatoxin B1 xMAP test is shown below

[0199]

[0200]

[0201] When the standard antigen (ug / ml) is 0.18ug / ml, the MFI value is 1230, so the lowest detection line of this method is 0.18ug / ml.

[0202] Among them, in S3.12, the standard vomitoxin antigen (original concentration was 8.2 mg / ml) was diluted 10-fold, and then a 2-fold serial dilution was performed. The sensitivity test of the diluted serum was performed using the established xMAP method. The results showed that the minimum detection line of this method was 102.5 ug / ml.

[0203] The sensitivity test table of the DON xMAP test is shown below.

[0204]

[0205] When the standard antigen is 102.5ug / ml, the MFI value is 1016, so the lowest detection line of this method is 102.5ug / ml.

[0206] Among them, the detection antibodies in S1.9, S2.9 and S3.9 were diluted into 5 concentration groups, namely 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml and 8ug / ml.

[0207] By setting up multiple concentration groups, the trend of the impact of antibody concentration on experimental results can be observed, which can eliminate experimental errors caused by accidental factors and improve the reliability and repeatability of experimental results.

[0208] Among them, SA-PE antibody was diluted into 4 concentration groups in S1.10, S2.10 and S3.10, namely 1ug / ml, 2ug / ml, 4ug / ml and 8ug / ml.

[0209] By setting different concentration gradients, the performance of SA-PE antibodies at different concentrations can be systematically evaluated, thereby determining its optimal working concentration under specific experimental conditions, thereby improving the accuracy and reliability of experimental results.

[0210] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0211] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology, characterized in that: The specific steps are as follows: S1. Establishment of the xMAP Detection Method for Zearalenone Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen onto magnetic beads and follow the instructions for specific operations. S1.

1. Validation of protein-coupled microspheres Select the microspheres coupled to the zearalenone antigen, oscillate and sonicate for about 20 seconds to resuspend the microspheres, then dilute the coupled microspheres with detection buffer to a final concentration of 50 microspheres / μL. At the same time, prepare a 4μg / mL IgG detection anti-antibody solution labeled with phycoerythrin with detection buffer, and dilute the detection antibody solution to 0.0625μg / mL at a dilution ratio of 1:2; then, prepare two columns of 50μL of microsphere solution and relative antibody in each well on a 96-well plate, add 50μL of reaction buffer to the well containing the microsphere solution as a blank control, and then add 50μL of diluted IgG detection anti-antibody solution to each well, and use Gently pipette the solution in the wells several times to mix the solution; cover the 96-well plate, place it on a shaker, and incubate at room temperature for 30 minutes; after the incubation is complete, place the 96-well plate on the Luminex magnetic separation plate, quickly and forcefully flip the plate, and pour the solution in the wells into the biowaste bin. Next, use a pipette to aspirate 100 μL of reaction buffer into each reaction well, gently aspirate and release it several times, and then discard the liquid in the well using the same method. Repeat this washing step twice; finally, use a pipette to aspirate 100 μL of reaction buffer, gently aspirate and release it several times to resuspend the microspheres, and use the Luminex instrument to read 50-75 μL of the reaction results according to the system manual; S1.

2. Zearalenone xMAP test (competitive immunoassay) Select the appropriate microsphere group that has been coupled to the antigen, vortex ultrasonic vibration for 20 seconds to resuspend the microspheres, then use PBS-TBN buffer to dilute the coupled microsphere group to a final concentration of 100 microspheres / species / μL of detection microsphere mixture, and use PBS-TBN buffer to dilute the competitive molecule to [IC70] or [IC80]; then add 25μL of PBS-TBN buffer to the background well, add 25μL of standard or sample to the corresponding well, add 25μL of diluted competitive molecule to each well, add 25μL of diluted corresponding antibody to each well, use a multichannel pipette to gently pipette several times to mix the reaction liquid, add 25μL of reaction microsphere mixture to each well, and use a multichannel pipette to gently pipette several times to mix the reaction liquid; cover the plate and use 8 00rpm shaking, incubate at room temperature for 60 minutes; after incubation, add 25ul diluted biotinylated anti-mouse IgG secondary antibody and incubate at room temperature for 60 minutes; use PBS-TBN buffer to dilute the SAPE reporter molecule to 4μg / mL, add 25μL diluted SAPE to each well, and then use a multichannel pipette to gently pipette several times to mix the reaction liquid, then cover the plate, shake at 800rpm, incubate at room temperature for 30 minutes, add 100μL of washing buffer (PBSTBN) to each well, do not stir the microspheres, and then repeat the multichannel pipette to gently pipette several times to mix the reaction liquid, wash twice in total, and finally add a final volume of 100μL reaction buffer to each well, and analyze 50-75μL of reaction solution using the Luminex instrument according to the operating instructions; S1.

3. Determination of the optimal coating concentration of zearalenone antigen The antigen was diluted to 1, 3, and 5 μg / 1x10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000; S1.

4. Optimization of the amount of detection antibody The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point. S1.

5. Determination of SA-PE Antibody Working Concentration The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test. S1.6 Specificity Test The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method; S1.7 Sensitivity test The standard zearalenone antigen (original concentration 6.8 mg / ml) was diluted 100-fold, followed by 2-fold serial dilutions. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control. S1.

8. Determination of the optimal amount of coating protein The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescent coded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of the three antigen amounts all reached above 10,000, indicating that the three coupled antibody amounts were all suitable. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 1ug; S1.

9. Determination of the optimal amount of antibody added The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml; S1.

10. Determination of the optimal SA-PE antibody working concentration The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml. S1.11 Specificity test The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method. The results showed that, except for zearalenone, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity. S1.12 Sensitivity test The sensitivity test of diluted serum was performed using the established xMAP method; S2. Establishment of xMAP detection method for aflatoxin B1 Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen with magnetic beads. The specific operation method is the same as step S1. S2.

1. Validation of protein-coupled microspheres Select microspheres coupled with aflatoxin B1 antigen, and the specific steps are the same as step S1.1; S2.

2. Aflatoxin B1 xMAP test (competitive immunoassay) Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2; S2.

3. Determination of the optimal coating concentration of aflatoxin B1 antigen Antigen was diluted to 1, 3, and 5 μg / 1 x 10 6 The recombinant antigen of the magnetic beads is coated on the magnetic beads and the protein coating is verified. The effective coating concentration is when the MFI value reaches more than 10,000; S2.

4. Optimization of the amount of detection antibody The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point. S2.

5. Determination of SA-PE Antibody Working Concentration The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test. S2.6 Specificity Test The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method; S2.7 Sensitivity test The standard aflatoxin B1 antigen (original concentration 4.6 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The diluted serum was subjected to sensitivity testing using the established xMAP method, and PBS was used as a blank control. S2.

8. Determination of the optimal amount of coating protein The secondary antibody (goat anti-mouse IgG-PE) was used to react with the fluorescence-encoded microspheres coupled with the antibody. The results showed that the MFI value increased with the increase of the antibody amount. However, when the secondary antibody reached saturation, the MFI values of 3μg and 5μg antigen amounts reached above 10,000, indicating that all three coupled antibody amounts are suitable. Considering various factors, the optimal coupled antibody amount for 1×106 fluorescence-encoded microspheres is 3ug. S2.

9. Determination of the optimal amount of antibody added The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml; S2.

10. Determination of the optimal SA-PE antibody working concentration The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml. S2.11 Specificity Test The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for aflatoxin B1, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity. S2.12 Sensitivity test The sensitivity test of diluted serum was performed using the established xMAP method; S3. Establishment of the xMAP Detection Method for Deoxynivalenol Antigen coating: Use Antibody Coupling Kit: Coat the purified antigen onto magnetic beads. The specific operation is the same as step S1. S3.

1. Validation of Protein-Coupled Microspheres Select microspheres coupled with vomitoxin antigen, and the specific steps are the same as step S1.1; S3.

2. DON xMAP test (competitive immunoassay) Select an appropriate set of antigen-coupled microspheres, following the same steps as step S1.2; S3.3 Determination of the optimal coating concentration of vomitoxin antigen Dilute the antigen and select 1, 3, and 5 μg / 1x 106 magnetic beads of recombinant antigen to coat the magnetic beads for protein coating verification. The effective coating concentration is when the MFI value reaches more than 10,000. S3.

4. Optimization of the amount of detection antibody The detection antibody was diluted into five concentration groups: 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody dosage was determined based on the median fluorescence intensity (MFI) obtained by the test. The optimal antibody dosage was the dosage that reached the plateau inflection point. S3.5 Determination of SA-PE Antibody Working Concentration The SA-PE antibody was diluted into four concentration groups: 1ug / ml, 2ug / ml, 4ug / ml, and 8ug / ml. The optimal antibody addition amount was determined based on the median fluorescence intensity (MFI) obtained by the test. S3.6 Specificity test The established xMAP method was used to detect zearalenone, aflatoxins, and vomitoxin to verify the specificity of the method; S3.7 Sensitivity test The standard vomitoxin antigen (original concentration 8.2 mg / ml) was diluted 10-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method, and PBS was used as a blank control. S3.

8. Determination of the optimal amount of coating protein The results showed that the MFI value increased with the increase of the amount of antibody. However, when the secondary antibody reached saturation, the MFI value of only 5 μg antigen reached more than 10,000, indicating that the amount of the three coupled antibodies can be used. Considering all factors, 1×10 6 The optimal amount of antibody coupled to each fluorescent coding microsphere is 5ug; S3.

9. Determination of the optimal amount of antibody added The detection antibody was diluted into 5 concentration groups, and the results showed that the optimal working concentration of the detection antibody was 4ug / ml; S3.

10. Determination of the Optimal SA-PE Antibody Working Concentration The SA-PE antibody was diluted into four concentration groups. The results showed that as the detection concentration increased, the MFI value also increased. Taking all factors into consideration, the optimal working concentration of the SA-PE antibody was 4ug / ml. S3.11 Specificity test The established xMAP method was used to detect zearalenone, aflatoxin B1, and vomitoxin to verify the specificity of the method. The results showed that, except for vomitoxin, all other molds were negative, and there was no cross-reaction, indicating that the established method has good specificity. S3.12 Sensitivity test The established xMAP method was used to perform sensitivity testing on diluted serum.

2. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: The specific operation method of antigen coating described in S1 is as follows: first, select magnetic beads and record the code of the laboratory magnetic beads, then vortex and sonicate for 20 seconds each to suspend the magnetic beads, then transfer 5.0x 106 magnetic beads to a USA Scientific microcentrifuge low binding rate test tube and centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads; after removing the supernatant, add 100μl double distilled water, vortex and sonicate for 20 seconds each to suspend, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, and use 80μl activation buffer (100mM pH 6.2 sodium dihydrogen phosphate) by vortexing and sonicating for 20 seconds each to suspend; then, 10 μl of 50 mg / mL sulfosuccinimide (Sulfo-NHS) and 10 μl of 50 mg / mL dicarbodiimide (EDC) were added to the suspended magnetic beads, and the reaction volume was 100 μl; protected from light, incubated at room temperature with gentle shaking for 20 minutes, and centrifuged at ≥12000xg for 3 minutes to precipitate the magnetic beads; the supernatant was removed and 250 μl of coupling buffer (50 mM, pH 7.0) was used. 5.0 μl MES buffer) was vortexed and sonicated for 20 seconds each to suspend, and then centrifuged at ≥12,000 x g for 3 minutes to pellet the magnetic beads. After that, the pellet was suspended with MES buffer of the same concentration and washed twice by centrifugation. After vortexing and sonicating with 100 μl MES buffer of the same concentration for 20 seconds each, 1, 3, and 5 μg of antigen were added to the suspended magnetic beads to a total volume of 500 μl. After vortexing to mix, the beads were incubated with shaking at room temperature for 2 hours in the dark. After the incubation is completed, centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads; remove the supernatant, vortex and sonicate with 500μL PBS-TBN buffer for 20 seconds each for suspension, then centrifuge at ≥12000xg for 3 minutes to precipitate the magnetic beads, remove the supernatant, vortex and sonicate with 1000μL PBS-TBN buffer for 20 seconds each for suspension, centrifuge again at ≥12000xg for 3 minutes to precipitate the magnetic beads, finally remove the supernatant, vortex and sonicate with 1000μL PBS, 10% BSA, 0.05% sodium azide, pH7.4 mixed buffer for 20 seconds each for suspension, and store the coupled microspheres in a 2-8℃ refrigerator away from light.

3. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: When diluting the coupled microsphere set into a final concentration of 100 microspheres / species / μL of the detection microsphere mixture using PBS-TBN buffer as described in S1.2, it should be noted that 25 μL of the detection microsphere mixture is required for each reaction.

4. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: When the reaction background is high as described in S1.2, you can choose to use a magnetic plate and carefully remove the supernatant by manual inversion or pipetting through a magnetic plate washer, taking care not to disturb the microspheres.

5. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: As described in S1.12, the standard zearalenone antigen (original concentration was 6.8 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method. The results showed that the minimum detection limit of this method was 0.0664 ug / ml.

6. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: As described in S2.12, the standard aflatoxin B1 antigen (original concentration was 4.6 mg / ml) was diluted 100-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method. The results showed that the minimum detection limit of this method was 0.18 ug / ml.

7. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: As described in S3.12, the standard vomitoxin antigen (original concentration was 8.2 mg / ml) was diluted 10-fold, followed by a 2-fold serial dilution. The sensitivity of the diluted serum was tested using the established xMAP method. The results showed that the minimum detection limit of this method was 102.5 ug / ml.

8. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: The detection antibodies were diluted into five concentration groups as described in S1.9, S2.9 and S3.9, namely 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml and 8ug / ml.

9. The method for detecting zearalenone, aflatoxins and vomitoxin based on xMAP technology according to claim 1, characterized in that: As described in S1.10, S2.10 and S3.10, the SA-PE antibody was diluted into four concentration groups: 1 ug / ml, 2 ug / ml, 4 ug / ml and 8 ug / ml, respectively.

Images