CN114957186B

CN114957186B - Antigen structural analogue, application thereof in immunoaffinity column capacity real-time monitoring, method and product

Info

Publication number: CN114957186B
Application number: CN202210702462.2A
Authority: CN
Inventors: 刘仁荣; 吴珂; 吴珊珊; 朱可迦; 马海灵; 姬文慧; 杨适菲; 危雅丽; 杨延花; 吴思婷
Original assignee: Jiangxi Science and Technology Normal University
Current assignee: Jiangxi Science and Technology Normal University
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-05-05
Anticipated expiration: 2042-06-21
Also published as: CN114957186A

Abstract

The invention discloses an antigen structural analogue, and application, a method and a product thereof in immunoaffinity column capacity real-time monitoring. The invention provides the antigen structural analogues for real-time monitoring of the immunoaffinity column for the first time, develops a series of antigen structural analogues and immunoaffinity column real-time monitoring products based on the antigen structural analogues, solves the technical problems which cannot be solved by industrial methods including national standards, and has huge application prospect in the field of immunoaffinity column quality inspection.

Description

Antigen structural analogue, application thereof in immunoaffinity column capacity real-time monitoring, method and product

Technical Field

The invention belongs to the field of immunoaffinity column capacity detection, and particularly relates to an antigen structure analogue, an application of the antigen structure analogue in immunoaffinity column capacity real-time monitoring, a method and a product of the antigen structure analogue.

Background

Immunoaffinity columns (Immunoaffinity column, IAC) are increasingly used as an important pretreatment means for analytical chemistry in the detection of food safety. The IAC separation principle is to separate and purify the analyte by immobilizing the antibody on an insoluble substrate and utilizing the specificity and reversibility of the affinity between the antigen and the antibody. IAC has the advantages of high sensitivity, high specificity, less time consumption, convenient use, solvent saving and the like, greatly improves the purification effect and detection sensitivity of the sample, and can obtain high-purity analytes through one-time simple treatment of IAC.

IAC-HPLC method combined with IAC is a classical method for detecting mycotoxins such as aflatoxin, has high sensitivity and precision, and has wide application in home and abroad. IAC is used for sample purification in national standard methods (such as GB5009.22-2016, GB5009.24-2016, GB5009.96-2016, GB5009.111-2016, GB5009.118-2016, GB5009.209-2016, etc.) for detecting AFT.

However, IAC has a significant drawback as a pretreatment method: that is, the column capacity of IAC (the amount of analyte that the antibody in the column can bind effectively, typically measured as the maximum amount of analyte added at a recovery rate of 80% or more) has a significant impact on the detection results, and if the column capacity is insufficient, it can result in a lower (lower recovery rate) sample detection result at a high concentration (exceeding the column capacity); furthermore, the antibody in IAC is a protein, and the production, storage and transport conditions of IAC affect the stability of IAC, thereby significantly affecting the column capacity. Therefore, in all standard methods involving IAC, spot check is required to monitor the column capacity of the IAC and strict regulations are imposed on the column capacity.

The method for measuring the column capacity is to combine IAC on the analyte with a certain concentration, and measure the concentration of the analyte after elution to determine the column capacity. The method for measuring the immunoaffinity column capacity of aflatoxin B1 (AFTB 1) comprises the following steps: 600ng of AFTB1 standard stock solution was added to 30mL of 1% Triton X-100 (or Tween-20) -PBS and thoroughly mixed. The same batch of 3 immunoaffinity columns was taken, and the loading amount of each column was 10mL (200 ng each). Loading, eluting, collecting eluent, drying to 1mL with nitrogen, fixing volume to 10mL with initial mobile phase, and separating with liquid chromatograph to determine AFTB1 content. And (3) result judgment: AFTB1 is more than or equal to 160ng (recovery rate is more than 80 percent), and is a usable commodity.

Since the eluting reagent of IAC generally adopts organic solvents such as methanol and acetonitrile, the activity of antibody can be destroyed, therefore, IAC is generally disposable as a consumable, and at present, almost all national standards (such as GB5009.22-2016, GB5009.24-2016, GB5009.96-2016, GB5009.111-2016, GB5009.118-2016, GB5009.209-2016, etc.) related to immunoaffinity columns can only monitor the column capacity by sampling (such as GB5009.22-2016: taking 3 immunoaffinity columns of the same batch respectively), and each IAC cannot be ensured to be qualified, but if the column capacity cannot be ensured, the experimental result must be greatly influenced, so a novel method for detecting the capacity of the IAC column is urgently needed.

Disclosure of Invention

Aiming at the technical problems which cannot be solved all the time in the industries (including the national standard method), the inventor considers that the accuracy of each measurement result can be ensured only by monitoring the column capacity (real-time monitoring) while detecting the sample. Based on this, the solution proposed by the inventors is that a structural analogue of an analyte, i.e. an antigen, is obtained by chemically modifying the analyte (or its structural analogue), which structural analogue is not present in nature and is not confused with other naturally occurring structural analogues, and which structural analogue retains the binding capacity for antibodies in IAC, but which binding capacity for antibodies is less than that of the analyte, i.e. the antigen, and which structural analogue is loaded separately in the same molar amount as the nominal column capacity of IAC, at a recovery of more than 80%; when the structural analogue and the analyte are loaded in the same molar quantity with the same IAC nominal column capacity, the preferential binding of the analyte to the antibody of the immunoaffinity column can be ensured, and the recovery rate of the analyte is more than 80 percent, i.e. the addition of the structural analogue does not interfere with the detection result of the actual analyte. Monitoring the internal standard by taking the structural analogue with a certain concentration as the column capacity, purifying (adding simultaneously or subsequently) IAC on the sample extracting solution, and when the sample does not contain the analyte, the structural analogue can be combined with the antibody, and detecting after elution can detect the column capacity and verify the effectiveness of the column capacity; when there is an analyte in the sample, since the binding capacity of the analyte to the antibody is greater than that of the structural analogue to the antibody, the analyte preferentially binds to the antibody, if there are redundant binding sites, by the structural analogue, and after elution, the amount of analyte and structural analogue are separately determined by HPLC or mass spectrometry separation detection, and the sum of the two can determine the column capacity and verify its effectiveness, so as to realize monitoring the column capacity (real-time monitoring) while detecting the sample.

Based on the innovative research, a series of antigen structural analogues meeting the requirements are synthesized, and the structure of the antigen structural analogues is shown as one of the formulas (1) - (16):

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wherein the compound with the structure of the formula (1) and the compound with the structure of the formula (2) are obtained by condensing amino groups in glycine, alanine, beta-aminopropionic acid, beta-aminobutyric acid, gamma-aminobutyric acid, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, histidine or ethylenediamine with carboxyl groups in OTA and OTB respectively;

the compound with the structure of formula (3), the compound with the structure of formula (4), the compound with the structure of formula (5) and the compound with the structure of formula (6) are obtained by esterification reaction of glutaric anhydride, succinic anhydride, acetic anhydride, propionic anhydride or butyric anhydride with alcohol hydroxyl groups of alpha-zearalenol, beta-zearalenol, alpha-zearalenol and beta-zearalenol respectively;

the compound with the structure of the formula (7) and the compound with the structure of the formula (8) are respectively obtained by esterification reaction of glutaric anhydride, succinic anhydride, acetic anhydride, propionic anhydride or butyric anhydride with 15-hydroxyl alcohol of 3-acetyldeoxynivalenol and 3-hydroxyl alcohol of 15-acetyldeoxynivalenol.

Wherein OTA-D1-21 (compound of formula (1)) and OTB-D1-21 (compound of formula (2)) are structural analogues of ochratoxin A (OTA), and the obtained structural analogues are sequentially named as D1-D21 according to the sequence from glycine to ethylenediamine.

The number of the obtained structural analogues from small to large is named as D3, 5, 7, 9, 11, the number of the compounds of the formulae (9), the compounds of the formulae (3), the formulae (ZEN-D3, 5, 7, 9, 11), the formulae (ZEN-D4, 6, 8, 10, 12), the number of the obtained structural analogues from small to large is named as D4, 6, 8, 10, 12), the number of the formulae (ZEN-D13, 15, 17, 19, 21, the number of the compounds of the formulae (5), the formulae (ZEN-D3, 5, 7, 9, 11), the order of the formulae (ZEN-D3, 5, 7, 9, 11), the resulting structural analogs were designated as D13, 15, 17, 19, 21), ZEN-D14, 16, 18, 20, 22 (compounds of the structure of formula (6)) in the order of glutaric anhydride to butyric anhydride, and as D14, 16, 18, 20, 22), ZEN-D23 (compounds of the structure of formula (10)) in the order of glutaric anhydride to butyric anhydride.

DON-D2 (compound of formula (11)), DON-D3, 5, 7, 9, 11 (compound of formula (7)), the resulting structural analogs are designated as D3, 5, 7, 9, 11 from small to large in the order glutaric anhydride to butyric anhydride, DON-D4, 6, 8, 10, 12 (compound of formula (8)), the resulting structural analogs are designated as D4, 6, 8, 10, 12 from small to large in the order glutaric anhydride to butyric anhydride, DON-D13 (compound of formula (12)), DON-D14 (compound of formula (13)) and Deoxynivalenol (DON).

AFB1-D1-D3 (in order, a compound of formula (14), a compound of formula (15), and a compound of formula (16)) are structural analogs of aflatoxin B1 (AFB 1) and aflatoxin M1 (AFM 1).

The antigen structural analogues meeting certain conditions (including the antigen structural analogues) can be applied to the real-time monitoring of the immunoaffinity column; the real-time monitoring is as follows: when the immunoaffinity column detects sample liquid of an object to be detected suspected of carrying antigen, detecting the column capacity of the immunoaffinity column at the same time; the certain condition is: when the immunoaffinity column is loaded with the antigen structural analogues with the same mole number as the nominal column capacity of the immunoaffinity column alone, the recovery rate is more than 80 percent, and when the immunoaffinity column is loaded with the antigen structural analogues with the same mole number as the nominal column capacity of the immunoaffinity column and the antigen simultaneously or subsequently, the recovery rate of the antigen is more than 80 percent.

Based on the above description, the invention also provides a real-time monitoring method for the capacity of the immunoaffinity column, which comprises the following steps: when an immunoaffinity column detects an antigen of a sample liquid of an object to be detected suspected of carrying the antigen, simultaneously or subsequently adding an antigen structural analogue, eluting, and detecting by LC, HPLC, LC-MS or HPLC-MS to obtain detection results of the antigen and the antigen structural analogue and detection results of the immunoaffinity column capacity measurement respectively; if the sample liquid of the object to be detected does not contain antigen, the recovery rate of the antigen structural analogue detection is more than 80%; if the sample liquid of the object to be detected contains the antigen, the recovery rate of the antigen is more than 80 percent, and the number of moles of the antigen plus the number of moles of the analogue of the antigen structure is the number of moles actually detected by the column capacity.

Meanwhile, based on the above description, the invention also provides an immunoaffinity column real-time monitoring product/set, namely comprising the immunoaffinity column and an antigen structure analogue which is added simultaneously or subsequently with the sample liquid of the to-be-detected object when the immunoaffinity column detects the sample liquid of the to-be-detected object suspected to carry the antigen; if the sample liquid of the object to be detected does not contain antigen, the recovery rate of the antigen structural analogue detection is more than 80%; if the sample liquid of the object to be detected contains the antigen, the recovery rate of the antigen is more than 80 percent, and the number of moles of the antigen plus the number of moles of the analogue of the antigen structure is the number of moles actually detected by the column capacity.

In general, the mole number of the added antigen structural analogue is the same as the nominal antigen mole number of the immunoaffinity column, so that the column capacity of the immunoaffinity column can be monitored at any time, but the immunoaffinity column capacity generally only has influence on the detection result when detecting samples with high antigen content, and has little influence on samples with low content. Therefore, preferably, the number of moles of the antigen structural analogue added can be smaller than the nominal number of moles of the antigen in the immunoaffinity column, the effectiveness of the immunoaffinity column is verified when the content of the sample is low or the immunoaffinity column is not contained, and the volume of the column is verified when the content of the sample is high (the number of moles of the antigen plus the number of moles of the antigen structural analogue is the number of moles actually detected by the volume of the column), so that the effect of saving the use amount of the antigen structural analogue can be achieved.

In the application, the real-time monitoring method and the real-time monitoring product/set, the antigen can be ochratoxin A, zearalenone, deoxynivalenol, aflatoxin M1 or aflatoxin B1. The antigen structural analogues of the well-described structure of the present invention (OTA-D1-21, OTB-D1-21, ZEN-D1, ZEN-D3-23, DON-D2-14, AFB 1-D1-3) can be used. Antibodies in the immunoaffinity column can be monoclonal antibodies, polyclonal antibodies, single chain antibodies, animal-derived antibodies such as mice, rabbits, sheep, horses, birds and the like, humanized antibodies, nucleic acid aptamers, molecularly imprinted antibodies, genetic engineering antibodies and the like. The molar amount of the structural antigen analogue added is less than or equal to the molar amount of the antigen column capacity of the immunoaffinity column.

The beneficial effects of the invention are as follows: the invention provides the antigen structural analogues for real-time monitoring of the immunoaffinity column for the first time, develops a series of antigen structural analogues and immunoaffinity column real-time monitoring products based on the antigen structural analogues, solves the technical problems which cannot be solved by industrial methods including national standards, and has huge application prospect in the field of immunoaffinity column quality inspection.

Drawings

FIG. 1 is a schematic diagram of the present invention;

FIG. 2 shows a mass spectrum [ M-H ] of OTA-D1] ^- 。

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention.

The principle of the invention is shown in figure 1. Monitoring the internal standard by taking a certain concentration of the antigen structural analogue as a column capacity, purifying (simultaneously adding or subsequently adding) IAC on a sample extracting solution, wherein the structural analogue can be combined with an antibody when no analyte exists in the sample, detecting after elution to determine the column capacity (1, 2 and 3 steps in figure 1, no analyte exists in the sample, adding 6 molecules of the structural analogue, 6 IAC are combined, 6 elution, the column capacity is 6, and the detection result is effective, and if the elution is less than 6, the column capacity is less than 6); when there is analyte in the sample, since the binding capacity of the analyte to the antibody is greater than that of the structural analogue to the antibody, the analyte preferentially binds to the antibody, if there is an excess binding site, the binding is performed by the structural analogue, the elution is followed by HPLC or mass spectrometry separation detection, and the number of the analyte and the structural analogue is determined separately, and the sum of the two can determine whether the column capacity is acceptable (as in

steps

4,5 and 6 in FIG. 1, 3 analytes in the sample, 6 structural analogue molecules are added, IAC binds to 3 analytes and 3 structural analogues, 3 analytes and 3 structural analogues are eluted, the detection result is 3, the addition of the structural analogue does not affect the detection result of the analyte, the column capacity is 6, and a usable product is obtained, and if the sum of the detection result after elution is less than 6, the column capacity is less than 6), so that the column capacity is monitored simultaneously with the detection of the sample (real-time monitoring).

Example 1:

1. synthesis of ochratoxin A structural analogue OTA-D1

The synthetic route for OTA-D1 is shown below:

the preparation method comprises the following steps:

weighed OTA (20 mg), N-hydroxysuccinimide (NHS) 50mg, dicyclohexylcarbodiimide (DCC) 100mg were dissolved in 10mL anhydrous THF and reacted at 16℃for 40 hours, followed by the addition of 100mg glycine for 24 hours (dissolved in 0.1M NaHCO) ₃ ) After the reaction is finished, separating and purifying by using a prepared thin plate chromatography to obtain a product OTA-D1 of 10mg; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of OTA-D1 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(1) OTA immunoaffinity column (product nominal column capacity >300ng, sampling inspection is qualified) rewarming for 30 minutes at room temperature;

(2) Taking 2 immunoaffinity columns, preparing 20mL of OTA standard products with the content of 0 and 300ng (prepared according to national standard GB 5009.96-2016), adding 300ng of OTA-D1 (for intuitionistic, uniformly displaying the same mass of column capacity nominal by the affinity column, and due to different molecular weights, the actual added amount is the mass of OTA-D1 converted into the same mole number of 300ng of OTA), loading and combining according to a procedure, and leaching; adding 2mL methanol to elute immunoaffinity column, nitrogen blowing, dissolving with mobile phase, loading onto high performance liquid chromatography or liquid chromatography tandem mass spectrometry, detecting and analyzing OTA and OTA-D1 respectively, adding 0ng OTA standard, and determining with immunoaffinity column OTA-D1 to obtain 272.5 (recovery rate) >80%) and 253.2 (recovery rate) respectively>80%) can be used for real-time immunoaffinity column capacity monitoring (determination of affinity binding constants of OTA and OTA-D1 to antibody of 7.32X10, respectively ⁸ mol ^-1 And 1.08X10 ⁸ mol ^-1 The binding constant ratio of OTA-D1/OTA was 2.12X10 ⁸ /7.32×10 ⁸ ＝0.29(<0.3 When the ratio is smaller than 0.3), the method can also be used for monitoring the capacity of the OTA immunoaffinity column in real time);

(3) 10 immune affinity columns are taken, 10mL of an OTA standard product or wheat sample with the content of 0, 50, 100, 200ng and 300ng is prepared, 10mL of an extracting solution (prepared according to national standard GB 5009.96-2016) is added into the immune affinity column for loading and combining according to the rules, 10mL of OTA-D1 ng is added simultaneously or later (the same molar quantity of the nominal column capacity is added, the actual adding quantity is the same molar quantity of OTA-D1 in terms of OTA 300ng due to different molecular weights, the same mass of the nominal column capacity of the affinity column is uniformly displayed for visual representation, the later embodiment also carries out the same conversion), and the immune affinity column is added into the immune affinity column for loading and combining according to the rules for leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after nitrogen blowing, the immunoaffinity column was dissolved in a mobile phase and subjected to high performance liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze the amounts of OTA and OTA-D1, respectively, and the results are shown in Table 1 and Table 2.

TABLE 1 direct labeling detection results for standard substances

TABLE 2 sample labeled extract detection results

In summary, the detection results of the standard substances and the sample labeled extracting solution are normal, the recovery rate is more than 80%, and the addition of 300ng of OTA-D1 can detect that the column capacity of each immunoaffinity column is more than 240ng, so that the immunoaffinity column is a qualified product, and the real-time monitoring of the column capacity is realized while the actual OTA content in the sample is detected.

Example 2:

1. synthesis of ochratoxin A structural analogues OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19, OTA-D20

The specific preparation method of the OTA-D2, the OTA-D3, the OTA-D4, the OTA-D5, the OTA-D6, the OTA-D7, the OTA-D8, the OTA-D9, the OTA-D10, the OTA-D11, the OTA-D12, the OTA-D13, the OTA-D14, the OTA-D15, the OTA-D16, the OTA-D17, the OTA-D18, the OTA-D19 and the OTA-D20 comprises the following steps: taking ochratoxin A as raw material, 19 groups of reaction principle and preparation process are same as in example 1, each group is prepared by dissolving weighed OTA (20 mg), N-hydroxysuccinimide (NHS) 50mg and Dicyclohexylcarbodiimide (DCC) 100mg in 10mL anhydrous THF sufficiently, reacting at 16 ℃ for 40 hours, adding 100mg (OTA-D2 alanine, OTA-D3 beta-aminopropionic acid, OTA-D4 beta-aminobutyric acid, OTA-D5 gamma-aminobutyric acid, OTA-D6 valine, OTA-D7 leucine, OTA-D8 isoleucine, OTA-D9 methionine, OTA-D10 tryptophan, OTA-D11 serine, OTA-D12 tyrosine, OTA-D13 cysteine, OTA-D14 phenylalanine, OTA-D15 asparagine, OTA-D16 glutamine and OTA-D17 threo Acid, OTA-D18 aspartic acid, OTA-D19 glutamic acid and OTA-D20 histidine) for 24 hours (dissolved in 0.1M NaHCO ₃ ) Separating and purifying by preparative thin plate chromatography after the reaction is finished to obtain products of OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20-20 mg; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the material by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum to obtain structural analogues of ochratoxin A, namely OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20.

Application of OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(2) Taking 38 immune affinity columns (2 in each group), preparing 20mL of OTA standard products with the content of 0 and 300ng (prepared according to national standard GB 5009.96-2016), adding OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20 ng (for intuitively, uniformly displaying the same mass of column capacity of the affinity column, the actual adding amount is OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D14, OTA-D15 and OTA-D17, and combining the same mass with the standard substances according to the standards of OTA-D17, OTA-D13, OTA-D14 and OTA-D17. Adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase or a liquid chromatography tandem mass spectrum after nitrogen blowing, respectively detecting and analyzing the amounts of the OTA and the OTA-D2, the OTA-D3, the OTA-D4, the OTA-D5, the OTA-D6, the OTA-D7, the OTA-D8, the OTA-D9, the OTA-D10, the OTA-D11, the OTA-D12, the OTA-D13, the OTA-D14, the OTA-D15, the OTA-D16, the OTA-D17, the OTA-D18, the OTA-D19 and the OTA-D20, adding 0ng of the immunoaffinity column OTA-D2, the OTA-D3, the OTA-D4, the OTA-D5, the OTA-D6, the OTA-D7, the OTA-D8, the OTA-D9, the OTA-D10, the OTA-D11, the OTA-D12, the OTA-D13, the OTA-D14, the OTA-D15, the OTA-D16, the OTA-D17, the OTA-D18, the OTA-D19 and the OTA-D20, and the results of the immunoaffinity column of 0ng of the OTA-D standard product, and the results of the OTA-D300 are more than 80 ng. The recovery rate is more than 80 percent, and the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) 190 immunoaffinity columns (19 groups of 10) are taken, 20mL of standard or wheat sample labeling extracting solution (prepared according to national standard GB 5009.96-2016) with the content of 0, 50, 100, 200ng and 300ng is prepared, and each group respectively adds OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20 ng (for visual purposes), the same mass as the nominal column capacity of the affinity column is shown in a unified way, and the actual addition amount is the mass of OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18, OTA-D19 and OTA-D20 converted into the same mole number of OTA 300ng, and the same mole number of OTA-D2, OTA-D3, OTA-D4, OTA-D5, OTA-D6, OTA-D7, OTA-D8, OTA-D9, OTA-D10, OTA-D11, OTA-D12, OTA-D13, OTA-D14, OTA-D15, OTA-D16, OTA-D17, OTA-D18 are added according to the procedure is added into the immune affinity column for loading and combined for rinsing.

(4) Adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry on the mixture, respectively detecting and analyzing the amounts of the OTA and the OTA-D2, the OTA-D3, the OTA-D4, the OTA-D5, the OTA-D6, the OTA-D7, the OTA-D8, the OTA-D9, the OTA-D10, the OTA-D11, the OTA-D12, the OTA-D13, the OTA-D14, the OTA-D15, the OTA-D16, the OTA-D17, the OTA-D18, the OTA-D19 and the OTA-D20, wherein the detection results of the standard products and the sample labeling extracting solution are all normal, the recovery rate is more than 80 percent, and the detection contents of the OTA-D2, the OTA-D3, the OTA-D4, the OTA-D5, the OTA-D6, the OTA-D7, the OTA-D8, the OTA-D9, the OTA-D10, the OTA-D11, the OTA-D12, the OTA-D13, the OTA-D14, the OTA-D15, the OTA-D16, the OTA-D17 and the OTA-D19 are detected in real-D15, the sample-D16, the OTA-D17, the OTA-D18 and the sample is detected in real-time, and the practical capacity is realized.

Example 3:

1. synthesis of ochratoxin A structural analogues OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19, OTB-D20

The specific preparation method of the OTB-D1, the OTB-D2, the OTB-D3, the OTB-D4, the OTB-D5, the OTB-D6, the OTB-D7, the OTB-D8, the OTB-D9, the OTB-D10, the OTB-D11, the OTB-D12, the OTB-D13, the OTB-D14, the OTB-D15, the OTB-D16, the OTB-D17, the OTB-D18, the OTB-D19 and the OTB-D20 comprises the following steps: the structural analogue ochratoxin B (OTB) of ochratoxin A is taken as raw material, the reaction principle and the preparation process are the same as those of example 1, 20 groups are taken, each group is prepared by weighing 100mg of weighed OTB (20 mg), 50mg of N-hydroxysuccinimide (NHS), 100mg of Dicyclohexylcarbodiimide (DCC), 10mL of anhydrous THF is fully dissolved, after the reaction is carried out for 40 hours at 16 ℃, 100mg of (OTB-D1 glycine, OTB-D2 alanine, OTB-D3 beta-aminopropionic acid, OTB-D4 beta-aminobutyric acid, OTB-D5 gamma-aminobutyric acid, OTB-D6 valine, OTB-D7 leucine, OTB-D8 isoleucine, OTB-D9 methionine, OTB-D10 tryptophan, OTB-D11, serine, OTB-D12 tyrosine, OTB-D13 cysteine, OTB-D14 phenylalanine, OTB-D15 asparagine, OTB-D16 glutamine, OTB-D17, OTB-D18 threonine, OTB-D18, OTB-D19D and 0.20M are added for reaction ₃ ) After the reaction is finished, the products OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19 and OTB-D20-20 mg are obtained by separating and purifying by using a preparative thin plate chromatography; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. Identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum to obtain ochratoxin A structural analogues OTB-D1, OTB-D2, OTB-D3 and OTB-D4、OTB-D5、OTB-D6、OTB-D7、OTB-D8、OTB-D9、OTB-D10、OTB-D11、OTB-D12、OTB-D13、OTB-D14、OTB-D15、OTB-D16、OTB-D17、OTB-D18、OTB-D19、OTB-D20。

Application of OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19 and OTB-D20 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(2) 40 immunoaffinity columns (2 in each group) were taken, each group was prepared to have a content of 0 and 300ng of OTA standard 20mL (prepared according to national standard GB 5009.96-2016), and OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19, OTB-D20 ng were added to each standard (for visualization, the same mass as the nominal column capacity of the affinity column is shown in unity, the actual addition amounts are the mass of OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19, OTB-D20 converted to the same number of moles of OTA 300ng, and the immunoaffinity column is added according to the protocol for loading and combining; adding 2mL of methanol to elute an immunoaffinity column, dissolving the immunoaffinity column on a mobile phase or a liquid chromatography tandem mass spectrum after nitrogen blowing, respectively detecting and analyzing the amounts of OTA and OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19 and OTB-D20, adding 0ng of OTA standard substance to the immunoaffinity column OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14 and OTB-D20, and determining the yields of the results of the immunoaffinity column to be equal to 80-D, wherein the results of the immunoaffinity column, OTB-D and OTB-D14, OTB-D and OTB-D20 are respectively: the recovery rate is more than 80 percent, and the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) 200 immunoaffinity columns (20 groups of 10) are taken, 20mL of OTA standard or rice sample labeling extracting solution with the content of 0, 50, 100, 200ng and 300ng (prepared according to national standard GB 5009.96-2016) is prepared, and OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19 and OTB-D20 ng are added into the standard or sample labeling extracting solution (for visual purposes, the same mass as the nominal column capacity of the affinity column is shown in unity, the actual addition amounts are the mass of OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19, OTB-D20 converted to the same number of moles of OTA 300ng, and the immunoaffinity column is added according to the protocol for loading and combining;

(4) Adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase by using a mobile phase after nitrogen blowing, respectively detecting and analyzing the amounts of OTA and OTB-D1, OTB-D2, OTB-D3, OTB-D4, OTB-D5, OTB-D6, OTB-D7, OTB-D8, OTB-D9, OTB-D10, OTB-D11, OTB-D12, OTB-D13, OTB-D14, OTB-D15, OTB-D16, OTB-D17, OTB-D18, OTB-D19 and OTB-D20, wherein the detection results of the standard and sample labeling extracting solutions are all normal, the recovery rate is more than 80 ng, and the column capacity of each immunoaffinity column can be detected to be more than 240ng by adding 300ng of OTB-D1, so that the real-time monitoring of the column capacity is realized while detecting the actual OTA content in the sample.

Example 4:

1. synthesis of ochratoxin A structural analogue OTA-D21

The specific preparation method of the OTA-D21 comprises the following steps:

the weighed OTA (20 mg), NHS (30 mg) and DCC (90 mg) are fully dissolved by 10mL anhydrous THF, and 60mg ethylenediamine is added for reaction for 24 hours after the reaction is carried out at 26 ℃, and 10mg of product is obtained by separation and purification through preparative thin plate chromatography after the reaction is finished; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And then the structure and purity of the substance are identified through nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of OTA-D21 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(2) Taking 2 immunoaffinity columns, preparing an OTA standard product with the content of 0 and 300ng (prepared according to national standard GB 5009.96-2016), adding 300ng of OTA-D21 (for intuitionistic, uniformly displaying the same mass of column capacity nominal by the affinity column, and due to different molecular weights, the actual added amount is the mass of OTA-D21 converted into the same mole number of 300ng of OTA), loading and combining according to a procedure, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of OTA and OTA-D21, wherein the measurement result of the immunoaffinity column OTA-D21 added with 0ng of OTA standard substance is 256.1 (the recovery rate is more than 80 percent), and the measurement result of the immunoaffinity column OTA added with 300ng of OTA standard substance is 246.1 (the recovery rate is more than 80 percent), so that the immunoaffinity column capacity real-time monitoring can be realized.

(3) Taking 10 immune affinity columns, preparing 20mL of standard or wheat sample adding standard extracting solution (prepared according to national standard GB 5009.96-2016) with the content of 0, 50, 100, 200ng and 300ng of OTA standard or wheat sample, adding 200ng of OTA-D21 (for intuitively, uniformly displaying the same mass of column capacity of the nominal affinity column, and the actual adding amount is the mass of OTA-D21 converted into the same mole number of OTA 200ng due to different molecular weights) into the standard or sample adding standard extracting solution, loading and combining the standard or sample adding OTA-D21 according to the procedure, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after nitrogen blowing, the immunoaffinity column was dissolved in a mobile phase and subjected to high performance liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze the amounts of OTA and OTA-D21, respectively, and the results are shown in Table 3 and Table 4.

TABLE 3 direct labeling detection results for standard substances

TABLE 4 sample labeled extract detection results

In conclusion, most of detection results of standard products and sample labeling extracting solutions are normal, column capacity verification is effective or qualified, the detection result of a high-concentration sample is abnormal as a usable product, the OTA recovery rate is less than 80%, the column capacity of the immunoaffinity column can be detected to be less than 240ng (80% of nominal 300 ng) by adding 200ng of OTA-D21, and the real-time monitoring of the column capacity is realized for unqualified products.

Example 5:

1. synthesis of ochratoxin A structural analogue OTB-D21

The specific preparation method of the OTB-D21 comprises the following steps: the structural analogue of ochratoxin A, OTB-D21, is obtained by taking the structural analogue of ochratoxin A, OTB (OTB), as a raw material, and adopting the same reaction principle and preparation process as in example 4.

Application of OTB-D21 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(2) Taking 2 immunoaffinity columns, preparing an OTA standard product with the content of 0 and 300ng (prepared according to national standard GB 5009.96-2016), adding OTB-D21 300ng (for intuitively, uniformly displaying the same mass of the nominal column capacity of the affinity column, the actual adding amount is OTB-D21 mass converted into the same mole number of OTA 300ng due to different molecular weights) into the standard product, adding the immunoaffinity column according to the specification, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of OTA and OTB-D21, wherein the measurement result of the immunoaffinity column OTB-D21 added with 0ng of OTA standard substance is 275.3 (the recovery rate is more than 80 percent), and the measurement result of the immunoaffinity column OTA added with 300ng of OTA standard substance is 257.1 (the recovery rate is more than 80 percent), so that the immunoaffinity column can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 10 immunoaffinity columns, preparing 20mL of standard or corn sample adding standard extracting solution (prepared according to national standard GB 5009.96-2016) with the content of 0, 50, 100, 200ng and 300ng, adding 300ng of OTB-D21 (for intuitively, uniformly displaying the same mass of column capacity of the nominal affinity column, the actual adding amount is OTB-D21 mass converted into the same mole number of OTA 300ng due to different molecular weights) into the standard or sample adding standard extracting solution, adding the same mass of OTB-D21 into the immunoaffinity column according to the procedure, loading and combining, and leaching;

(4) Adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column by using a mobile phase after nitrogen blowing, and respectively detecting and analyzing the amounts of OTA and OTB-D21 by using a high performance liquid chromatography or a liquid chromatography tandem mass spectrum, wherein the detection results of the standard and sample labeling extracting solutions are all normal, the recovery rate is more than 80%, and adding 300ng of OTB-D21 can detect that the column capacity of each immunoaffinity column is more than 240ng, so that the immunoaffinity column is a qualified product, and the real-time monitoring of the column capacity is realized while detecting the actual OTA content in the sample.

Example 6:

1. synthesis of ochratoxin A structural analogue OTA-D22

The specific preparation method of the OTA-D22 comprises the following steps:

weighed OTA (20 mg), NHS (30 mg) and DCC (80 mg) were dissolved in 10mL anhydrous THF and reacted at 27℃for 36 hours, 100mg aminocaproic acid (0.1M NaHCO) was added ₃ ) After the reaction is completed for 24 hours, separating and purifying by using a prepared thin plate chromatography to obtain 12mg of product OTA-D; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And then the structure and purity of the substance are identified through nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of OTA-D22 in real-time monitoring of ochratoxin A immunoaffinity column capacity

(2) Taking 2 immunoaffinity columns, preparing 20mL of OTA standard products with the content of 0 and 300ng (prepared according to national standard GB 5009.96-2016), adding 300ng of OTA-D22 (for intuitionistic, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the mass of OTA-D22 converted into the same mole number of 300ng of OTA due to different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of OTA and OTA-D22, adding 0ng of OTA standard substance to obtain an immunoaffinity column OTA-D22 measuring result of 275.5 (recovery rate > 80%), adding 300ng of OTA standard substance to obtain an immunoaffinity column OTA measuring result of 86.8 (recovery rate < 80%), and testing the immunoaffinity column capacity for real-time monitoring.

(3) Taking 10 immune affinity columns, preparing 20mL (prepared according to national standard GB 5009.96-2016) of an OTA standard or wheat sample with the content of 0, 50, 100, 200ng and 300ng, adding OTA-D22 300ng into the standard or sample labeled extract, adding the immune affinity columns according to the procedure, loading and combining, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after nitrogen blowing, the immunoaffinity column was dissolved in a mobile phase and subjected to high performance liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze the amounts of OTA and OTA-D22, respectively, and the results are shown in Table 5 and Table 6.

Table 5 direct labelling test results for standard

TABLE 6 sample labeled extract detection results

In summary, when 300ng of OTA-D22 is used as a column capacity monitor, the column capacity of all immunoaffinity columns can be detected to be more than 240ng (80% of nominal 300 ng) as a qualified product, but the detection result of part of standard products and sample labeling extracting solutions is abnormal, and the recovery rate is less than 80%, so that the OTA-D22 can interfere with the normal combination of OTA and antibodies, and cannot be accurately detected, and cannot be used for real-time monitoring of the column capacity.

Example 7:

1. synthesis of zearalenone structural analogue ZEN-D1

The synthetic route for ZEN-D1 is shown below:

the preparation method comprises the following steps:

20mg of zearalenone is dissolved in analytically pure anhydrous pyridine, and 60mg of carboxymethyl hydroxylamine semi-hydrochloride is added for reaction at 25 ℃ for 24 hours; after the reaction is finished, separating and purifying by using a prepared thin plate chromatography to obtain 15mg of a product ZEN-D1; the product was purified by preparative liquid chromatography (0.00001 g) after adding NHS (30 mg) and DCC (90 mg) and fully dissolving in 10mL of anhydrous THF, reacting at 26℃for 24 hours, adding 60mg of ethylenediamine and reacting for 24 hours. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of ZEN-D1 in ZEN immunoaffinity column capacity real-time monitoring

(1) The ZEN immunoaffinity column (the nominal column capacity of the product is more than 1000ng, and the spot inspection is qualified) is rewarmed for 30 minutes at room temperature;

(2) Taking 2 immunoaffinity columns, preparing 20mL of ZEN standard substances with the content of 0 and 1000ng, adding 1000ng of ZEN-D1 (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the ZEN-D1 mass converted into the same mole number of 1000ng of ZEN because of different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase by using a mobile phase after nitrogen blowing or carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amount of ZEN and ZEN-D1, adding 0ng of ZEN standard substance to obtain an immunoaffinity column ZEN-D1 measuring result of 947.3 (the recovery rate is more than 80 percent), adding 1000ng of ZEN standard substance to obtain an immunoaffinity column ZEN measuring result of 912.8 (the recovery rate is more than 80 percent), and carrying out real-time monitoring on the column capacity of the immunoaffinity column.

(3) 10 immune affinity columns are taken, 10mL of ZEN standard products or corn sample standard adding extracting solutions with the content of 0, 5, 25, 200ng and 1000ng (according to the measurement of zearalenone in national food safety standard of GB 5009.209-2016) are prepared, 1000ng of ZEN-D1 are added into the standard products or the sample standard adding extracting solutions, and the immune affinity columns are added according to the regulations for loading and combining, leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or lower with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography tandem mass spectrometry, and the results of the analysis of ZEN and ZEN-D1 were shown in tables 7 to 8.

Table 7 ZEN-D1 group standard direct labeling detection results

Table 8 ZEN-D1 sets of sample labeled extract detection results

In sum, the detection results of the ZEN-D1 group standard products and the sample labeled extracting solutions are all normal, the recovery rate is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 800ng (80% of the nominal 1000 ng) by adding 1000ng of ZEN-D1, and the real-time monitoring of the column capacity is realized while the actual ZEN content in the sample is detected as a qualified product.

Example 8:

1. synthesis of zearalenone structural analogue ZEN-D2

The specific preparation method of ZEN-D2 comprises the following steps:

20mg of zearalenone is dissolved in analytically pure anhydrous pyridine, 30mg of carboxymethyl hydroxylamine semi-hydrochloride is added and reacted for 24 hours at 25 ℃; after the reaction is finished, separating and purifying by using a prepared thin plate chromatography to obtain 15mg of a product ZEN-D2; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of ZEN-D2 in ZEN immunoaffinity column capacity real-time monitoring

(2) Taking 2 immunoaffinity columns, preparing 20mL of ZEN standard substances with the content of 0 and 1000ng, adding 1000ng of ZEN-D2 (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the ZEN-D2 mass converted into the same mole number of 1000ng of ZEN because of different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase by using a mobile phase after nitrogen blowing or carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amount of ZEN and ZEN-D2, adding 0ng of ZEN standard substance to obtain an immunoaffinity column ZEN-D2 measuring result of 908.3 (the recovery rate is more than 80 percent), adding 1000ng of ZEN standard substance to obtain an immunoaffinity column ZEN measuring result of 563.8 (the recovery rate is less than 80 percent), and testing the immunoaffinity column capacity for real-time monitoring.

(3) Taking 10 immune affinity columns, preparing 20mL of ZEN standard products or corn sample labeling extracting solutions with the contents of 0, 5, 25, 200ng and 1000ng (according to GB 5009.209-2016-national food safety standard-determination of zearalenone in food), respectively adding ZEN-D2 1000ng into the standard products or the sample labeling extracting solutions, adding the immune affinity columns according to the regulations, loading and combining, and leaching;

(4) The immunoaffinity column was eluted with 1.5mL of methanol, and after drying at 55℃or lower with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography tandem mass spectrometry, ZEN and ZEN-D2 were detected and analyzed, respectively, and the results are shown in tables 9 to 10.

Table 9 direct labeling test results of standard substances

Table 10 sample labeled extract detection results

In summary, when 1000ng of ZEN-D2 is used as the column capacity monitor, it is possible to detect that the column capacity of all immunoaffinity columns is greater than 800ng (80% of the nominal 1000 ng), which is a qualified product, but the detection recovery rate of part of the high concentration standard and sample addition-labeled extract is less than 80%, so ZEN-D2 interferes with the normal binding of ZEN and antibody, resulting in inaccurate detection, and thus cannot be used for real-time monitoring of column capacity.

Example 9:

1. synthesis of zearalenone structural analogue ZEN-D3/ZEN-D4

The synthetic route for ZEN-D3/ZEN-D4 is shown below:

the preparation method comprises the following steps:

dissolving 20mg of zearalenone structural analogue alpha-zearalenol/beta-zearalenol in anhydrous THF, adding 50mg of 4-Dimethylaminopyridine (DMAP) solution and 50mg of glutaric anhydride, reacting at 20 ℃ for 52 hours, and separating and purifying by using a prepared thin plate chromatography after the reaction is finished to obtain 10-20mg of each of the products ZEN-D3/ZEN-D4; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of ZEN-D3/ZEN-D4 in real-time monitoring of ZEN immunoaffinity column capacity

(2) Taking 4 immune affinity columns (two of ZEN-D3/ZEN-D4 groups), preparing 20mL of ZEN standard substances with the content of 0 and 1000ng, adding 1000ng of ZEN-D3/ZEN-D4 (for intuitionism, uniformly displaying the same mass of column capacity nominal by the affinity column, and due to different molecular weights, the actual adding amount is the mass of ZEN-D3/ZEN-D4 converted into the same mole number of 1000ng of the ZEN), adding the immune affinity column according to the rules, loading, combining and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column by using a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of ZEN and ZEN-D3/ZEN-D4, wherein the immunoaffinity column ZEN-D3/ZEN-D4 with 0ng of ZEN standard substance is determined to be 898.1 and 954.0 (recovery rate > 80%), and the immunoaffinity column ZEN with 1000ng of ZEN standard substance is determined to be 932.7 and 964.8 (recovery rate > 80%), so that the immunoaffinity column can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 20 immune affinity columns (10 in each of ZEN-D3/ZEN-D4 groups), preparing 20mL of ZEN standard or corn sample standard addition extracting solution with the content of 0, 5, 25, 200ng and 1000ng (according to GB 5009.209-2016-national food safety standard-determination of zearalenone in food), adding 1000ng of ZEN-D3/ZEN-D4 into the standard or sample standard addition extracting solution, loading and combining according to the procedure, and leaching;

(4) The immunoaffinity column was eluted with 1.5mL of methanol, and after drying at 55℃or lower with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography tandem mass spectrometry, ZEN and ZEN-D3/ZEN-D4 were detected and analyzed, respectively, and the results are shown in tables 11 to 14.

Table 11 ZEN-D3 group standard direct labeling detection result

Table 12 ZEN-D3 sets of sample labeled extract detection results

Table 13 ZEN-D4 group standard direct labeling detection results

Table 14 ZEN-D4 sets of sample labeled extract detection results

In sum, the detection results of the ZEN-D3/ZEN-D4 standard products and the sample labeling extracting solution are normal, the recovery rate is more than 80%, and the column capacity of each immunoaffinity column can be detected to be more than 800ng (80% of nominal 1000 ng) by adding 1000ng of ZEN-D3/ZEN-D4, so that the real-time monitoring of the column capacity is realized while the actual ZEN content in the sample is detected as a qualified product.

Example 10:

1. synthesis of zearalenone structural analogues ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11, ZEN-D12

The synthesis principle of the compounds ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 is the same as that of example 9, and the zearalenone structural analogue alpha-zearalenol/beta-zearalenol is taken as a raw material to carry out esterification modification on the alcoholic hydroxyl groups, but the added reagents are succinic anhydride, acetic anhydride, propionic anhydride and butyric anhydride respectively. Specific preparation is the same as in example 9: dissolving 20mg of zearalenone structural analogue alpha-zearalenol/beta-zearalenol in anhydrous THF, adding 50mg of 4-Dimethylaminopyridine (DMAP) solution and 50mg of (succinic anhydride, acetic anhydride, propionic anhydride and butyric anhydride), reacting for 52 hours at 20 ℃, and separating and purifying by using prepared thin plate chromatography after the reaction is finished to obtain products ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 of 10-20mg respectively; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum. .

Application of ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 in real-time monitoring of ZEN immunoaffinity column capacity

(2) Taking 16 immune affinity columns (8 groups, each group comprises 2) and each group is prepared with the content of 0 and 1000ng of ZEN standard substance of 20mL, adding ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 1000ng (for intuitionistic, the same mass of column capacity which is shown as the nominal of the affinity column is unified, and due to different molecular weights, the actual addition amount is ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 with the same molar number converted into ZEN of 1000ng, and loading and combining the immune affinity columns according to the rules, leaching; adding 1.5-2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of ZEN and ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12, adding 0ng of ZEN standard substance of the immunoaffinity column ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12, wherein the recovery rate of the measurement results of the immunoaffinity column ZEN with the addition of 1000ng of the ZEN standard substance is more than 80 percent, and the immunoaffinity column capacity real-time monitoring can be realized.

(3) Taking 80 immune affinity columns (8 groups of 10) respectively, preparing 10mL of ZEN standard products or corn sample labeling extracting solutions with the content of 0, 5, 25, 200ng and 1000ng (according to the determination of zearalenone in GB 5009.209-2016 food safety national standard food), respectively adding ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 simultaneously or subsequently into the standard products or the sample labeling extracting solutions, respectively 1000ng, adding the immune affinity columns according to the procedure for loading and combining, and leaching;

(4) After adding 2mL of methanol to elute the immunoaffinity column and blow-drying the column with nitrogen below 55 ℃, 1.0mL of mobile phase is used for dissolving residues, and the residues are respectively detected and analyzed by liquid chromatography or liquid chromatography tandem mass spectrometry, wherein the results show that the ZEN and ZEN-D5, ZEN-D6, ZEN-D7, ZEN-D8, ZEN-D9, ZEN-D10, ZEN-D11 and ZEN-D12 are respectively detected and analyzed: the detection results of the various standard substances and sample-marked extracting solutions of the ZEN-D5, the ZEN-D6, the ZEN-D7, the ZEN-D8, the ZEN-D9, the ZEN-D10, the ZEN-D11 and the ZEN-D12 are all normal, the recovery rate is more than 80 percent, and the column capacity of each immunoaffinity column can be detected to be more than 800ng (80 percent of nominal 1000 ng) for qualified products by adding 1000ng of the ZEN-D5, the ZEN-D6, the ZEN-D7, the ZEN-D8, the ZEN-D9, the ZEN-D10, the ZEN-D11 and the ZEN-D12, and the real-time monitoring of the column capacity is realized while the actual ZEN content in the sample is detected.

Example 11:

1. synthesis of zearalenone structural analogues ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21, ZEN-D22

The principles of zearalenone structural analogues ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22 are the same as those of example 9, and the zearalenone structural analogues alpha-zearalanol/beta-zearalanol are used as raw materials to carry out esterification modification on the alcoholic hydroxyl groups of the zearalenone structural analogues, but the added reagents are glutaric anhydride, succinic anhydride, acetic anhydride, propionic anhydride and butyric anhydride respectively. The specific preparation is the same as that of the example: dissolving 20mg of zearalenone structural analogue alpha-zearalanol/beta-zearalanol in anhydrous THF, adding 50mg of 4-Dimethylaminopyridine (DMAP) solution and 50mg of (glutaric anhydride, succinic anhydride, acetic anhydride, propionic anhydride and butyric anhydride), carrying out reflux reaction for 52 hours at 30 ℃, and separating and purifying by using prepared thin plate chromatography after the reaction is finished to obtain 10-20mg of products ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21, ZEN-D22 immunoaffinity column capacity real-time monitoring

(2) Taking 20 immune affinity columns (10 groups, each group comprises 2) and preparing 20mL of ZEN standard substances with the content of 0 and 1000ng, adding ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22 into each group respectively, wherein 1000ng (for visualization, the same mass of column capacity is uniformly displayed as the affinity column nominal, and due to different molecular weights, the actual addition amounts are ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22 with the same molar number converted into ZEN 1000 ng) according to the rules, and loading and combining according to the rules; adding 1.5mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of ZEN and ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22, adding 0ng of ZEN standard sample of the immunoaffinity column ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22, wherein the recovery rate of the measurement results of the immunoaffinity column ZEN added with 1000ng of the ZEN standard sample is more than 80%, and the immunoaffinity column capacity real-time monitoring can be used.

(3) Taking 100 immune affinity columns (10 groups of 10) and preparing 10mL of ZEN standard products or corn sample labeling extracting solutions with the content of 0, 5, 25, 200ng and 1000ng (according to the determination of zearalenone in GB 5009.209-2016 food safety national standard food), respectively adding 1000ng of ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19, ZEN-D20, ZEN-D21 and ZEN-D22 into the standard products or the sample labeling extracting solutions simultaneously or subsequently, and adding the immune affinity columns according to the rules for loading and combining, and leaching;

(4) After adding 2mL of methanol to elute the immunoaffinity column and blow-drying the column with nitrogen below 55 ℃, 1.0mL of mobile phase is used for dissolving residues, and the residues are respectively detected and analyzed by liquid chromatography or liquid chromatography tandem mass spectrometry, wherein the results show that the ZEN and ZEN-D13, ZEN-D14, ZEN-D15, ZEN-D16, ZEN-D17, ZEN-D18, ZEN-D19 and ZEN-D20 are respectively detected and analyzed: the detection results of the standard substances and the sample-marked extracting solutions of the ZEN-D13, the ZEN-D14, the ZEN-D15, the ZEN-D16, the ZEN-D17, the ZEN-D18, the ZEN-D19 and the ZEN-D20 are all normal, the recovery rate is more than 80 percent, and by adding 1000ng of the ZEN-D13, the ZEN-D14, the ZEN-D15, the ZEN-D16, the ZEN-D17, the ZEN-D18, the ZEN-D19, the ZEN-D20, the ZEN-D21 and the ZEN-D22, the column capacity of each immunoaffinity column is more than 800ng (80 percent of the nominal 1000 ng) which is a qualified product, and the real-time monitoring of the column capacity is realized while the actual ZEN content in a sample is detected.

Example 12:

1. synthesis of zearalenone structural analogue ZEN-D23

The preparation method of the zearalenone structural analogue ZEN-D23 by taking the zearalenone structural analogue zearalenone as a raw material comprises the following steps: 20mg of zearalenone is dissolved in analytically pure anhydrous pyridine, and 60mg of carboxymethyl hydroxylamine semi-hydrochloride is added for reaction at 25 ℃ for 24 hours; after the completion of the reaction, 15mg of ZEN-D was obtained as a product by separation and purification by preparative thin plate chromatography, and the product was purified by preparative liquid chromatography and then precisely quantified (0.00001 g). And identifying the structure and purity (purity is more than 98%) of the substance by nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of ZEN-D23 in ZEN immunoaffinity column capacity real-time monitoring

(2) Taking 2 immunoaffinity columns, preparing 20mL of ZEN standard substances with the content of 0 and 1000ng, adding ZEN-D21 1000ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is ZEN-D23 mass converted into the same mole number of ZEN 1000ng due to different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 1.5-2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase by using a mobile phase after nitrogen blowing or carrying out liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amounts of ZEN and ZEN-D23, adding 0ng of ZEN standard sample to the immunoaffinity column ZEN-D23, measuring the recovery rate of the result to be more than 80 percent, adding 1000ng of ZEN standard sample to the immunoaffinity column ZEN, measuring the recovery rate to be more than 80 percent, and carrying out real-time monitoring on the capacity of the immunoaffinity column.

(3) 10 immune affinity columns are taken, 10mL of ZEN standard products or corn sample standard adding extracting solutions with the content of 0, 5, 25, 200ng and 1000ng (according to the measurement of zearalenone in national food safety standard of GB 5009.209-2016) are prepared, 1000ng of ZEN-D23 is added into the standard products or the sample standard adding extracting solutions, and the immune affinity columns are added according to the regulations for loading and combining, leaching;

(4) Adding 2mL of methanol to elute the immunoaffinity column, drying the immunoaffinity column by nitrogen below 55 ℃, dissolving residues by using 1.0mL of mobile phase, and carrying out liquid chromatography or liquid chromatography tandem mass spectrometry measurement to respectively detect and analyze ZEN and ZEN-D23, wherein the detection results of each standard product and sample labeled extraction liquid are all normal, the recovery rate is more than 80%, and by adding 1000ng of ZEN-D23, the column capacity of each immunoaffinity column is more than 800ng (80% of nominal 1000 ng) and the real-time monitoring of the column capacity is realized while detecting the actual ZEN content in a sample.

Example 13:

1. synthesis of deoxynivalenol structural analogue DON-D1

The specific preparation method of DON-D1 comprises the following steps:

taking 20mg of DON standard substance, adding 80mg of 4-Dimethylaminopyridine (DMAP) and 100mg of glutaric anhydride into the solution, dissolving the solution in anhydrous THF, carrying out reflux reaction at 36 ℃ for 48 hours, and separating and purifying the solution by using a prepared silica gel plate after the reaction to obtain 16mg of DON-D; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. The structure and purity (purity greater than 98%) of the material were then identified by nuclear magnetic resonance, mass spectrometry and ultraviolet spectroscopy.

Application of DON-D1 in real-time monitoring of DON immunoaffinity column capacity

(1) DON immunoaffinity column (product nominal column capacity >2000ng, spot inspection is qualified) rewarming for 30 minutes at room temperature;

(2) Taking 2 immunoaffinity columns, preparing 20mL of DON standard products with the content of 0 and 2000ng, adding DON-D1 to 2000ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is DON-D1 mass converted into the same mole number of DON 2000ng due to different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 1.5-2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the quantity of DON and DON-D1, wherein the determination result of the immunoaffinity column DON-D1 added with 0ng of DON standard substance is 1808.6 (the recovery rate is more than 80 percent), and the determination result of the immunoaffinity column DON added with 2000ng of DON standard substance is 938.9 (the recovery rate is less than 80 percent), so that the method is used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 10 immune affinity columns, preparing 20mL (according to GB 5009.111-2016) of DON standard products or wheat sample labeling extracting solutions with the content of 0, 50, 250, 1000ng and 2000ng, adding 2000ng DON-D1 into the standard products or the sample labeling extracting solutions, loading and combining the immune affinity columns according to the regulations, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or below with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography-tandem mass spectrometry, and DON-D1 were detected and analyzed, respectively, with the results shown in tables 15 to 16.

Table 15 direct labeling test results of standard substances

Table 16 sample labeled extract test results

In summary, the detection result of part of the high-concentration standard substance and the sample labeling extracting solution is abnormal, the recovery rate of the part of the standard substance and the sample labeling extracting solution is less than 80%, and the column capacity of all immunoaffinity columns can be detected to be more than 1600ng (80% of nominal 2000 ng) by adding 2000ng of DON-D1, so that the DON-D1 can interfere the normal combination of DON and antibodies, so that the actual DON content in the sample cannot be accurately detected, and the method cannot be used for real-time monitoring of the column capacity.

Example 14:

1. synthesis of deoxynivalenol structural analogue DON-D2

The preparation principle of DON-D2 is as above, and the specific method comprises the following steps:

adding 20mg of DON and 100mg of N, N' -Carbonyl Diimidazole (CDI) into anhydrous THF solution, reacting at 30 ℃ for 24 hours, evaporating the reaction solution in a rotary way, adding 6mL of 0.01M PBS (pH 7.0) and 10mL of DMF, adding 20-40mg of ethylenediamine, reacting at 45 ℃ for 24 hours, and separating and purifying by using preparative thin plate chromatography after the reaction is finished to obtain the product DON-D2 mg; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. The structure and purity (purity greater than 98%) of the material were then identified by nuclear magnetic resonance, mass spectrometry and ultraviolet spectroscopy.

Application of DON-D2 in real-time monitoring of DON immunoaffinity column capacity

(2) Taking 2 immunoaffinity columns, preparing 20mL of DON standard products with the content of 0 and 2000ng, adding DON-D2 to 2000ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is DON-D2 mass converted into the same mole number of DON 2000ng due to different molecular weights), adding the immunoaffinity column according to the rules, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the quantity of DON and DON-D2, wherein the determination result of the immunoaffinity column DON-D2 added with 0ng of DON standard substance is 1912.0 (the recovery rate is more than 80 percent), and the determination result of the immunoaffinity column DON added with 2000ng of DON standard substance is 1957.8 (the recovery rate is more than 80 percent), so that the immunoaffinity column can be used for real-time monitoring of the column capacity of the immunoaffinity column.

(3) Taking 10 immune affinity columns, preparing 20mL (according to GB 5009.111-2016) of DON standard products or wheat sample labeling extracting solutions with the content of 0, 50, 250, 1000ng and 2000ng, adding 2000ng DON-D2 into the standard products or the sample labeling extracting solutions, loading and combining the immune affinity columns according to the regulations, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or below with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography-tandem mass spectrometry, and DON-D2 were detected and analyzed, respectively, as shown in tables 17 to 18.

Table 17 direct labeling detection result of standard substance

Table 18 sample labeled extract detection results

In sum, the detection results of the standard substances and the sample labeled extracting solution are normal, the recovery rate is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 1600ng (80% of the nominal 2000 ng) by adding 2000ng DON-D2, and the real-time monitoring of the column capacity is realized while the actual DON content in the sample is detected as a qualified product.

Example 15:

1. synthesis of deoxynivalenol structural analogue DON-D3/DON-D4

The specific preparation method of DON-D3/DON-D4 comprises the following steps:

adding 80mg of 4-Dimethylaminopyridine (DMAP) and 100mg of glutaric anhydride into 20mg of DON structural analogues of 3-acetyldeoxynivalenol (3-acetyldeoxynivalenol, 3-ACDON) or 15-acetyldeoxynivalenol (15-acetyldeoxynivalenol, 15-ACDON), dissolving in anhydrous THF, carrying out reflux reaction at 45 ℃ for 48 hours, and separating and purifying the product DON-D3/DON-D4/10 mg by using a prepared silica gel plate after the reaction; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. The structure and purity (purity greater than 98%) of the material were then identified by nuclear magnetic resonance, mass spectrometry and ultraviolet spectroscopy.

Application of DON-D3/DON-D4 in real-time monitoring of DON immunoaffinity column capacity

(2) Taking 4 immunoaffinity columns (2 in DON-D3/DON-D4 groups), preparing 20mL of DON standard substances with the content of 0 and 2000ng, adding 2000ng of DON-D3/DON-D4 (for intuitionism, uniformly displaying the same mass of column capacity nominal by the affinity column, and due to different molecular weights, the actual adding amount is DON-D3/DON-D4 mass converted into 2000ng of the same mole number) into the immunoaffinity column, loading, combining and leaching according to the rules; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the quantity of DON and DON-D3/DON-D4, wherein the measurement result of the immunoaffinity column DON-D3/DON-D4 added with 0ng of DON standard substance is 1893.7/1845.2 (the recovery rate is more than 80 percent), and the measurement result of the immunoaffinity column DON added with 2000ng of DON standard substance and 2000ng of DON-D3/DON-D4 is 1879.3/1798.2 (the recovery rate is more than 80 percent), so that the immunoaffinity column can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 20 immune affinity columns (10 in DON-D3/DON-D4 groups) and preparing 20mL (according to GB 5009.111-2016) of DON standard substances or wheat sample labeling extracting solutions with the content of 0, 50, 250, 1000ng and 2000ng, adding 2000ng of DON-D3 into the standard substances or the sample labeling extracting solutions, adding the immune affinity columns according to the regulations, loading and combining, and leaching; adding DON-D4 1000ng into the other 10 standard substances or sample labeled extracting solutions, adding an immunoaffinity column according to the procedure for loading and combining, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or below with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography-tandem mass spectrometry, and DON-D3/DON-D4 were detected and analyzed, respectively, with the results shown in tables 19 to 22.

Table 19 DON-D3 group standard direct labeling detection results

Table 20 DON-D3 sample labeled extract detection results

Table 21 DON-D4 standard direct labeling detection results

Table 22 DON-D4 sample labeled extract detection results

In sum, the detection results of the DON-D3 standard products and the sample labeled extracting solution are normal, the recovery rate is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 1600ng (80% of the nominal 2000 ng) by adding 2000ng DON-D3, and the real-time monitoring of the column capacity is realized while the actual DON content in the sample is detected as a qualified product; the detection results of the DON-D4 group standard products and sample labeled extracting solutions are normal, the recovery rate is more than 80%, the effectiveness of each immunoaffinity column can be detected by adding 1000ng of DON-D4, the capacity of the actual measurement column is more than 1600ng (80% of the nominal 2000 ng) for 1000ng/2000ng high-concentration standard products and samples, and the real-time monitoring of the column capacity is realized while the actual DON content in the samples is detected.

Example 16:

1. synthesis of deoxynivalenol structural analogues DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10, DON-D11/DON-D12

The specific preparation method of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12 comprises the following steps:

adding 80mg of 4-Dimethylaminopyridine (DMAP) and 100mg of solution of 3-acetyldeoxynivalenol (3-acetyldeoxynivalenol, 3-ACDON) or 15-acetyldeoxynivalenol (15-acetyldeoxynivalenol, 15-ACDON) serving as structural analogues of DON into anhydrous THF (succinic anhydride, acetic anhydride, propionic anhydride and butyric anhydride), and carrying out reflux reaction at 50 ℃ for 48 hours, and separating and purifying the obtained products by using a silica gel plate prepared above to obtain DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12/10 mg; the method comprises the steps of carrying out a first treatment on the surface of the Accurate quantification (0.00001 g) after purification by preparative liquid chromatography. The structure and purity (purity greater than 98%) of the material were then identified by nuclear magnetic resonance, mass spectrometry and ultraviolet spectroscopy.

Application of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12 in real-time monitoring of DON immunoaffinity column capacity

(2) Taking 16 immune affinity columns (DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10, DON-D11/DON-D12 groups, each group of 2) respectively, preparing 20mL of DON standard substance with the content of 0 and 2000ng, adding 2000ng of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10, DON-D11/DON-D12 into each column (for intuitiveness, the same mass of column capacity which is displayed as the nominal affinity column is unified, and the actual adding amount is DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12 in terms of equivalent mole number of DON 2000ng is converted into DON-D, and the same mass) according to the rules, adding immune affinity columns into the column for eluting and combining; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column on a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry on the mobile phase, wherein the recovery rate is more than 80 percent, and the immunoaffinity column DON measurement results of adding 2000ng of DON standard and 2000ng of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10, DON-D11/DON-D12 are more than 80 percent, and the immunoaffinity column DON measurement results of adding 0ng of DON standard are more than 2000ng of DON standard and 2000ng of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12, so that the recovery rate of the immunoaffinity column can be used for real-time monitoring.

(3) Taking 80 immune affinity columns (10 in each group of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12), preparing 20mL (according to GB 5009.111-2016) of DON standard or wheat sample standard extracting solution with the content of 0, 50, 250, 1000ng and 2000ng, adding DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12 into the standard or sample standard extracting solution, respectively, and loading and combining according to the rules;

(4) Adding 2mL of methanol to elute the immunoaffinity column, drying the immunoaffinity column by nitrogen below 55 ℃, dissolving residues by using 1.0mL of mobile phase, and carrying out liquid chromatography or liquid chromatography tandem mass spectrometry measurement to detect and analyze DON and DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12 respectively, wherein the results show that the detection results of each standard product and sample added with the standard extract are normal, the recovery rate is more than 80%, and the real-time monitoring of the column capacity can be realized while the actual DON content in the detected sample is realized by adding 2000ng of DON-D5/DON-D6, DON-D7/DON-D8, DON-D9/DON-D10 and DON-D11/DON-D12.

Example 17:

1. Synthesis of deoxynivalenol structural analogue DON-D13/DON-D14

The preparation principle of DON-D13/DON-D14 is the same as that of DON-D2 of the embodiment, and the specific preparation method comprises the following steps: adding 20mg of DON structural analogue 3-acetyldeoxynivalenol or 15-acetyldeoxynivalenol and 100mg of CDI into anhydrous THF solution, reacting at 30 ℃ for 24 hours, rotary steaming and pumping out reaction liquid, adding 6mL of 0.01M PBS (pH 7.0) and 10mL of DMF, adding 20-40mg of ethylenediamine, reacting at 35 ℃ for 24 hours, and separating and purifying by using a prepared thin plate chromatography to obtain 15-17mg of DON-D13/DON-D14; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. The structure and purity (purity greater than 98%) of the material were then identified by nuclear magnetic resonance, mass spectrometry and ultraviolet spectroscopy.

Application of DON-D13/DON-D14 in real-time monitoring of DON immunoaffinity column capacity

(2) Taking 2 immunoaffinity columns, preparing 20mL of DON standard products with the content of 0 and 2000ng, adding DON-D13/DON-D14 2000ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and due to different molecular weights, the actual adding amount is DON-D13/DON-D14 mass converted into the same mole number of 2000ng of DON), adding the immunoaffinity column according to the regulations, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the quantity of DON and DON-D13/DON-D14, wherein the recovery rate of the determination result of the immunoaffinity column DON-D13/DON-D14 added with 0ng of DON standard substance is more than 80%, and the recovery rate of the determination result of the immunoaffinity column DON added with 2000ng of DON standard substance is more than 80%, so that the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 20 immune affinity columns (10 in DON-D13/DON-D14 groups), preparing 20mL of DON standard products or wheat sample labeling extracting solutions with the content of 0, 50, 250, 1000ng and 2000ng (according to GB 5009.111-2016), adding DON-D13/DON-D14 2000ng into the standard products or the sample labeling extracting solutions, loading and combining the immune affinity columns according to the regulations, and leaching;

(4) Adding 2mL of methanol to elute the immunoaffinity column, drying the immunoaffinity column by nitrogen below 55 ℃, dissolving residues by using 1.0mL of mobile phase, and carrying out liquid chromatography or liquid chromatography tandem mass spectrometry to determine, respectively detecting and analyzing DON and DON-D13/DON-D14, wherein the result shows that the detection results of each standard product and sample added with the standard extracting solution are normal, the recovery rate is more than 80%, and the column capacity of each immunoaffinity column can be detected to be more than 1600ng (80% of nominal 2000 ng) by adding 2000ng of DON-D13/DON-D14, so that the real-time monitoring of the column capacity is realized while the actual DON content in the sample is detected as a qualified product.

Example 18:

1. synthesis of aflatoxin B1 structural analogue AFB1-D1

The specific preparation method of the AFB1-D1 comprises the following steps:

20mg of aflatoxin B1 is added into analytically pure anhydrous pyridine, 100mg of carboxymethyl hydroxylamine semi-hydrochloride is added, and the mixture is reacted for 24 hours at 25 ℃; separating and purifying by using a prepared thin plate chromatography after the reaction is finished to obtain 18mg of AFB 1-D1; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And then the structure and purity (purity is more than 98%) of the material are identified through nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of AFB1-D1 in AFB1 immunoaffinity column capacity real-time monitoring

(1) AFB1 immunoaffinity column (product nominal column capacity >200ng, spot inspection is qualified) rewarming for 30 minutes at room temperature;

(2) Taking 2 immunoaffinity columns, preparing 20mL of AFB1 standard products with the content of 0 and 200ng, adding 200ng of AFB1-D1 (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the mass of AFB1-D1 converted into 200ng of AFB1 with the same mole number due to different molecular weights), adding the immunoaffinity columns according to the regulations, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the amounts of AFB1 and AFB1-D1, wherein the determination result of the immunoaffinity column AFB1-D1 added with 0ng of AFB1 standard substance is 190.1 (the recovery rate is more than 80 percent), and the determination result of the immunoaffinity column AFB1 added with 200ng of AFB1 standard substance is 176.6 (the recovery rate is more than 80 percent), so that the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Preparing 20mL of AFB1 standard or barley sample with the content of 0, 2, 10, 50 and 200ng by adding 20-mL of the standard extracting solution (according to GB 5009.22-2016), adding 200ng of AFB1-D1, adding the immunoaffinity column according to the procedure, loading and combining, and leaching;

(4) After adding 2mL of methanol to elute the immunoaffinity column and blow-drying the column with nitrogen at a temperature below 55 ℃, 1.0mL of mobile phase is used for dissolving residues, and the residues are subjected to liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze AFB1 and AFB1-D1 respectively, and the results are shown in tables 23-24.

Table 23 standard direct labeling detection result

Table 24 sample labeled extract test results

In sum, the detection results of the standard substances and the sample added with the standard extracting solution are normal, the recovery rate of the sample is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 160ng (80% of the nominal 200 ng) by adding 200ng of AFB1-D1, and the real-time monitoring of the column capacity is realized while the actual AFB1 content in the sample is detected as a qualified product.

Example 19:

1. synthesis of aflatoxin B1 structural analogue AFB1-D2

The specific preparation method of the AFB1-D2 comprises the following steps:

AFB1 structural analogue AFB1-D2 is prepared by taking AFB1 structural analogue aflatoxin B2 as a raw material. 20mg of aflatoxin B2 is added into analytically pure anhydrous pyridine, 100mg of carboxymethyl hydroxylamine semi-hydrochloride is added, and the mixture is reacted for 24 hours at 25 ℃; separating and purifying by using a prepared thin plate chromatography after the reaction is finished to obtain 15mg of AFB 1-D2; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And then the structure and purity (purity is more than 98%) of the material are identified through nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of AFB1-D2 in AFB1 immunoaffinity column capacity real-time monitoring

(2) Taking 2 immunoaffinity columns, preparing 20mL of AFB1 standard products with the content of 0 and 200ng, adding 200ng of AFB1-D2 (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the mass of AFB1-D2 converted into 200ng of AFB with the same mole number due to different molecular weights), adding the immunoaffinity columns according to the regulations, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amounts of AFB1 and AFB1-D2, wherein the determination result of the immunoaffinity column AFB1-D2 added with 0ng of AFB1 standard substance is 178.2 (the recovery rate is more than 80 percent), and the determination result of the immunoaffinity column AFB1 added with 200ng of AFB1 standard substance is 169.8 (the recovery rate is more than 80 percent), so that the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Preparing 20mL of AFB1 standard or millet sample with the content of 0, 2, 10, 50 and 200ng by taking 10 immunoaffinity columns (according to GB 5009.22-2016), adding 200ng of AFB1-D2, loading and combining the immunoaffinity columns according to the procedure, and leaching;

(4) After adding 2mL of methanol to elute the immunoaffinity column and blow-drying the column with nitrogen at a temperature below 55 ℃, 1.0mL of mobile phase is used for dissolving residues, and the residues are subjected to liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze AFB1 and AFB1-D2 respectively, and the results are shown in tables 25-26.

Table 25 standard direct labelling detection result

Table 26 sample labeled extract test results

In conclusion, except that the standard product addition recovery rate is lower than 80%, the detection results of other standard products and sample addition extracting solutions are normal, the sample recovery rate is higher than 80%, the column capacity of the affinity column is lower than 160ng through adding 200ng of AFB1-D2, the column capacities of other immunoaffinity columns are higher than 160ng (80% of nominal 200 ng) and are qualified products, and real-time monitoring of the column capacity is realized while the actual AFB1 content in the sample is detected.

Example 20

1. Synthesis of aflatoxin B1 structural analogue AFB1-D3

The specific preparation method of the AFB1-D3 comprises the following steps:

AFB1 structural analogue AFB1-D3 is prepared by taking AFB1 structural analogue aflatoxin B2a as a raw material. 20mg of aflatoxin B2a is added into analytically pure anhydrous pyridine, 100mg of carboxymethyl hydroxylamine semi-hydrochloride is added, and the mixture is reacted for 24 hours at 25 ℃; separating and purifying by using a prepared thin plate chromatography after the reaction is finished to obtain 15mg of AFB 1-D3; accurate quantification (0.00001 g) after purification by preparative liquid chromatography. And then the structure and purity (purity is more than 98%) of the material are identified through nuclear magnetic resonance, mass spectrum, fluorescence spectrum and ultraviolet spectrum.

Application of AFB1-D3 in AFB1 immunoaffinity column capacity real-time monitoring

(2) Taking 2 immunoaffinity columns, preparing 20mL of AFB1 standard products with the content of 0 and 200ng, adding 200ng of AFB1-D3 (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is the mass of AFB1-D3 converted into 200ng of AFB with the same mole number due to different molecular weights), adding the immunoaffinity columns according to the regulations, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column with a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amounts of AFB1 and AFB1-D3, wherein the recovery rate of the measurement results of the immunoaffinity column AFB1-D3 added with 0ng of AFB1 standard is more than 80%, and the recovery rate of the measurement results of the immunoaffinity column AFB1 added with 200ng of AFB1 standard is more than 80%, so that the real-time monitoring of the capacity of the immunoaffinity column can be realized.

(3) Preparing 20mL of AFB1 standard or wheat sample with the content of 0, 2, 10, 50 and 200ng by taking 10 immunoaffinity columns (according to GB 5009.22-2016), adding 100ng of AFB1-D3, loading and combining the immunoaffinity columns according to the procedure, and leaching;

(4) After adding 2mL of methanol to elute the immunoaffinity column and blow-drying the column with nitrogen at a temperature below 55 ℃, 1.0mL of mobile phase is used for dissolving residues, and the residues are subjected to liquid chromatography or liquid chromatography tandem mass spectrometry to detect and analyze AFB1 and AFB1-D3 respectively, and the results are shown in tables 27-28.

Table 27 standard direct labelling detection result

Table 28 sample labeled extract test results

The detection results of the standard and sample labeled extraction solutions are normal, the recovery rate is more than 80%, the effectiveness of each immunoaffinity column can be detected by adding 100ng of AFB1-D3, the detection column capacity of the high-concentration standard and the high-concentration labeled sample is more than 160ng, and the high-concentration standard and the high-concentration labeled sample are qualified products, so that the real-time monitoring of the column capacity is realized while the actual AFB1 content in the sample is detected.

Example 21:

1. synthesis of aflatoxin M1 structural analogue AFM1-D1

The specific preparation method of the AFM1-D1 comprises the following steps: as in example 18, AFM1-D1 is AFB1-D1

Application of AFM1-D1 in AFM1 immunoaffinity column capacity real-time monitoring

(1) AFM1 immunoaffinity column (product nominal column capacity >100ng, sampling inspection is qualified) rewarming for 30 minutes at room temperature;

(2) Taking 2 immunoaffinity columns, preparing 10mL of AFM1 standard products with the content of 0 and 100ng, adding AFM1-D1 ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is AFM1-D1 mass converted into the same mole number of AFM1 ng due to different molecular weights), adding the immunoaffinity columns according to the regulation, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column by using a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry to respectively detect and analyze the quantity of AFM1 and AFM1-D1, wherein the measurement result of the immunoaffinity column AFM1-D1 added with 0ng of AFM1 standard substance is 90.2 (the recovery rate is more than 80 percent), and the measurement result of the immunoaffinity column AFM1 added with 100ng of AFM1 standard substance is 86.3 (the recovery rate is more than 80 percent), so that the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 10 immune affinity columns, preparing 20mL of AFM1 standard products or wheat sample labeling extracting solutions with the content of 0, 1, 5, 25 and 100ng (according to GB 5009.24-2016), adding 100ng of AFM1-D1 into the standard products or the sample labeling extracting solutions, adding the immune affinity columns according to the regulations, loading and combining, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or lower with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography-tandem mass spectrometry, and the results of the analysis of AFM1 and AFM1-D1 were shown in tables 27 to 28, respectively.

Table 29 standard direct labeling detection result

Table 30 sample labeled extract detection results

In sum, the detection results of the standard substances and the sample addition marked extracting solution are normal, the sample recovery rate is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 80ng (80% of the nominal 100 ng) by adding 100ng of AFM1-D1, and the real-time monitoring of the column capacity is realized while the actual AFM1 content in the sample is detected as a qualified product.

Example 22:

1. synthesis of aflatoxin M1 structural analogue AFM1-D2

The specific preparation method of AFM1-D2 comprises the following steps: as in example 19, AFM1-D2 is AFB1-D2

Application of AFM1-D2 in AFM1 immunoaffinity column capacity real-time monitoring

(2) Taking 2 immunoaffinity columns, preparing 10mL of AFM1 standard products with the content of 0 and 100ng, adding AFM1-D2 ng (for intuitionism, uniformly displaying the same mass of the nominal column capacity of the affinity column, and the actual adding amount is AFM1-D2 mass converted into the same mole number of AFM1 ng due to different molecular weights), adding the immunoaffinity columns according to the regulation, loading and combining, and leaching; adding 2mL of methanol to elute the immunoaffinity column, dissolving the immunoaffinity column by using a mobile phase after nitrogen blowing, and carrying out high performance liquid chromatography or liquid chromatography tandem mass spectrometry, respectively detecting and analyzing the amounts of AFM1 and AFM1-D2, wherein the determination result of the immunoaffinity column AFM1-D2 of 0ng of AFM1 standard substance is 97.1 (the recovery rate is more than 80%), and the determination result of the immunoaffinity column AFM1 of 100ng of AFM1 standard substance is 96.5 (the recovery rate is more than 80%), so that the method can be used for real-time monitoring of the capacity of the immunoaffinity column.

(3) Taking 10 immune affinity columns, preparing 20mL of AFM1 standard products or wheat sample labeling extracting solutions with the content of 0, 1, 5, 25 and 100ng (according to GB 5009.24-2016), adding 100ng of AFM1-D2 into the standard products or the sample labeling extracting solutions, adding the immune affinity columns according to the regulations, loading and combining, and leaching;

(4) The immunoaffinity column was eluted with 2mL of methanol, and after drying at 55℃or lower with nitrogen, the residue was dissolved in 1.0mL of a mobile phase and analyzed by liquid chromatography or liquid chromatography-tandem mass spectrometry, and the results of the analysis of AFM1 and AFM1-D2 were shown in tables 29 to 30, respectively.

Table 31 direct labeling detection result of standard substance

Table 32 sample labeled extract test results

In sum, the detection results of the standard substances and the sample addition marked extracting solution are normal, the sample recovery rate is more than 80%, the column capacity of each immunoaffinity column can be detected to be more than 80ng (80% of the nominal 100 ng) by adding 100ng of AFM1-D2, and the real-time monitoring of the column capacity is realized while the actual AFM1 content in the sample is detected as a qualified product.

The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be construed as being limited to the above embodiments as long as the technical effects of the present invention are achieved by the same means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.

Claims

1. An antigen structural analogue, which is characterized in that the structure is shown as one of the formulas (1) - (2):

Formula (1),>

(2),

wherein the compound with the structure shown in the formula (1) and the compound with the structure shown in the formula (2) are respectively obtained by condensing amino groups in glycine, alanine, beta-aminopropionic acid, beta-aminobutyric acid, gamma-aminobutyric acid, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, threonine, aspartic acid, glutamic acid and histidine with carboxyl groups in OTA and OTB;

the structure of the OTA is as follows:

；

the structure of the OTB is as follows:

。

2. the use of an antigen structural analogue according to claim 1 for non-diagnostic or therapeutic purposes in real-time immunoaffinity column monitoring, characterized in that when the immunoaffinity column detects a sample fluid of an analyte suspected to carry an antigen, the column capacity of the immunoaffinity column is detected by the antigen structural analogue at the same time, i.e. the column capacity is monitored in real time; when the immunoaffinity column is loaded with the antigen structural analogues with the same mole number as the nominal column capacity of the immunoaffinity column alone, the recovery rate is more than 80 percent, and when the immunoaffinity column is loaded with the antigen with the same mole number as the nominal column capacity of the immunoaffinity column and the antigen structural analogues simultaneously or sequentially, the recovery rate of the antigen is more than 80 percent.

3. A method for real-time monitoring of immunoaffinity column capacity for non-diagnostic or therapeutic purposes, comprising the steps of: when an immunoaffinity column detects sample liquid of an object to be detected suspected of carrying antigen, simultaneously or subsequently adding the antigen structural analogue of claim 1 with the mole number of 10-100% of the nominal column capacity of the immunoaffinity column, eluting, and detecting by LC, HPLC, LC-MS or HPLC-MS to obtain detection results of the antigen and the antigen structural analogue and detection results of the immunoaffinity column capacity; if the sample liquid of the object to be detected does not contain antigen, the recovery rate of the antigen structural analogue detection is more than 80%; if the sample liquid of the object to be detected contains the antigen, the recovery rate of the antigen is more than 80 percent, and the sum of the mole number of the antigen and the mole number of the structural analogue of the antigen is the mole number actually detected by the column capacity.

4. The method of claim 3, wherein the number of moles of the structural antigen analogue added is less than or equal to the nominal number of moles of the antigen column capacity of the immunoaffinity column.

5. An immunoaffinity column real-time monitoring product for non-diagnostic or therapeutic purposes, comprising an immunoaffinity column and the structural antigen analogue of claim 1 added simultaneously or subsequently with a sample fluid of an analyte when the immunoaffinity column detects the sample fluid of the analyte suspected of carrying the antigen; if the sample liquid of the object to be detected does not contain antigen, the recovery rate of the antigen structural analogue detection is more than 80%; if the sample liquid of the object to be detected contains the antigen, the recovery rate of the antigen is more than 80 percent, and the sum of the mole number of the antigen and the mole number of the antigen structural analogue is the mole number actually detected by the column capacity.