Zearalenone immunomagnetic bead as well as preparation method and application thereof

Abstract

The invention relates to the technical field of zearalenone detection, in particular to zearalenone immunomagnetic beads as well as a preparation method and application thereof. The method specifically comprises the following steps: (1) cleaning N-hydroxysuccinimide activated agarose magnetic beads with ethanol, removing ethanol, adding a 2-morpholinoethanesulfonic acid buffer solution and a ZEN monoclonal antibody for mixed incubation, and discarding an upper solution; and (2) adding a glycine-containing buffer solution into the incubated magnetic beads, mixing, sealing, carrying out magnetic separation, and leaching to obtain the ZEN immunomagnetic beads. According to the method, IMBs high-flux full-automatic purification is combined with a high performance liquid chromatograph, the method which is rapid and convenient and can automatically treat ZEN in feed and feed raw material samples in batches is established, and the first feed industry association group standard about the IMBs purification technology is researched and formulated on the basis of the method; and a method reference is provided for prevention and control of fungaltoxin pollution in basic detection laboratories and feed enterprises.

Description

Technical Field

[0001] The present invention relates to the technical field of zearalenone detection, and in particular to zearalenone immunomagnetic beads and a preparation method and application thereof. Background Art

[0002] Zearalenone (ZEN), commonly known as F-2 toxin, is mainly produced by Fusarium graminearum and can widely contaminate crops such as corn, soybeans, and wheat. It is one of the most common mycotoxins in food and feed. ZEN has acute and chronic toxicity, immunotoxicity, reproductive toxicity, and "three-hazard" (carcinogenic, teratogenic, and mutagenic) effects on humans and animals. It is a Class 3 carcinogen announced by the World Health Organization's International Agency for Research on Cancer. In addition, because ZEN has a similar chemical structure to estradiol, it can bind to the body's estrogen receptors to produce estrogen-like effects and has obvious endocrine disrupting activity. It is also an environmental endocrine disruptor (Endocrine-disrupting chemicals, EDCs) announced by the World Wildlife Fund. ZEN has a wide range of contamination in feed. Hao et al. analyzed 9,392 samples of feed and feed raw materials from 2017 to 2021 and found that ZEN was detected in more than half of the feed samples (detection rate 56.29%), with an average detection content of 183.81 μg / kg, and most of them were composite contamination of more than two mycotoxins. Zeng Rui et al. monitored and analyzed three mycotoxins, ZEN, ochratoxin A, and chrysophagatoxin in 232 poultry compound feeds in South China, and found that ZEN was detected in all feed samples (detection rate 100%), with a detection content of 1.00-53.61 μg / kg (average 8.59 μg / kg).

[0003] In addition, the discovery of hidden toxins such as ZEN-14-glucoside (ZEN-14G) and ZEN-14-sulfate (ZEN-14S) also increases its exposure risk. In view of the toxic effects of ZEN on farmed animals and its widespread contamination in feed, the establishment of an accurate, rapid, and high-throughput quantitative analysis method for ZEN in feed is of great significance for the exposure assessment and risk prevention and control of mycotoxins in feed.

[0004] At present, the commonly used detection methods for ZEN in feed mainly include rapid detection methods such as colloidal gold test strips, enzyme-linked immunosorbent assay (ELISA), and immunolateral flow chromatography, as well as instrumental confirmation analysis methods such as gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Rapid detection technologies such as ELISA, colloidal gold test strips, and immunolateral flow chromatography are prone to false positives due to cross-reaction of antibodies; GC and GC-MS often require derivatization, which is complicated in pretreatment and the stability of the derivatized products is also poor. HPLC is the most commonly used quantitative analysis technology for mycotoxins due to its advantages such as simple operation, stability, reliability, and good reproducibility. In addition, LC-MS / MS is increasingly widely used for qualitative and quantitative detection of mycotoxins in feed, but its large-scale application in the feed industry is limited due to its expensive instruments, complex operation, and the inherent matrix effect of the electrospray ionization source (ESI), which can easily affect the accuracy of the quantitative results. Before HPLC and LC-MS / MS quantitative analysis, the sample needs to be purified. Common purification techniques for toxin analysis include solid-phase extraction (SPE), QuEChERS, and immunoaffinity column (IAC). The national standard currently in effect in the feed industry, "Determination of Zearalenone in Feed by Immunoaffinity Column Purification-High Performance Liquid Chromatography" (GB / T28716-2012), uses IAC for sample purification. Although IAC has better selectivity and purification effects than traditional SPE and QuEChERS methods, and is less affected by interference from various sample matrices, it is less stable due to the influence of antibodies, is expensive, has high detection costs, and the purely manual processing process is time-consuming.

[0005] Immunomagnetic beads (IMBs) are a magnetic carrier separation technology developed in the late 1970s. They have the high specificity of immunology and the remarkable characteristics of magnetic nanomaterials such as superparamagnetism, small size, and diversified modification. They can quickly capture and separate target compounds under the action of an external magnetic field, avoiding cumbersome solid-liquid separation processes such as filtration or centrifugation, thus realizing a rapid sample pretreatment technology that integrates extraction, purification, and concentration. IMBs were initially mainly used for the separation and purification of biological macromolecules such as nucleic acids, proteins, and microorganisms. In recent years, they have attracted widespread attention due to their advantages such as simple operation, short time consumption, and batch automation, and have gradually been applied to the detection of small molecules such as mycotoxins and veterinary drug residues. Chen et al. used N-hydroxysuccinimide (NHS) activated magnetic Fe3O4 nanomaterials and coupled them with aflatoxin M1 (AFM1) monoclonal antibody to prepare immunomagnetic beads, and established a confirmatory detection method for AFM1 residues in milk by combining LC-MS / MS, achieving rapid processing of 24 milk samples within 13 minutes. Wang et al. used CNBr activated agarose gel (CNBr) coupled with mixed monoclonal antibodies of aflatoxin, fumonisin, T-2 toxin and cyclopiazonic acid (CPA) to successfully construct an immunomagnetic bead purification UPLC-MS / MS simultaneous confirmation analysis method for seven fungal toxins, AFB1, AFB2, AFG1, AFG2, FB1, T-2 and CPA. In general, compared with IAC, immunomagnetic bead purification has lower costs and can be fully automated, freeing the hands of experimental personnel and significantly improving work efficiency and detection throughput.

[0006] Although IMBs separation technology has been partially applied in the field of food safety research in recent years, its related research in feed quality and safety analysis is still scarce. Therefore, how to provide a rapid determination method for the harmful substance zearalenone in feed to solve the above technical problems is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] The purpose of the present invention is to provide a zearalenone immunomagnetic bead and its preparation method and application. The present invention uses IMBs high-throughput fully automatic purification combined with high-performance liquid chromatography to establish a fast, convenient, batch-automatic method for processing ZEN in feed and feed raw material samples, and on this basis, the first group standard of the Feed Industry Association on IMBs purification technology has been studied and formulated, in order to provide a method reference for grassroots testing laboratories and feed companies to prevent and control fungal toxin contamination.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing zearalenone immunomagnetic beads, comprising the following steps:

[0010] (1) washing the N-hydroxysuccinimide activated agarose magnetic beads with ethanol, removing the ethanol, adding 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody, mixing and incubating, and discarding the upper solution;

[0011] (2) adding a buffer containing glycine to the magnetic beads after incubation in step (1), mixing and blocking, separating by magnetic absorption, and obtaining ZEN immunomagnetic beads after elution.

[0012] Preferably, in step (1), the ratio of agarose magnetic beads, 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody is 0.8-1.2 mL: 0.8-1.2 mL: 1.8-2.2 mg; the incubation time is 100-140 min, the temperature is 36-38° C., and the shaking speed is 180-220 rpm.

[0013] Preferably, in step (1), the volume ratio of the agarose magnetic beads to ethanol is 1:8-12; the ethanol is anhydrous ethanol; and the washing time is 4-6 minutes.

[0014] Preferably, in step (2), the volume ratio of the glycine-containing buffer to the agarose magnetic beads is 0.8-1.2:0.8-1.2; the content of glycine in the glycine-containing buffer is 2-3% (m:V); the blocking time is 50-70 min, the temperature is 36-38° C., and the speed of the shaker is 180-220 rpm; the elution method is: eluting 2-3 times with PBS buffer containing 0.1-0.2% (V:V) Tween-20 and PBS buffer in sequence.

[0015] The present invention also provides immunomagnetic beads obtained by the method.

[0016] Preferably, the particle size of the immunomagnetic beads is 10 nm to 2 μm.

[0017] The present invention also provides a kit for detecting zearalenone, comprising a chamber A, a chamber B, a chamber C, a chamber D and a chamber E; the chamber A is a sample to be tested, the chamber B is the immunomagnetic beads according to claim 5 or 6, the chamber C is a PBS cleaning solution, the chamber D is a water cleaning solution, and the chamber E is a methanol eluent.

[0018] The invention also provides application of the kit in the detection of zearalenone.

[0019] Preferably, the method for using the kit is: purifying the immunomagnetic beads and then performing HPLC determination.

[0020] Preferably, the instrument used for purification is a fully automatic mycotoxin purification instrument;

[0021] The purification method is as follows: 1) the magnetic rod transfers the immunomagnetic beads from the B bin to the A bin, mixes with the magnetic rod for 10 to 14 minutes, and magnetically absorbs for 55 to 65 seconds; 2) the magnetic rod transfers the immunomagnetic beads to the C bin, washes for 55 to 65 seconds, and magnetically absorbs for 55 to 65 seconds; 3) the immunomagnetic beads are transferred to the D bin, washed for 55 to 65 seconds, and magnetically absorbed for 55 to 65 seconds; 4) the immunomagnetic beads are transferred to the E bin for elution, eluted for 55 to 65 seconds, and after magnetic absorption for 55 to 65 seconds, the immunomagnetic beads are transferred to the B bin, and the purification procedure is completed;

[0022] The HPLC assay conditions were set as follows: chromatographic column: Agilent Zorbax SB-C18 column; column temperature: 38-42° C.; injection volume 45-55 μL; mobile phase: acetonitrile / water=55 / 45, isocratic elution; flow rate 0.7-0.9 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention aims to establish a method for rapid determination of zearalenone in feed by high-throughput fully automatic purification of immunomagnetic beads combined with high-performance liquid chromatography. First, N-hydroxysuccinimide (NHS) activated agarose magnetic beads were incubated with zearalenone monoclonal antibodies to prepare ZEN immunomagnetic beads, which were characterized by scanning electron microscopy and vibrating sample magnetometer. Feed samples were extracted with 80% acetonitrile water, purified by fully automatic immunomagnetic beads, and determined by high-performance liquid chromatography-fluorescence detector, and quantified by external standard method.

[0025] The results showed that the linear relationship of zearalenone was good in the concentration range of 0.01-1.0 μg / mL (R2≥0.9999), the detection limit of the method was 2 μg / kg, and the quantification limit was 10 μg / kg; the average recovery rate of zearalenone at four added concentration levels of 0.01, 0.02, 0.10 and 1.0 mg / kg was 86.90%-100.27%, the intra-batch relative standard deviation (RSD) was 3.18%-9.88%, and the inter-batch RSD was 4.94%-9.40%.

[0026] Compared with traditional solid phase extraction and immunoaffinity column purification methods, immunomagnetic bead purification integrates extraction, separation, purification and concentration in one, which can realize fully automated processing, significantly improve work efficiency and detection throughput, avoid result deviation caused by human operation, and is suitable for the determination of zearalenone in large-scale feed samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 Schematic diagram of the fully automatic fungal toxin purification instrument and the ZEN immunomagnetic bead purification kit (bin A: PBST sample diluent; bin B: immunomagnetic bead solution; bin C: PBS cleaning solution; bin D: water cleaning solution; bin E: methanol eluent).

[0029] Figure 2 This is the scanning electron microscopy (SEM) image of ZEN immunomagnetic beads.

[0030] Figure 3 Magnetic hysteresis loops of NHS-activated agarose beads and ZEN immunomagnetic beads.

[0031] Figure 4 The effect of different reaction times on the enrichment of zearalenone by immunomagnetic beads (n=6).

[0032] Figure 5 This is the chromatogram of blank corn.

[0033] Figure 6 This is the chromatogram of corn with 0.01 mg / kg zearalenone added.

[0034] Figure 7 This is the chromatogram of corn with 0.10 mg / kg zearalenone added. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing zearalenone immunomagnetic beads, comprising the following steps:

[0036] (1) washing the N-hydroxysuccinimide activated agarose magnetic beads with ethanol, removing the ethanol, adding 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody, mixing and incubating, and discarding the upper solution;

[0037] (2) adding a buffer containing glycine to the magnetic beads after incubation in step (1), mixing and blocking, separating by magnetic absorption, and obtaining ZEN immunomagnetic beads after elution.

[0038] In the present invention, the ratio of agarose magnetic beads, 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody in step (1) is 0.8-1.2 mL: 0.8-1.2 mL: 1.8-2.2 mg; preferably 0.9-1.1 mL: 0.9-1.1 mL: 1.9-2.1 mg; more preferably 1 mL: 1 mL: 2 mg.

[0039] In the present invention, the incubation time is 100-140 min, the temperature is 36-38°C, and the shaking speed is 180-220 rpm; preferably, the incubation time is 110-130 min, the temperature is 37°C, and the shaking speed is 190-210 rpm; further preferably, the incubation time is 120 min, the temperature is 37°C, and the shaking speed is 200 rpm; more preferably.

[0040] In the present invention, the volume ratio of the agarose magnetic beads to ethanol in step (1) is 1:8-12; preferably 1:9-11; and more preferably 1:10.

[0041] In the present invention, the ethanol is anhydrous ethanol;

[0042] In the present invention, the cleaning time is 4 to 6 minutes, preferably 5 minutes.

[0043] In the present invention, the volume ratio of the glycine-containing buffer to the agarose magnetic beads in step (2) is 0.8-1.2:0.8-1.2; preferably 0.9-1.1:0.9-1.1; and more preferably 1:1.

[0044] The content of glycine in the glycine-containing buffer is 2-3% (m:V), preferably 2% (m:V).

[0045] In the present invention, the sealing time is 50 to 70 minutes, the temperature is 36 to 38°C, and the shaking speed is 180 to 220 rpm; preferably, the sealing time is 54 to 66 minutes, the temperature is 37°C, and the shaking speed is 190 to 210 rpm; further preferably, the sealing time is 60 minutes, the temperature is 37°C, and the shaking speed is 200 rpm.

[0046] In the present invention, the elution method is: eluting 2-3 times with PBS buffer containing 0.1-0.2% (V:V) Tween-20 and PBS buffer successively; preferably, eluting 2 times with PBS buffer containing 0.1% (V:V) Tween-20 and PBS buffer successively.

[0047] The present invention also provides immunomagnetic beads obtained by the method.

[0048] In the present invention, the particle size of the immunomagnetic beads is 10 nm to 2 μm, preferably 2 μm.

[0049] The present invention also provides a kit for detecting zearalenone, comprising a chamber A, a chamber B, a chamber C, a chamber D and a chamber E; the chamber A is a sample to be tested, the chamber B is the immunomagnetic beads according to claim 5 or 6, the chamber C is a PBS cleaning solution, the chamber D is a water cleaning solution, and the chamber E is a methanol eluent.

[0050] The invention also provides application of the kit in the detection of zearalenone.

[0051] In the present invention, the method for using the kit is: purifying the immunomagnetic beads and then performing HPLC determination.

[0052] In the present invention, the instrument used for purification is a fully automatic mycotoxin purification instrument;

[0053] In the present invention, the purification method is as follows: 1) the magnetic rod transfers the immunomagnetic beads from the B bin to the A bin, the magnetic rod mixes for 10 to 14 minutes, and the magnetic rod is attracted for 55 to 65 seconds; 2) the magnetic rod transfers the immunomagnetic beads to the C bin, washes for 55 to 65 seconds, and the magnetic rod is attracted for 55 to 65 seconds; 3) the immunomagnetic beads are transferred to the D bin, washed for 55 to 65 seconds, and the magnetic rod is attracted for 55 to 65 seconds; 4) the immunomagnetic beads are transferred to the E bin for elution, eluted for 55 to 65 seconds, and the immunomagnetic beads are transferred to the B bin after 55 to 65 seconds of magnetic attraction, and the purification procedure is ended; preferably, the purification method is as follows: 1) the magnetic rod transfers the immunomagnetic beads from the B bin to the A bin, the magnetic rod mixes for 11 to 13 minutes, and the magnetic rod is attracted for 57 to 63 seconds; 2) the magnetic rod transfers the immunomagnetic beads to the C bin, washes for 57 ~63s, magnetic attraction 57~63s; 3) the immunomagnetic beads are transferred to the D warehouse, washed for 57~63s, and magnetically attracted for 55~65s; 4) the immunomagnetic beads are transferred to the E warehouse for elution, eluted for 57~63s, and magnetically attracted for 57~63s before transferring the immunomagnetic beads to the B warehouse, and the purification procedure is completed; the purification method is further preferably as follows: 1) the magnetic rod transfers the immunomagnetic beads from the B warehouse to the A warehouse, the magnetic rod mixes for 12min, and magnetically attracted for 60s; 2) the magnetic rod transfers the immunomagnetic beads to the C warehouse, washed for 60s, and magnetically attracted for 60s; 3) the immunomagnetic beads are transferred to the D warehouse, washed for 60s, and magnetically attracted for 60s; 4) the immunomagnetic beads are transferred to the E warehouse for elution, eluted for 60s, and magnetically attracted for 60s before transferring the immunomagnetic beads to the B warehouse, and the purification procedure is completed.

[0054] In the present invention, the HPLC determination conditions are set as follows: chromatographic column: Agilent Zorbax SB-C18 column; column temperature: 38-42°C; injection volume 45-55 μL; mobile phase: acetonitrile / water = 55 / 45, isocratic elution; flow rate 0.7-0.9 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm; preferably, the HPLC determination conditions are set as follows: chromatographic column: Agilent Zorbax SB-C18 column; column temperature: 39-41°C; injection volume 47-53 μL; mobile phase: acetonitrile / water = 55 / 45, isocratic elution; flow rate 0.8 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm; further preferably, the HPLC determination conditions are set as follows: chromatographic column: Agilent Zorbax SB-C18 column; column temperature: 40°C; injection volume 50 μL; mobile phase: acetonitrile / water = 55 / 45, isocratic elution; flow rate 0.8 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1

[0057] 1 Materials and methods

[0058] 1.1 Instruments and Equipment

[0059] Agilent 1100 high performance liquid chromatograph: equipped with fluorescence detector; fully automatic mycotoxin purification instrument: Suzhou Beaver Biomedical Engineering Co., Ltd.; ZEN immunoaffinity column: Beijing Huaan Maike Biotechnology Co., Ltd.; nitrogen generator: Shanghai Anpu Experiment Technology Co., Ltd.; N-EVAP 112 nitrogen blower: Organomation, USA; CrestUltrasonics CP 2600D ultrasonic cleaner: Beijing Tianlin Hengtai Technology Co., Ltd.; Milli-Q plus ultrapure water instrument: Millipore, USA; BS210S analytical balance: Sartorius, Germany; MS1 vortex mixer: IKA, Germany; 3K15 high-speed refrigerated centrifuge: Sigma, Germany.

[0060] 1.2 Reagents and Materials

[0061] N-hydroxysuccinimide (NHS)-activated agarose magnetic beads (particle size of approximately 2 μm) were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.; ZEN monoclonal antibody was provided by Beijing Weideweikang Biotechnology Co., Ltd.; ZEN standard solution (100 μg / mL) was purchased from Qingdao Puruibang Bioengineering Co., Ltd.; acetonitrile and methanol (chromatographic grade) were from Fisher, USA; the experimental water was first-class water (≥18.2 MΩ) purified by Milli-Q; sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl), Tween-20, etc. were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; nylon filter membrane (0.45 μm) was purchased from Shanghai Titan Technology Co., Ltd.

[0062] 2-Morpholineethanesulfonic acid (MES) buffer (0.1 mol / L, pH 5.5-6.5) was purchased from Sigma, USA; MES buffer containing 2% glycine: 2.0 g of glycine was weighed, dissolved in MES buffer and fixed to 100 mL.

[0063] Phosphate buffer (PBS, pH 7.0): weigh 8.0 g NaCl, 1.2 g Na2HPO4, 0.2 g KH2PO4 and 0.2 g KCl, dissolve them in water and make up to 1000 mL; PBS buffer containing 0.1% Tween-20 (PBST): take 1 mL Tween-20 and dilute to 1000 mL with PBS solution.

[0064] Feed, feed ingredients and pet feed samples were provided by the National Feed Quality Inspection and Testing Center (Beijing).

[0065] 1.3 Experimental methods

[0066] 1.3.1 Preparation of Zearalenone Immunomagnetic Beads

[0067] 1 mL of N-hydroxysuccinimide (NHS) activated agarose beads was removed, 10 mL of anhydrous ethanol was added and shaken for 5 min, the upper ethanol solution was discarded by magnetic separation, 1 mL of 2-morpholineethanesulfonic acid (MES) buffer and 2 mg of ZEN monoclonal antibody were added, vortexed and mixed, and incubated at 37°C, 200 rpm shaker for 2 h. The upper solution was discarded by magnetic separation, 1 mL of MES buffer containing 2% glycine was added, vortexed and mixed, and the beads were blocked at 37°C, 200 rpm shaker for 1 h. After magnetic separation, the ZEN immunomagnetic beads were washed twice with PBS buffer (PBST) containing 0.1% Tween-20 and PBS buffer, respectively, and then the ZEN immunomagnetic beads were stored in PBS buffer at 4°C for later use.

[0068] 1.3.2 Sample extraction

[0069] Refer to the national standard "Determination of zearalenone in feed by immunoaffinity column purification-high performance liquid chromatography" (GB / T 28716-2012): weigh 10g of feed sample (accurate to 0.01g) in a 50mL centrifuge tube, accurately add 20mL of 80% acetonitrile aqueous solution, vortex mix for 30s, place in an ultrasonic cleaner for ultrasonic extraction for 20min, take out and shake 2 to 3 times during the period, place in a low-temperature high-speed centrifuge at 10000r / min for 5min, and take the supernatant for use.

[0070] 1.3.3 Automatic purification of immunomagnetic beads

[0071] Take 1-2 mL of the supernatant and add it to the sample well A of the ZEN Immunomagnetic Purification Kit. Start the automatic mycotoxin purification instrument and run the ZEN purification program. The automatic mycotoxin purification instrument and the ZEN Immunomagnetic Purification Kit are shown in Figure 2. Figure 1 The ZEN immunomagnetic bead fully automatic purification procedure is as follows: (1) The magnetic rod transfers the immunomagnetic beads from well B to sample well A, mixes with the magnetic rod for 12 minutes, and magnetically absorbs for 60 seconds; (2) The magnetic rod transfers the immunomagnetic beads to washing well C, washes for 60 seconds, and magnetically absorbs for 60 seconds; (3) The magnetic beads are transferred to washing well D, washes for 60 seconds, and magnetically absorbs for 60 seconds; (4) The magnetic beads are transferred to well E for elution, eluted for 60 seconds, and magnetically absorbed for 60 seconds before transferring the magnetic beads to magnetic bead recovery well B, and the purification procedure is completed.

[0072] After purification, transfer the eluate from well E to nitrogen blown to near dryness at 50°C, reconstitute with 0.5mL 10% acetonitrile / water, vortex mix, and filter through a 0.45μm filter before HPLC determination. The automatic purification time of zearalenone in a single kit is about 20min, and 10 samples can be processed online simultaneously.

[0073] 1.3.4 HPLC instrument conditions

[0074] Column: Agilent Zorbax SB-C 18 Column (4.6 mm × 250 mm, particle size 5 μm); column temperature: 40 ° C; injection volume 50 μL; mobile phase: acetonitrile / water = 55 / 45, isocratic elution; flow rate 0.8 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm.

[0075] 2 Results and discussion

[0076] 2.1 Characterization of ZEN Immunomagnetic Beads

[0077] The prepared ZEN immunomagnetic beads were characterized and tested using scanning electron microscopy (SEM). Figure 2As shown in the figure, the black opaque core is agarose magnetic beads, and the lighter outer layer is ZEN monoclonal antibody. The magnetic beads have a regular spherical structure and good dispersion. It can be seen that ZEN monoclonal antibody has successfully bound to the surface of NHS agarose magnetic beads. The magnetic strength was further tested using a vibrating sample magnetometer (VSM) ( Figure 3 ), the magnetic saturation intensity of NHS agarose beads is about 40emu / g, and the magnetic saturation intensity after combining with ZEN monoclonal antibodies is reduced to about 32emu / g. It can be seen that ZEN monoclonal antibodies have been successfully modified on the surface of NHS agarose beads, and the hysteresis loops of agarose beads and ZEN immunomagnetic beads prepared in the study are symmetrical around the origin, and the magnetization curve overlaps with the demagnetization curve.

[0078] In summary, the present invention successfully prepared ZEN immunomagnetic beads. In addition, no coercivity and remanence phenomena were observed during the experiment, indicating that the prepared ZEN immunomagnetic beads have superparamagnetism.

[0079] 2.2 Selection of immunomagnetic bead size

[0080] The size of the particle size has a great influence on the performance index of the immunomagnetic beads. The present invention first compares two magnetic beads of different particle sizes (based on the above step 1.3.1, the same below), nano-scale magnetic beads (particle size of about 10nm) and micro-scale magnetic beads (particle size of about 2μm), and the results are shown in Table 1. It can be seen that the micro-scale magnetic beads of ZEN have a higher load and recovery rate than the nano-scale. The reason for the analysis may be that the monoclonal antibody is usually a few nanometers, and the coupling amount with the nano-scale magnetic beads is small. At the same time, the dispersion of the nano-magnetic beads after coupling is also poor, which is not conducive to the subsequent immune affinity reaction, resulting in a low recovery rate. Relatively speaking, micro-scale magnetic beads have a higher antibody load and dispersion, so micro-scale magnetic beads are selected for subsequent experiments.

[0081] Table 1 Comparison of performance of immunomagnetic beads with different particle sizes (n=6)

[0082]

[0083]

[0084] 2.3 Optimization of immunomagnetic bead reaction time

[0085] IMBs need to fully bind to the target in the sample to achieve the best separation effect. The reaction time between IMBs and the sample extract is the key to magnetic bead purification. Therefore, IMBs were first reacted with a ZEN standard solution with a concentration of 1.0 μg / mL, and the binding efficiency at different reaction times (1, 3, 5, 8, 10, 15 and 20 min) was investigated. The results are shown in Figure 2. Figure 4 As shown. It can be seen that when the reaction time is above 10 min, the adsorption of ZEN by IMBs tends to be stable, and the recovery rate is between 99.1% and 100.1%, indicating that the 10-min reaction time can meet the requirements for the interaction between IMBs and the target. Considering the influence of different sample matrices in the actual detection process, we appropriately extended the reaction time to 12 min to ensure that IMBs can achieve good separation and enrichment effects on different sample matrices.

[0086] 2.4 Linear correlation, detection limit and quantification limit of the method

[0087] The ZEN standard solution (100 μg / mL) was diluted step by step to prepare 6 standard working solutions with different concentration gradients (0.01, 0.05, 0.10, 0.20, 0.50 and 1.0 μg / mL). The standard curve was drawn with the concentration of the standard working solution as the abscissa and the chromatographic peak area as the ordinate. The results are shown in Table 2. The results show that ZEN has a good linear relationship in the concentration range of 0.01-1.0 μg / mL, and the correlation coefficient (R 2 )≥0.9999, which can meet the requirements of detection and analysis. With 3 times the signal-to-noise ratio (S / N) as the limit of detection (LOD) and 10 times the S / N as the limit of quantification (LOQ), the LOD and LOQ of the method are 2μg / kg and 10μg / kg respectively. The LOQ of the method can meet the detection requirements of the mandatory national standard "Feed Hygiene Standard" (GB 13078-2017) on the limit of ZEN in feed and feed raw materials (≤0.1~1.5mg / kg).

[0088] Table 2 ZEN standard curve, detection limit and quantification limit

[0089]

[0090] 2.5 Method accuracy and precision

[0091] Representative blank samples were selected, including feed raw materials such as corn meal, whole distillers dried grains (DDGS), soybean meal, peanut meal, wheat flour, pig, chicken, fish compound feed, concentrated feed and cattle and sheep concentrate supplements, as well as pet feed such as dog and cat food, and the recovery test was carried out. Taking into account the relevant requirements of the US FDA "Bioanalytical Method Validation-Guidance for Industry" (US Food and Drug Administration (US FDA). Bioanalytical Method Validation-Guidance for Industry, 2018. http: / / www.fda.gov / Drugs / Guidance Compliance Regulatory Information / Guidances / default.htm) and the "Feed Hygiene Standard" (GB 13078-2017) on the limit of ZEN in feed and feed ingredients, four added concentration levels of LOQ, 2×LOQ, 10×LOQ and 100×LOQ were set, and 6 parallels were set for each concentration. The samples were processed and determined according to the above method. The accuracy and precision results of the method are shown in Table 3, and the representative HPLC chromatograms are shown in Table 3. Figure 5 to Figure 7 It can be seen that at the addition levels of 0.01, 0.02, 0.10 and 1.0 mg / kg, the average intra-batch recovery of the method was 86.90%-100.27%, and the intra-batch RSD was 3.18%-9.88%; the average inter-batch recovery was 88.70%-99.59%, and the inter-batch RSD was 4.94%-9.40%, indicating that the accuracy and precision of this method are good, and it is suitable for the accurate and rapid determination of ZEN in feed.

[0092] Table 3 Results of accuracy and precision determination of the method

[0093]

[0094]

[0095]

[0096] 2.6 Actual sample measurement

[0097] A total of 223 samples of feed and feed raw materials were collected for inspection, including compound feed, concentrated feed, concentrate supplement and pet feed, as well as corn flour, soybean meal, DDGS, peanut meal, palm kernel meal, rice bran meal, wheat flour and other feed raw materials. The content of ZEN was determined by the fully automatic immunomagnetic bead purification combined with HPLC established by the present invention. As shown in Table 4, the detection rate and content of ZEN in feed raw materials are both high, among which the detection rate of corn flour is 93.8%, and the highest detection content is 4380 μg / kg. According to the limit (≤0.5 mg / kg) of the "Feed Hygiene Standard" (GB 13078-2017), the excess rate is 9.4%. It is worth noting that the detection content, detection rate and excess rate of ZEN in compound feed are all high, indicating that it is necessary to strengthen the prevention and control of mycotoxins in feed products.

[0098] Table 4 Detection content of zearalenone in feed samples

[0099]

[0100]

[0101] 2.7 Comparison with immunoaffinity column cleanup

[0102] With reference to the national standard "Determination of Zearalenone in Feed by Immunoaffinity Column Purification-High Performance Liquid Chromatography" (GB / T 28716-2012), 4 certified reference materials (CRM) of corn flour, wheat flour, animal feed, and pet dog food purchased from FAPAS in the United Kingdom and 6 feed and feed raw material samples provided by the National Feed Quality Inspection and Testing Center (Beijing) were analyzed and tested by immunoaffinity column purification method and immunomagnetic bead purification method established by the present invention, and the results are shown in Table 5. The two groups of data were statistically analyzed by two-tailed T test, and the results showed that there was no significant difference between the two groups of results, indicating that the immunomagnetic bead purification method established by the present invention is well consistent with the immunoaffinity column purification method specified in the current effective national standard (such as GB T 28718-2012). However, the fully automatic mycotoxin purification instrument used in conjunction with the immunomagnetic bead purification method can process 10 samples at a time, and the single purification process takes about 20 minutes, with an average processing time of only about 2 minutes per sample, thus achieving rapid high-throughput detection and analysis of zearalenone in feed. Therefore, when processing large batches of feed samples, fully automatic immunomagnetic bead purification can significantly improve work efficiency and reduce detection bias.

[0103] Table 5 Comparison of purification results of immunomagnetic beads and immunoaffinity columns (n=6)

[0104]

[0105]

[0106] In summary, it is not difficult to see that the present invention has established a method for determining the content of zearalenone in feed by fully automatic immunomagnetic bead purification combined with high performance liquid chromatography. Zearalenone has a good linear relationship in the concentration range of 0.01 to 1.0 μg / mL, the method detection limit is 2 μg / kg, and the quantitative limit is 10 μg / kg; the average recovery rate of zearalenone at four concentration addition levels (0.01, 0.02, 0.10 and 1.0 mg / kg) is 86.90% to 100.27%, and the relative standard deviation is 3.18% to 9.88%. The method is simple to operate, can automatically process samples in batches, significantly improve work efficiency, and is suitable for quantitative analysis of zearalenone in large batches of feed samples.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing zearalenone immunomagnetic beads, characterized in that: The steps include: (1) washing the N-hydroxysuccinimide activated agarose magnetic beads with ethanol, removing the ethanol, adding 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody, mixing and incubating, and discarding the upper solution; (2) adding a buffer containing glycine to the magnetic beads after incubation in step (1), mixing and blocking, separating by magnetic absorption, and obtaining ZEN immunomagnetic beads after elution.

2. The preparation method according to claim 1, characterized in that: In step (1), the ratio of agarose magnetic beads, 2-morpholineethanesulfonic acid buffer and ZEN monoclonal antibody is 0.8-1.2 mL: 0.8-1.2 mL: 1.8-2.2 mg; the incubation time is 100-140 min, the temperature is 36-38° C., and the rotation speed of the shaker is 180-220 rpm.

3. The preparation method according to claim 1, characterized in that: In step (1), the volume ratio of agarose magnetic beads to ethanol is 1:8-12; the ethanol is anhydrous ethanol; and the cleaning time is 4-6 minutes.

4. The preparation method according to claim 1, characterized in that: The volume ratio of the glycine-containing buffer to the agarose magnetic beads in step (2) is 0.8-1.2:0.8-1.2; the content of glycine in the glycine-containing buffer is 2-3% (m:V); the blocking time is 50-70 min, the temperature is 36-38° C., and the rotation speed of the shaking table is 180-220 rpm; the elution method is: eluting 2-3 times with PBS buffer containing 0.1-0.2% (V:V) Tween-20 and PBS buffer in sequence.

5. Immunomagnetic beads obtained by the method according to any one of claims 1 to 4.

6. The immunomagnetic beads according to claim 5, characterized in that The particle size of the immunomagnetic beads is 10 nm to 2 μm.

7. A kit for detecting zearalenone, characterized in that: It comprises warehouse A, warehouse B, warehouse C, warehouse D and warehouse E; warehouse A is the sample to be tested, warehouse B is the immunomagnetic beads according to claim 5 or 6, warehouse C is PBS cleaning solution, warehouse D is water cleaning solution, and warehouse E is methanol eluent.

8. Use of the kit according to claim 7 in the detection of zearalenone.

9. The use according to claim 7, characterized in that: The method of using the kit is: purifying the immunomagnetic beads described in claim 5 or 6 and then performing HPLC determination.

10. The use according to claim 9, characterized in that: The instrument used for the purification is a fully automatic mycotoxin purification instrument; The purification method is as follows: 1) transferring the immunomagnetic beads from bin B to bin A using a magnetic rod, mixing with the magnetic rod for 10 to 14 minutes, and magnetically absorbing for 55 to 65 seconds; 2) Transfer the immunomagnetic beads to chamber C with a magnetic rod, wash for 55-65 seconds, and magnetically absorb for 55-65 seconds; 3) Transfer the immunomagnetic beads to chamber D, wash for 55-65 seconds, and magnetically absorb for 55-65 seconds; 4) The immunomagnetic beads are transferred to chamber E for elution for 55 to 65 seconds. After magnetic absorption for 55 to 65 seconds, the immunomagnetic beads are transferred to chamber B, and the purification process is completed; The HPLC assay conditions were set as follows: chromatographic column: Agilent Zorbax SB-C18 column; column temperature: 38-42° C.; injection volume 45-55 μL; mobile phase: acetonitrile / water=55 / 45, isocratic elution; flow rate 0.7-0.9 mL / min; fluorescence detector: excitation wavelength 274 nm, emission wavelength 440 nm.

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