Monoclonal antibody for monospecific resistance to zearalenone-14 glucoside and application

Abstract

The invention discloses a monospecific monoclonal antibody for resisting zearalenone-14 glucoside and application of the monospecific monoclonal antibody. An immunogen and a coating antigen are used; the monoclonal antibody capable of monospecifically recognizing the zearalenone-14 glucoside and the rapid detection kit for detecting the zearalenone-14 glucoside are obtained through the technologies of mouse immunization, serum detection, cell fusion, monoclonal screening, ascites induced antibody and the like. The method is suitable for single-specificity trace residue detection of the zearalenone-14 glucoside in samples such as corn, rice, millet, wheat and oat, has very high recognition sensitivity to the zearalenone-14 glucoside, and realizes rapid, large-batch and low-cost detection of the zearalenone-14 glucoside.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, and particularly relates to a monospecific monoclonal antibody against zearalenone-14-glucoside and application thereof. BACKGROUND

[0002] Zearalenone-14-glucoside (Z14G) is a masked mycotoxin produced by plants after being contaminated by molds through endogenous metabolism, which belongs to the glycosidation modification product of zearalenone (ZEN). Its molecular formula is C 24 H 32 O9, and its relative molecular mass is 464.51. Z14G mainly exists in corn, wheat, barley and other cereals, and is formed by glycosidation of the 14th phenolic hydroxyl group of ZEN by UDP-glucose transferase (UGTs) in plants. As a Phase II metabolic form of ZEN, Z14G has high polarity and metabolic stability in crops and is not easily detected by conventional detection methods, and is therefore referred to as a "masked mycotoxin".

[0003] The potential harm of Z14G lies in its release of the parent toxin ZEN in the animal digestive tract by intestinal microorganisms or enzymatic hydrolysis, thereby indirectly exerting toxic effects. ZEN belongs to a non-steroidal estrogenic toxin, which can interfere with the endocrine function of the animal body after being ingested, causing reproductive disorders, miscarriage or precocious puberty and other problems. Studies have shown that the residues of Z14G in food have a certain stability and persistence, and can enter the animal body through the feed chain, ultimately affecting human health. Experiments have also found that Z14G can affect the hormone levels of mammals and interfere with their normal physiological metabolic processes. Although the toxicity of Z14G mainly depends on the release of the parent ZEN, it has a higher food safety risk due to its difficulty in detection.

[0004] In order to protect public health, different countries and international organizations have regulated the maximum residue limit (MRL) of ZEN in corn, wheat and related livestock products, which is usually between 0.02 and 0.5 mg / kg, but the regulation of masked toxins such as Z14G is still not perfect. The conventional detection methods of Z14G include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and other technologies, although these methods have high sensitivity and strong accuracy, but they usually rely on expensive equipment and complex pretreatment steps, which are not conducive to on-site rapid screening. Therefore, the development of sensitive, convenient and low-cost detection means is of great significance for food safety. SUMMARY

[0005] The main purpose of the present application is to provide a monospecific monoclonal antibody against zearalenone-14-glucoside and application, aiming to prepare a monoclonal antibody with strong specificity by synthesizing an immunogen and a coating antigen, and establish a corresponding enzyme-linked immunoassay method, so as to realize rapid, large-scale and low-cost detection of zearalenone-14-glucoside.

[0006] To achieve the above-mentioned purpose, in the first aspect, the present application provides a monospecific monoclonal antibody against zearalenone-14-glucoside, and the structure of zearalenone-14-glucoside is shown as formula I, and the monospecific monoclonal antibody against zearalenone-14-glucoside is characterized in that the immunizing hapten and the coating hapten of the monospecific monoclonal antibody against zearalenone-14-glucoside are both prepared by reacting zearalenone-14-glucoside with carboxymethoxylamine hemisalt, and the structure is shown as formula II.

[0007]

[0008] The immunogen is obtained by reacting the immunizing hapten with bovine serum albumin, and the coating antigen is obtained by reacting the coating hapten with chicken egg white albumin;

[0009] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows:

[0010] QVQLKQSGPGLVQPSQSLSLTCTVSDLSFTNYGVHWIRQSPEKGLEWL GMIWSAGNTDYNAAFISRLKIIKDSSKSQVFFTMNSLQTDDTAIYYCATYDF AFWGQGTLVTVSA.

[0011] The amino acid sequence of the light chain variable region of the monoclonal antibody is as follows:

[0012] DVVMTQTPLTLSVTFGQLASISCKSSQNLLYSNGETYLNWLLQRPGQS PKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCVQCTHFP CTFGGGTKLEIK.

[0013] In the present application, the immunogen is obtained by coupling the immunizing hapten with the carrier protein BSA, and the coating antigen is obtained by coupling the coating hapten with the carrier protein OVA, the serum is detected by using the ELISA method, the spleen of the mouse with good immunization effect is taken out and fused with tumor cells, the hybridoma cell strain is screened and obtained, and finally the monoclonal antibody is induced by ascites.

[0014] In a second aspect, the present application provides a use of a monospecific monoclonal antibody against zearalenone-14-glucoside in the preparation of a kit for detecting zearalenone-14-glucoside.

[0015] In a third aspect, the present application provides a kit comprising the monospecific monoclonal antibody against zearalenone-14-glucoside as described above.

[0016] In a third aspect, the present application provides a kit comprising the monospecific monoclonal antibody against zearalenone-14-glucoside as described above.

[0017] In a fourth aspect, the present application provides an enzyme-linked immunoassay method for monospecific detection of zearalenone-14-glucoside residues in food, which uses the monospecific monoclonal antibody against zearalenone-14-glucoside as described above, and which comprises the following steps:

[0018] Step S10, diluting the coating agent and coating the solid phase carrier;

[0019] Step S20, diluting the monoclonal antibody and preparing a monoclonal antibody working solution;

[0020] Step S30, extracting the sample to be tested with an organic solvent and preparing a sample solution to be tested;

[0021] Step S40, adding the sample solution to be tested and the monoclonal antibody working solution to the solid phase carrier and incubating;

[0022] Step S50, adding horseradish peroxidase-labeled goat anti-mouse IgG (enzyme-labeled secondary antibody) and incubating;

[0023] Step S60, adding a substrate solution and a stop solution to develop color, measuring the OD value with an enzyme-labeled instrument and calculating the content of zearalenone-14-glucoside.

[0024] In the present application, the concentration of the diluted coating agent is 0.5 μg / mL, the concentration of the monoclonal antibody working solution is 0.4 μg / mL, and the color development times of the monoclonal antibody, enzyme-labeled secondary antibody and substrate solution are 45 min, 35 min and 15 min, respectively.

[0025] The present application has the following beneficial effects:

[0026] 1. Specificity: The monospecific monoclonal antibody against zearalenone-14-glucoside in the application can achieve monospecific detection of zearalenone-14-glucoside, and the cross-reactivity rate with other zearalenone and its derivatives (including zearalenone, zearalenone-16-glucoside, zearalenone-14-sulfate, a-zearalanol, β-zearalanol, a-zearalenol and β-zearalenol) is less than 11%.

[0027] 2. Sensitivity: The monospecific monoclonal antibody against zearalenone-14-glucoside in the application has high recognition sensitivity to zearalenone-14-glucoside, and the detection sensitivity is further improved by optimizing the detection conditions, so that the IC 50 value reaches 0.093 μg / L, and the minimum detection limit in corn, rice, millet, wheat and oat reaches 0.012 μg / kg. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings shown.

[0029] Figure 1 is the standard curve for detecting zearalenone-14-glucoside by enzyme-linked immunoassay in embodiment 3 of the application, wherein the X axis is the logarithm of the concentration of the zearalenone-14-glucoside standard solution, and the Y axis is the inhibition rate, i.e. the absorbance value of the blank well minus the absorbance value of the standard solution, and then divided by the absorbance value of the blank well [(OD0-OD x ) / OD0] x 100%.

[0030] The implementation, functional features and advantages of the application will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0032] At present, in the field of zearalenone-14-glucoside residue detection, the enzyme-linked immunosorbent method based on immune analysis is the most mature and the highest recognized, which is simple to operate and low in cost, and is very suitable for screening of large quantities of samples. The present application realizes rapid, large quantity and low cost detection of zearalenone-14-glucoside by synthesizing immunogen and coating antigen, preparing monoclonal antibody with strong specificity, and establishing corresponding enzyme-linked immune detection method.

[0033] Example 1: Preparation of zearalenone-14-glucoside immunogen and coating antigen

[0034] 1. Preparation of immunizing hapten:

[0035] 4mg of zearalenone-14-glucoside and 9mg of carboxymethoxylamine hemi-hydrochloride were weighed into a reaction bottle, 2mL of pyridine was added, and stirring was carried out at 55℃ water bath for 24 hours, TLC was used to monitor the completion degree of the reaction, after the reaction was completed, nitrogen was used for blowing dry, and then it was redissolved in 3mL of DMF ultrapure water. After adjusting the pH to 8 using 0.1M NaOH solution, equal volume of ethyl acetate was used for extraction, and the extraction was repeated three times, the ethyl acetate phase was collected, and nitrogen was used for blowing dry, thereby obtaining the immunizing hapten.

[0036] 2. Preparation of coating hapten:

[0037] 4mg of zearalenone-14-glucoside and 9mg of carboxymethoxylamine hemi-hydrochloride were weighed into a reaction bottle, 2mL of pyridine was added, and stirring was carried out at 55℃ water bath for 24 hours, TLC was used to monitor the completion degree of the reaction, after the reaction was completed, nitrogen was used for blowing dry, and then it was redissolved in 3mL of DMF ultrapure water. After adjusting the pH to 8 using 0.1M NaOH solution, equal volume of ethyl acetate was used for extraction, and the extraction was repeated three times, the ethyl acetate phase was collected, and nitrogen was used for blowing dry, thereby obtaining the coating hapten.

[0038] 3. Preparation of immunogen:

[0039] The prepared zearalenone-14-glucoside immunizing hapten was dissolved in 1mL of DMF, 4mg of NHS and 6mg of EDC were added, and stirring was carried out at room temperature for 12h; 10mg of BSA was weighed and dissolved in 2mL of PBS (pH=7.4). The above-mentioned activated solution was added dropwise to the BSA solution, and stirring was carried out while adding, and stirring was carried out at 4℃ for 12h. The above-mentioned reaction solution was loaded into a dialysis bag, and PBS was used for dialysis at 4℃ for 3-5d, the dialysis liquid was changed three times a day, and centrifugation was carried out at 10000r / min for 15min, and the supernatant was the required immunogen, which was stored in a-20℃ refrigerator for storage.

[0040] 4. Preparation of coating antigen:

[0041] The prepared zearalenone-14 glucoside hapten was dissolved in 1 mL DMF, 4 mg of NHS and 6 mg of EDC were added, and stirring was carried out at room temperature for 12 h. 10 mg of OVA was weighed and dissolved in 2 mL of PBS (pH = 7.4). The above-mentioned activated solution was added dropwise to the OVA solution, stirring was carried out while adding, and stirring was carried out at 4°C for 12 h. The above-mentioned reaction solution was loaded into a dialysis bag, and dialysis was carried out with PBS at 4°C for 3-5 days, the dialysis solution was changed three times a day, centrifugation was carried out at 10,000 r / min for 10 min, and the supernatant was the required coating antigen, which was stored in a refrigerator at -20°C for storage.

[0042] Example 2: Preparation of monospecific zearalenone-14 glucoside monoclonal antibody:

[0043] 1. Animal immunization:

[0044] BALB / c mice were immunized with the zearalenone-14 glucoside immunogen prepared in Example 1, and the serum of the tail tip was collected on the 7th day after the fourth immunization for detection to determine whether the prepared zearalenone-14 glucoside antigen had immunocompetence. 5-6 week-old BALB / c mice were selected for immunization, and the above-prepared 1 mg / mL immunogen was emulsified with an equal amount of Freund's adjuvant, and then the mice were immunized by subcutaneous multi-point injection, 10 μg per mouse. Freund's complete adjuvant was used for the first immunization, Freund's incomplete adjuvant was used for the booster immunization, and physiological saline was used to dilute the immunogen to the corresponding concentration for intraperitoneal injection for the challenge immunization; the first immunization and the second immunization were separated by 3 weeks, and the booster immunization was separated by 2 weeks. Blood was collected one week after the third immunization to detect the serum titer and inhibition; the mouse with good titer and inhibition was selected for challenge immunization three days before fusion.

[0045] 2. Cell fusion:

[0046] Three days after the challenge immunization, cell fusion was carried out according to the conventional PEG (molecular weight 2000) method, and the specific steps were as follows:

[0047] a. The mouse was anesthetized with ether, and immediately after the mouse was sacrificed by cervical dislocation, it was soaked in 75% alcohol for disinfection for about 5 min, and the spleen of the mouse was taken out under sterile conditions, ground with a homogenizer, and passed through a 40 μm cell screen to obtain a spleen cell suspension, which was collected, centrifuged (1200 rpm, 8 min), and the internal connective tissue was removed and centrifuged, and the spleen cells were diluted to a certain volume, counted, and reserved for use; SP2 / 0 cells were collected:

[0048] b. 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 complete medium containing 10% fetal bovine serum in a 5% CO2 incubator. The required SP2 / 0 tumor cell count before fusion should reach 1-4 × 10⁻⁶ cells / year. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected during fusion, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0049] c. Mix spleen cells and SP2 / 0 cells at a counting ratio of 1:10, centrifuge, and then fuse with 50% PEG for 1 min. After that, add RPMI-1640 basal culture medium from slow to fast to terminate the reaction. After centrifugation, suspend the cells in RPMI-1640 selection culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add the medium to a 96-well cell culture plate, and incubate at 37°C and 5% CO2.

[0050] 3. Cell screening:

[0051] Three days after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening. The screening consisted of two steps: first, positive cell wells were selected using indirect ELISA; second, the inhibitory effect on positive cells was determined using indirect competitive ELISA with zearalenone-14-glucoside as a standard. Cell wells showing good inhibition against both the standard and the standard were selected for subcloning using limiting dilution, and the same method was used for detection. The experiment was repeated three times to obtain a hybridoma cell line that produces a monoclonal antibody against zearalenone-14-glucoside. The hybridoma cell line was named Z14G-1C1 and deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: C2025304 and deposit date of 2025-10-09.

[0052] 4. Preparation of monoclonal antibodies:

[0053] 8-10 week old BALB / c mice were injected intraperitoneally with 0.5 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Hybridoma cells, from the seventh day, collect ascites, using the mouse monoclonal antibody subtype identification kit from Wuhan Three Eagle Biotechnology Co., Ltd. The subtype of the monoclonal antibody obtained by the application is identified, and the determination result is heavy chain IgG1 subtype and light chain Kappa subtype. After BCA protein concentration kit determination, the concentration of the antibody is 24 mg / mL. The collected ascites is placed in a-20℃ refrigerator for preservation for standby.

[0054] Example 3: Development of enzyme-linked immunoassay rapid detection kit:

[0055] 1. Establishment of enzyme-linked immunosorbent assay and condition optimization:

[0056] 1.1 Preparation of reagents (the reagents used in this example are prepared by the following method unless otherwise specified);

[0057] Phosphate buffer solution (phosphate-buffered saline, PBS, pH 7.4): weigh 8.00g NaCl, 0.20g KH2PO4, 2.90g Na2HPO4·12H2O, 0.20g KCl, a small amount of deionized water, dissolve, and make up to 1000mL (note: in this paper, unless otherwise specified, the PBS is pH 7.4);

[0058] Coating solution: weigh 1.59g Na2CO3, 2.93g NaHCO3, a small amount of deionized water, dissolve, and make up to 1000mL;

[0059] Washing solution: 8.0g NaCl, 0.2g KH2P04, 2.9g Na2HPO4·12H2O, 0.2g KCl, 0.5mL Tween20, add three times distilled water to 1000mL, adjust pH to 7.4;

[0060] Blocking solution: weigh 10.00g ovalbumin in 1000mL phosphate buffer solution;

[0061] Substrate A solution: weigh 160mg TMB, add 10ml dimethylacetamide, dissolve and mix evenly;

[0062] Substrate B solution: weigh 13.70g citric acid, 10.14g trisodium citrate and 282.00mg hydrogen peroxide, a small amount of deionized water, dissolve, and make up to 1000mL;

[0063] Substrate mixture: take 10mL of substrate B solution, add 100μL of substrate A solution, mix well, and use immediately;

[0064] Termination solution: measure 100mL of concentrated sulfuric acid, and slowly add to 800mL of deionized water.

[0065] 1.2, Determination of coating concentration and antibody working concentration:

[0066] The coating concentration and antibody working concentration were determined according to the square array titration method. The above-synthesized coating was selected for coating, diluted with CBS to 0.8, 0.4, 0.2, 0.1, 0.05 and 0.025 μg / mL 6 concentrations, then each concentration was added longitudinally to the enzyme-labeled plate, 100 μL per well, 4°C coated overnight, washed 3 times. 250 μL of 1% OVA solution was added to the enzyme-labeled well, incubated at 37°C for 90 min to block the non-specific binding sites, and washed 3 times. 50 μL of PBS was added to the enzyme-labeled well, and the monoclonal antibody prepared in Example 2 was diluted with PBS to 240, 80, 40, 26.6, 20, 16, 13.3, 11.4 ng / mL respectively, a total of 8 concentrations, added to the enzyme-labeled plate horizontally, 50 μL per well, removed after incubation at 37°C for 40 min, washed 3 times. The HRP-labeled goat anti-mouse secondary antibody was diluted with PBS to a working concentration of 1:10000 (batch number PA174421, Thermo Fisher Scientific), 100 μL per well, incubated at 37°C for 50 min, then removed, washed 3 times. 100 μL of color developing solution was added to the enzyme-labeled well, and color was developed at 37°C for 15 min in the dark, 50 μL of stop solution was added, and the optical density value (OD value) was determined at 450 nm wavelength by the enzyme-labeled instrument. The combination of antigen coating concentration and antibody dilution corresponding to the well with OD value between 1.8-2.2 and significantly different from the adjacent well value was selected.

[0067] The experimental results are shown in Table 1. The coating concentration was determined to be 0.4 μg / mL, the antibody working concentration was 16 ng / mL, the OD value of the blank well was 1.938, and the OD value was between 1.8-2.2 and significantly different from the adjacent value. The next step of optimization was carried out. 450

[0068] Table 1 Zearalenone-14 glucoside monoclonal antibody square array titration

[0069]

[0070] 1.3, Establishment of standard curve:

[0071] The zearalenone-14 glucoside standard solution was prepared into 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192 ng / L 14 serial standard concentrations, and the zearalenone-14 glucoside standard curve was established according to the conditions of the optimized enzyme-linked immunosorbent method. The Lg[c(Z14G)] was taken as the abscissa, and [(OD0-OD x ) / OD0]x100% was taken as the ordinate to draw the linear regression standard curve and calculate the IC​50 Value. Result as follows Figure 1 As shown. The regression equation of the fitted standard straight line is y = a + bx, where a = -35.56, b = 43.45, and R0 = 0. 2 =0.992, IC 50 The value was 0.093 μg / L.

[0072] 1.4 Cross-reactivity test:

[0073] Seven zearalenones and their derivatives, namely zearalenone-16-glucoside, zearalenone-14-sulfate, α-zearalenol, β-zearalenol, and α-zearalenol, were formulated to appropriate concentrations. Standard curves for these drugs were established under optimized conditions, and IC50 values ​​were calculated. 50 IC50 of zearalenone-14 glucoside standard 50 The cross-reactivity rate of other compounds was compared and calculated using the standard value. The results are shown in Table 2. Among the seven known zearalenones and their derivatives, only α-zearalenol had a cross-reactivity rate of 10.37%, while the rest were all below 10%. Therefore, this antibody can guarantee single-specific recognition of zearalenone-14 glucoside.

[0074] Table 2 Cross-reactivity rates of monoclonal antibodies and analogs

[0075] Zearalenone and its derivatives names IC 50 (μg / L) CR (%) Zearalenone-14 glucoside 0.093 100 Zearalenone-16 glucoside 27.75 0.335 Zearalenone-14 sulfate 8.96 1.038 Zearalenone 1.03 9.029 a-Zearalanol 18.87 0.493 β-Zearalanol 21.96 0.424 a-Zearalenol 0.897 10.370 β-Zearalenol 24.86 0.374

[0076] 2. Assembly of the enzyme-linked immunosorbent assay (ELISA) rapid test kit:

[0077] 2.1 The rapid detection kit of the present invention consists of the following parts:

[0078] (1) Solid-phase carrier (ELISA plate) coated with zearalenone-14-glucoside coating agent;

[0079] (2) Five bottles of zearalenone-14-glucoside standard solution with concentrations of 0.025, 0.05, 0.1, 0.2 and 0.4 μg / L, respectively;

[0080] (3) Concentrated anti-zearalenone-14-glucoside monoclonal antibody solution 24 mg / mL;

[0081] (4) Horseradish peroxidase (HRP) labeled working solution of goat anti-mouse IgG antibody;

[0082] (5) Concentrated phosphate buffer: 80.0g NaCl, 2.0g KH2PO4, 29.0g Na2HPO4·12H2O, 2.0g KCl, add deionized water to 1000mL;

[0083] (6) Concentrated washing solution: 80.0 g NaCl, 2.0 g KH2PO4, 29.0 g Na2HPO4·12H2O, 2.0 g KCl, add double distilled water to 1000 mL; 5 mL Tween 20, add deionized water to 1000 mL;

[0084] (7) Substrate A solution: accurately weigh 160 mg TMB, dissolve in 10 mL dimethylacetamide and mix well;

[0085] (8) Substrate B solution: accurately weigh 13.70 g citric acid, 10.14 g trisodium citrate and 282.00 mg hydrogen peroxide, dissolve in a small amount of deionized water, and dilute to 1000 mL; substrate mixture solution: accurately pipette 10 mL of substrate B solution, add 100 μL of substrate A solution, mix well, and prepare immediately before use;

[0086] (9) Stop solution: accurately measure 100 mL of concentrated sulfuric acid, and slowly add it to 800 mL of deionized water.

[0087] 2.2, Preparation of enzyme-labeled plate:

[0088] Dilute the coating agent to 0.4 μg / mL with the coating solution, add 100 μL per well, and incubate overnight at 4°C. Remove the coating solution, wash each well 3 times with 250 μL of PBST washing solution, pat dry, then add 250 μL of 1% OVA blocking solution per well, incubate at 37°C for 1.5 h, pour out the liquid in the wells, wash 3 times, pat dry, and store in a vacuum-sealed tin foil paper.

[0089] 3, Determination procedure of enzyme-linked immunosorbent rapid detection kit:

[0090] 3.1, Preparation of reagents:

[0091] (1) Sample extraction solution: 50% ethanol. Take 500 mL of ethanol, add ultrapure water to 1000 mL, and mix well.

[0092] (2) Phosphate buffer: dilute the concentrated phosphate buffer provided in the kit with deionized water 10 times before use;

[0093] (3) Washing solution: dilute the washing solution provided in the kit with triple distilled water 10 times before use;

[0094] (4) Monoclonal antibody working solution: dilute the monoclonal antibody concentrate with phosphate buffer to a final concentration of 16 ng / mL.

[0095] 3.2, Sample pretreatment:

[0096] Corn, rice, millet, wheat and oat: 1 g ± 0.01 g sample was taken in a 10 mL centrifuge tube, 5 mL of extractant was added, shaken for 5 min, ultrasonic for 15 min, 8000 r / min, 4℃ centrifugation for 10 min; take 5 mL of supernatant, dilute 20 times and then detect.

[0097] 3.3, determination step:

[0098] (1) sample addition: 50 μL of zearalenone-14-glucoside standard solution or sample extract was added to the micro-well of the enzyme-labeled plate, then 50 μL of monoclonal antibody working solution was added, and placed in a wet box, incubated at 37℃ for 40 min;

[0099] (2) washing: pour out the liquid in the well, add 250 μL of washing solution to each well, wash 3 times and pat dry;

[0100] (3) addition of horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody working solution: 100 μL of horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody working solution was added to each well, and placed in a wet box, incubated at 37℃ for 50 min;

[0101] (4) washing: pour out the liquid in the well, add 250 μL of washing solution to each well, wash 3 times and pat dry;

[0102] (5) addition of substrate: 100 μL of substrate mixture was added to each well, and placed in a wet box, incubated at 37℃ for 15 min;

[0103] (6) addition of stop solution: 50 μL of stop solution was added to each well;

[0104] (7) determination: the optical density value (OD value) of each well was determined by enzyme-labeled instrument at 450 nm wavelength.

[0105] 3.4, result judgment:

[0106] Standard curve:

[0107] The measured inhibition rate [(OD0-OD x ) / OD0] × 100% was taken as the ordinate, and Lg[c(Z14G)] was taken as the abscissa to draw a standard curve, and linear regression was carried out to obtain the regression equation.

[0108] Calculation of the concentration of zearalenone-14-glucoside in the sample:

[0109] The inhibition rate of the sample was calculated, and the regression equation of the standard curve was calculated to calculate the concentration of zearalenone-14-glucoside in the sample.

[0110] 4, sensitivity, precision, accuracy and repeatability test of the kit:

[0111] 4.1 Sensitivity test of the reagent kit:

[0112] IC with standard curve 50 The limits of detection (LODs) for corn, rice, millet, wheat, and oats were used as sensitivity indicators for the detection kit of this invention. Zearalenone-14 glucoside standard was diluted to six concentrations: 16, 32, 64, 128, 256, and 512 ng / L. Each concentration was tested in five replicates. The assay was repeated five times using the enzyme-linked immunosorbent assay (ELISA) method, and the IC50 values ​​from the five assays were collected. 50 The LOD (Limit of Detection) is determined through the following steps: OD values ​​of 20 blank samples of corn, rice, millet, wheat, and oats are measured; the corresponding zearalenone-14 glucoside concentration is calculated based on the regression equation of the standard curve; then, the mean (X) and standard deviation (SD) of the zearalenone-14 glucoside concentration are calculated; the lowest limit of detection is calculated using the formula Z = X + 3 × SD; and the limit of quantitation (LOQ) in corn, rice, millet, wheat, and oats is calculated using the formula Z = X + 10 × SD. The IC of this invention... 50 The value was 0.093 μg / L. The limits of detection for zearalenone-14 glucoside in corn, rice, millet, wheat and oats are detailed in Table 3.

[0113] Table 3. Detection limits of zearalenone-14-glucoside in actual samples.

[0114] Sample LOD (ng / kg) LOQ (ng / kg) Corn 12.63 20.03 Rice 13.75 23.72 Millet 13.97 22.19 Wheat 19.95 35.88 Oats 17.62 24.47

[0115] 4.2 Precision test of the reagent kit:

[0116] Zearalenone-14-glucoside standard was diluted to six concentrations: 16, 32, 64, 128, 256, and 512 ng / L. Each concentration was tested in five replicates. The assay was repeated five times using the enzyme-linked immunosorbent assay (ELISA) method. The measured values ​​of each concentration of standard solution were calculated using the regression equation of the standard curve. The intra- and inter-plate coefficients of variation were calculated. The results are shown in Table 4.

[0117] Table 4. Intra- and inter-plate coefficients of variation of the standard curve

[0118]

[0119] 4.3 Accuracy and repeatability tests of the reagent kit of this invention:

[0120] Different drug concentrations were added to corn, rice, millet, wheat, and oats, and their recoveries and coefficients of variation were calculated according to Formula 1. The results are shown in Table 5. The average recoveries for the five samples ranged from 82.10% to 113.1%, and the coefficients of variation were less than 11.2%.

[0121] Recovery rate (%) = measured concentration / added concentration x 100% (Formula 1)

[0122] Table 5 Addition recovery rate of zearalenone-14-glucoside in actual samples

[0123]

[0124] The beneficial effects of the present application are:

[0125] 1. Specificity: The monospecific monoclonal antibody against zearalenone-14-glucoside of the present application can achieve monospecific detection of zearalenone-14-glucoside, and the cross reaction rate with other structurally similar zearalenone and its derivatives (including zearalenone, zearalenone-16-glucoside, zearalenone-14-sulfate, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol) is less than 1%.

[0126] 2. Sensitivity: The monospecific monoclonal antibody against zearalenone-14-glucoside of the present application has high recognition sensitivity for zearalenone-14-glucoside, and the detection sensitivity is further improved by optimizing the detection conditions, so that the IC 50 value reaches 0.093 μg / L, and the minimum detection limit in corn, rice, millet, wheat and oat reaches 0.012 μg / kg.

[0127] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation made under the inventive concept of the present application, or direct / indirect application in other related technical fields is included in the patent protection scope of the present application.

Claims

1. A monospecific monoclonal antibody against zearalenone-14-glucoside, characterized in that, The structure of the zearalenone-14-glucoside is shown as formula I, and the hybridoma cell strain is named hybridoma cell strain Z14G-1C1, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C2025304, and the preservation date is October 9, 2025; The immunizing hapten and the coating hapten of the monospecific monoclonal antibody against zearalenone-14-glucoside are prepared by reacting zearalenone-14-glucoside with carboxymethoxylamine hemihydrochloride, and the structure is shown as formula II; The immunogen is obtained by reacting the immunizing hapten with bovine serum albumin, and the coating antigen is obtained by reacting the coating hapten with chicken egg white albumin.

2. Use of the monospecific monoclonal antibody against zearalenone-14-glucoside according to claim 1 in the preparation of a kit for detecting zearalenone-14-glucoside.

3. A kit characterized in that, The kit comprises the monospecific monoclonal antibody against zearalenone-14-glucoside according to claim 1.

4. The kit of claim 3, wherein The kit is an enzyme-linked immunosorbent kit for monospecific detection of zearalenone-14-glucoside.

5. A single specificity enzyme-linked immunoassay method for detecting zearalenone- 14-glucoside residues in foodstuffs, characterized in that, The method adopts the monospecific monoclonal antibody against zearalenone-14-glucoside according to claim 1, and the method comprises the following steps: Step S10, diluting the coating antigen and coating a solid phase carrier; Step S20, diluting the monoclonal antibody and preparing a monoclonal antibody working solution; Step S30, extracting the sample to be tested with an organic solvent and preparing a sample solution to be tested; Step S40, adding the sample solution to be tested and the monoclonal antibody working solution to the solid phase carrier and incubating; Step S50, adding horseradish peroxidase-labeled goat anti-mouse IgG and incubating; Step S60, adding a substrate solution and a stop solution for color development, measuring the OD value with an enzyme-labeled instrument, and calculating the content of zearalenone-14-glucoside.

6. The single-specific enzyme-linked immunoassay method for detecting zearalenone- 14-glucoside residues in food according to claim 5, characterized in that, The concentration of the diluted coating antigen is 0.4 μg / mL, the concentration of the monoclonal antibody working solution is 16 ng / mL, and the color development times of the monoclonal antibody, horseradish peroxidase-labeled goat anti-mouse IgG, and the substrate solution are 40 min, 50 min, and 15 min, respectively.

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