Colorimetric / fluorescent immunoassay kits and methods for zearalenone

Abstract

This invention discloses a colorimetric / fluorescent immunoassay kit and method for detecting zearalenone (ZEN). The kit includes an ELISA plate, zearalenone antigen, zearalenone monoclonal antibody, horseradish peroxidase-labeled IgG antibody, substrate solution, and H₂SO₄. The substrate solution includes PPD, PTA-NH₂, H₂O₂, and buffer. Furthermore, this kit is used to establish a novel colorimetric / fluorescent dual-mode immunoassay method for ZEN detection based on PTA-NH₂ and PPD. The detection method developed in this invention has advantages such as simple operation, high sensitivity and specificity, and applicability to ZEN detection in real samples, providing a new method for detecting ZEN residues in grains.

Description

Technical Field

[0001] This invention belongs to the field of zearalenone (ZEN) detection technology, specifically relating to a detection kit and a new detection method for zearalenone based on colorimetric / fluorescence dual-mode immunoassay using diaminoterephthalic acid (PTA-NH2) and p-phenylenediamine (PPD). Background Technology

[0002] Zearalenone (ZEN) is a toxic secondary metabolite produced by Fusarium fungi. It primarily contaminates cereal crops such as corn, sorghum, wheat, and barley, and is not easily destroyed during food and feed processing. ZEN exhibits mutagenic, teratogenic, carcinogenic, nephrotoxic, immunotoxic, and genotoxic properties. It can enter the human or animal body through contaminated crops such as cereals, endangering the health of any animal. Currently, most countries have very strict regulations on the ZEN content in food, cereals, and feed. For example, Australia stipulates that the ZEN content in cereals cannot exceed 0.05 mg / kg, Italy stipulates that the ZEN content in cereals and cereal products cannot exceed 0.1 mg / kg, and France stipulates that the ZEN content in vegetable oils and cereals must be less than 0.2 mg / kg.

[0003] The analysis and determination of zearalenone in zearalenone generally employs thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). TLC is cumbersome and time-consuming, with poor sensitivity and specificity. It requires a large variety and quantity of organic solvents during extraction, easily polluting the environment and posing significant health risks. While conventional HPLC is more sensitive than TLC, its cumbersome sample pretreatment procedures and complex operation limit its widespread adoption. LC-MS, the international standard analytical method of ZEN, offers good accuracy, sensitivity, and repeatability; however, the need for specialized analysts to operate the instrument and the complex sample preparation methods make it unsuitable for large-scale, rapid on-site analysis.

[0004] Therefore, there is a need to develop highly sensitive, rapid, and simple methods to detect the residual ZEN content in food samples and feed products. Summary of the Invention

[0005] Based on this, the present invention aims to provide a new, sensitive, accurate, simple and easy-to-use dual-mode immunoassay for the detection of ZEN.

[0006] The specific technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a colorimetric / fluorescent dual-mode immunoassay kit for zearalenone, comprising an enzyme-labeled plate, zearalenone antigen, zearalenone monoclonal antibody (Ab1), horseradish peroxidase-labeled IgG (denoted as HPR-IgG) antibody, substrate solution and H2SO4, wherein the substrate solution comprises PPD, PTA-NH2, H2O2 and buffer.

[0008] Preferably, in the above-mentioned detection kit, the zearalenone antigen is a complete antigen of bovine serum albumin conjugated with ZEN (denoted as ZEN-BSA).

[0009] Based on the above-mentioned detection kit, the second aspect of this invention constructs a colorimetric / fluorescence dual-mode immunoassay method for zearalenone, specifically including the following steps:

[0010] S1. Coat the zearalenone antigen onto an ELISA plate;

[0011] S2. Add the sample to be tested and Ab1 to the enzyme-labeled plate prepared in S1 at the same time, incubate, wash and pat dry;

[0012] S3. Add HPR-IgG, incubate, then wash and pat dry.

[0013] S4. After adding the substrate solution and incubating, first record the fluorescence intensity at 426 nm under 360 nm excitation light, then add H2SO4 and record the absorbance at 415 nm.

[0014] S5. Calculate the concentration of zearalenone in the sample based on fluorescence intensity, absorbance, and standard curve.

[0015] Preferably, in the above detection method, step S1 specifically involves: diluting the zearalenone antigen with CBS buffer, adding it to an ELISA plate for incubation, washing it with PBST buffer (PBS buffer + Tween 20), and then patting it dry.

[0016] In the above detection method, the optimal working concentration for coating with zearalenone antigen and the optimal working concentration of Ab1 in step S2 can be determined by checkerboard titration, specifically by measuring fluorescence intensity and OD. 415 The optimal working concentrations of ZEN-BSA and Ab1 were determined based on the large value mutations and the low antigen-antibody concentrations. Under the conditions of one embodiment of the present invention, the optimal working concentrations of both ZEN-BSA and Ab1 were 250 ng / mL.

[0017] More preferably, in the above detection method, the incubation conditions of zearalenone antigen in the ELISA plate are: incubation at room temperature (20-30℃) for 50-100 min, with 60 min being the optimal incubation time.

[0018] Preferably, in the above detection method, the sample preparation method is as follows: add methanol-water solution to the ZEN-contaminated sample, extract by ultrasonication, and centrifuge to obtain the supernatant. In actual detection, if the ZEN content in the sample is too high, the supernatant can be diluted to a suitable concentration with PBS buffer before detection.

[0019] Preferably, in the above detection method, step S3 specifically involves: diluting HPR-IgG with PBST buffer, adding it to the ELISA plate, incubating at room temperature, washing and patting dry. The optimal working concentration of HPR-IgG can be selected based on the lowest fluorescence intensity and the OD of the positive wells. 415 Average value / negative pore OD 415 (P / N value) (P (positive) is the signal value after adding the sample in the normal experiment, and N (negative) is the signal value without adding the antibody, but with everything else added. Since there is no antibody, there is no reaction, so it is the background control signal value). The maximum corresponding HRP-IgG concentration. Under the conditions of one embodiment of the present invention, the optimal working concentration of HRP-IgG is 140 ng / mL. Preferably, in the above detection method, the substrate solution is specifically a 10 mM PBS buffer containing 50 mM PPD, 2 μM PTA-NH2, and 4 mM H2O2.

[0020] Preferably, in the above detection method, the incubation time of the substrate solution in step S4 should be selected as the optimal substrate reaction time, corresponding to the time with the minimum fluorescence intensity and the maximum P / N value. In one embodiment of the present invention, the optimal incubation time of the substrate solution at room temperature is 20 min.

[0021] Preferably, in the above detection method, the standard curve in step S5 includes a fluorescence standard curve equation and a colorimetric standard curve equation, wherein:

[0022] The standard curve equation for the fluorescence method is plotted with the logarithm of the zearalenone concentration on the x-axis and F / F0 on the y-axis; F represents the fluorescence intensity value obtained when using different concentrations of zearalenone standards as test samples (F...). 426 F0 is the fluorescence intensity value obtained when using a standard with a zearalenone concentration of zero as the test sample. 426 In one embodiment of the present invention, the curve equation is Y = 1.310 + 0.135X, R 2 =0.996, detection range is 0.012 ng / mL to 3.125 ng / mL;

[0023] The colorimetric standard curve equation uses the logarithm of zearalenone concentration as the x-axis and the inhibition rate as the y-axis; inhibition rate (%) = [1 - (OD200%). A -OD 对照) / OD A ]×100%, OD 对照 The absorbance value (OD) obtained when using a standard with a zearalenone concentration of zero as the sample to be tested. 415 ), OD A The absorbance values ​​(OD) obtained when using zearalenone standards of different concentrations as test samples are shown. 415 In one embodiment of the present invention, the equation of the curve is Y = 49.517 + 32.884X, R 2 =0.995, detection range: 0.048ng / mL~3.125ng / mL.

[0024] This invention develops a dual-mode immunoassay for ZEN by combining the ic-ELISA mode with PPD and PTA-NH2. Specifically, free ZEN in the test sample competes with ZEN antigen (such as ZEN-BSA) coated in the wells of the ELISA plate for binding to Ab1. After the addition of HRP-IgG, a ZEN antigen@Ab1@HRP-IgG complex is formed, thus the ZEN concentration in the test sample is negatively correlated with the HRP concentration. In the presence of HRP and H2O2, PPD is oxidized to PPDox. PPDox has a broad absorption peak at 450 nm, and this peak effectively overlaps with the fluorescence emission spectrum of PTA-NH2. Therefore, PPDox quenches the fluorescence of PTA-NH2 under the action of IFE. PPDox is terminated by H2SO4 to produce a yellow quinone compound, p-benzoquinone (PBQ), which can be detected by colorimetric method.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: the detection kit and detection method developed by the present invention can simultaneously detect ZEN using both fluorescence and colorimetric signals, and the detection results are mutually corroborative, resulting in higher accuracy; the detection process is simple and easy to perform, suitable for rapid analysis of large-scale samples and on-site, and can be used for the detection of ZEN in actual maize samples, and the sample pretreatment operation is simple; in addition, the data from the examples show that the dual-mode immunoassay method developed by the present invention has good selectivity and high specificity for the detection of ZEN. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the principle of the fluorescence colorimetric dual-signal system based on PTA-NH2 and PPD for detecting ZEN developed in this invention;

[0027] Figure 2 This is a comparison of the fluorescence emission spectrum of PTA-NH2 and the UV-Vis absorption spectrum of PPDox in Example 1;

[0028] Figure 3The changes in fluorescence intensity of PTA-NH2 under different treatment methods in Example 1;

[0029] Figure 4 The UV-Vis absorption spectra of PPD under different processing methods in Example 1 are shown.

[0030] Figure 5 The images show the UV-Vis absorption and fluorescence emission spectra (360 nm excitation light) of different concentrations of HRP-IgG in Example 1.

[0031] Figure 6 This is a graph showing the effect of different incubation and coating conditions on fluorescence intensity and P / N value in Example 2;

[0032] Figure 7 This is a graph showing the effect of different concentrations of HRP-IgG on fluorescence intensity and P / N value in Example 2;

[0033] Figure 8 This is a graph showing the effect of different substrate incubation times on light intensity and P / N ratio in Example 2;

[0034] Figure 9 This is the standard curve plotted using the fluorescence method in Example 2;

[0035] Figure 10 This is the colorimetric standard curve plotted in Example 2;

[0036] Figure 11 This is a comparison chart of the detection results of ZEN and other fungal toxins using the method of the present invention in Example 3. Detailed Implementation

[0037] To make the technical problems, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0038] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0039] Example 1

[0040] The assay kit provided in this example includes an ELISA plate, ZEN-BSA, Ab1, HRP-IgG, substrate solutions (PPD, PTA-NH2, H2O2, and 10mM PBS buffer), and H2SO4. Additionally, it includes other auxiliary reagents such as CBS buffer (10mM, pH 9.6) for ZEN-BSA dilution and PBST buffer (10mM PBS buffer, 5% Tween 20) for ELISA plate washing and HRP-IgG dilution.

[0041] The principle of detecting ZEN using the above-mentioned test kit is as follows: Figure 1 As shown, ZEN-BSA competitively binds to Ab1 in the test sample and then binds to HRP-IgG to form a ZEN-BSA@Ab1@HRP-IgG complex; PPD forms PPDox, which can quench PTA-NH2 fluorescence in the presence of HRP and H2O2, and PPDox generates yellow PBQ under the action of H2SO4; therefore, the concentration of ZEN in the test sample can be confirmed by measuring fluorescence intensity and absorbance.

[0042] This example further verifies the feasibility of the above detection principle through the following experimental process:

[0043] (1) The fluorescence emission spectrum of PTA-NH2 and the UV-Vis absorption spectrum of PPDox have effective overlap.

[0044] Prepare two sets of samples: ① 2 μM PTA-NH2, ② 50 mM PPD + 10 ng / mL HRP-IgG + 4 mM H2O2. React each set of samples at room temperature for 5 min to allow for complete reaction. Then, add 200 μL of each sample to a 96-well microplate. Using a microplate reader, measure the fluorescence emission spectrum of sample ① at 400–700 nm under 360 nm excitation light; measure the UV-Vis absorption spectrum of sample ② at 350–700 nm.

[0045] The results are as follows Figure 2 As shown: The UV-Vis absorption spectrum of PPDox has a broad characteristic absorption peak at 450 nm, which effectively overlaps with the fluorescence emission spectrum of PTA-NH2, satisfying the conditions for IFE to occur.

[0046] (2) The fluorescence intensity of PTA-NH2 is affected by the generation of PPDox.

[0047] Prepare three sets of samples: ① 50 μL 10 μM PTA-NH2 + 150 μL PBS buffer (10 mM pH = 7.4), ② 50 μL 10 μM PTA-NH2 + 50 μL 50 mM PPD + 100 μL PBS buffer, ③ 50 μL 10 μM PTA-NH2 + 50 μL 50 mM PPD + 40 μL 10 ng / mL HRP-IgG + 10 μL 2 mM H2O2 + 50 μL PBS buffer. Measure the fluorescence emission spectra of the microplate at 400–600 nm under 360 nm excitation light using a microplate reader.

[0048] The results are as follows Figure 3 As shown: The presence of PPD alone does not affect the fluorescence intensity of PTA-NH2. However, when it is oxidized to PPDox, an IFE effect occurs, quenching the fluorescence of PTA-NH2. ① represents the fluorescence intensity of PTA-NH2 alone. ② represents the fluorescence intensity of PTA-NH2 + PPD; the presence of PPD has almost no effect on the fluorescence intensity of PTA-NH2. ③ represents the fluorescence intensity of PTA-NH2 + PPDox; in the presence of H2O2, PPD is oxidized to PPDox by HRP. Due to the high overlap between its absorption spectrum and the fluorescence emission spectrum of PTA-NH2, the fluorescence of PTA-NH2 is quenched based on IFE.

[0049] (3) PPDox is terminated by sulfuric acid to form a yellow quinone compound PBQ.

[0050] Prepare three sets of samples: ① 100 μL 50 mM PPD + 200 μL PBS buffer; ② 100 μL 50 mM PPD + 80 μL 10 ng / mL HRP-IgG + 20 μL 2 mM H2O2 + 100 μL PBS buffer; ③ 100 μL 50 mM PPD + 80 μL 10 ng / mL HRP-IgG + 20 μL 2 mM H2O2 + 100 μL 2 M H2SO4. Add 200 μL of each set to a 96-well microplate and measure the UV-Vis absorption spectrum from 370 to 700 nm using a microplate reader.

[0051] The results are as follows Figure 4 As shown: PPD is oxidized to PPDox in the presence of HRP and H2O2, with a weak absorption peak at 450 nm; after the reaction is terminated by the addition of H2SO4, a yellow quinone compound PBQ is generated, with a strong absorption peak at 415 nm; the inset shows actual photographs of PPD, PPDox, and PBQ under visible light.

[0052] (4) Effect of HRP-IgG concentration on PPDox generation and PTA-NH2 fluorescence intensity.

[0053] Prepare six sets of samples: 50 μL 10 μM PTA-NH2 + 50 μL 50 mM PPD + 40 μL HRP-IgG (① 40, ② 20, ③ 10, ④ 5, ⑤ 2.5, ⑥ 0 ng / mL) + 10 μL 2 mM H2O2. In a 96-well microplate, use a microplate reader to measure the UV-Vis absorption spectrum at 400–600 nm (A) and the fluorescence emission spectrum at 400–600 nm under 360 nm excitation light (B).

[0054] The results are as follows Figure 5 As shown, with the increase of HRP-IgG concentration, the absorption peak at 400–600 nm gradually increases. Figure 5 A), the fluorescence intensity at 426 nm gradually decreases ( Figure 5 B) indicates that the formation of PPDox is positively correlated with the concentration of HRP-IgG, while the fluorescence intensity of PTA-NH2 is negatively correlated with the formation of PPDox.

[0055] Example 2

[0056] Based on the detection kit provided in Example 1, this example constructs a detection method for ZEN by optimizing parameters such as the concentrations of ZEN-BSA, Ab1, and HRP-IgG, the ZEN-BSA coating conditions, and the substrate incubation time.

[0057] This example includes the following experimental procedure:

[0058] (1) Optimization of ZEN-BSA and Ab1 concentrations.

[0059] In this example, the checkerboard titration method was used to determine the optimal working concentrations of ZEN-BSA and Ab1. ZEN-BSA was diluted with CBS buffer to six different concentrations (2000, 1000, 500, 250, 125, 62.5 ng / mL) and coated into six columns of a 96-well microplate, incubated overnight at 4°C. After washing three times with PBST buffer and drying, eight different concentrations of Ab1 diluted with PBS buffer (1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 ng / mL) were added to eight rows of the microplate, incubated at room temperature for 1 hour, and then washed and dried. Subsequently, 100 μL of HRP-IgG diluted with PBST buffer was added to each well, incubated at room temperature for 1 hour, and then washed and dried. Add 150 μL of 10 mM PBS buffer, containing 50 mM PPD, 2 μM PTA-NH2, and 4 mM H2O2, and incubate at room temperature for 30 min. Record the fluorescence intensity (F) at 426 nm using a microplate reader under 360 nm excitation light. 426The reaction was terminated with 50 μL of 2M H2SO4, and the absorbance value (OD) at 415 nm was recorded using a microplate reader. 415 ). F 426 and OD 415 The optimal working concentrations of ZEN-BSA and Ab1 were determined based on the large value mutations and the low antigen-antibody concentrations.

[0060] F obtained by chessboard titration 426 The values ​​and P / N values ​​are shown in Tables 1 and 2, F 426 The optimal working concentrations for ZEN-BSA and Ab1 were determined to be those with larger variations in P / N values ​​and relatively lower antigen-antibody concentrations. The final optimal working concentrations for both ZEN-BSA and Ab1 were 250 ng / mL.

[0061] Table 1. Determination of optimal working concentrations of ZEN-BSA and Ab1 using fluorescence method.

[0062]

[0063] Table 2. Determination of optimal working concentrations of ZEN-BSA and Ab1 by colorimetric method.

[0064]

[0065]

[0066] (2) Optimization of ZEN-BSA encapsulation conditions.

[0067] Based on the optimal working concentrations of ZEN-BSA and Ab1 determined in step (1), the optimal coating conditions for ZEN-BSA are further determined.

[0068] Five different incubation and coating conditions were set: incubation at room temperature (25℃) for 20 min, 40 min, 60 min, 80 min, 100 min, and incubation at 4℃ for 12 h. Three replicate wells and one negative control well were grouped together. The remaining steps were the same as step (1). The coating condition corresponding to the minimum fluorescence intensity and the maximum P / N value was determined as the optimal incubation and coating condition for ZEN-BSA.

[0069] The results are as follows Figure 6As shown: Under room temperature coating conditions, the P / N value gradually increases from 20 min to 60 min with increasing time, but further increases in time do not significantly increase the P / N value; the P / N value is very small at 4℃. Similarly, under room temperature coating conditions, the fluorescence intensity at 426 nm gradually decreases from 20 min to 60 min with increasing time, but further increases in time do not significantly decrease the fluorescence intensity; the fluorescence intensity is very high at 4℃. Therefore, based on the principle of saving time, room temperature for 60 min is selected as the optimal incubation and coating condition for ZEN-BSA in this example.

[0070] (3) Optimization of HRP-IgG concentration.

[0071] Based on the conditions optimized in steps (1) and (2), the optimal working concentration of HRP-IgG was further optimized.

[0072] Six different HRP-IgG concentrations were set: 60, 80, 100, 120, 140, and 160 ng / mL. Three replicate wells and one negative control well were used as a group. The remaining steps were the same as the detection steps described above. The HRP-IgG concentration corresponding to the lowest fluorescence intensity and the highest P / N value was determined as the optimal working concentration of HRP-IgG.

[0073] The results are as follows Figure 7 As shown: When the HRP-IgG concentration is 60–140 ng / mL, the P / N ratio gradually increases with increasing HRP-IgG concentration; however, when the HRP-IgG concentration exceeds 140 ng / mL, the P / N ratio shows no significant change. Similarly, when the HRP-IgG concentration is 60–140 ng / mL, the fluorescence intensity at 426 nm gradually decreases with increasing HRP-IgG concentration; however, when the HRP-IgG concentration exceeds 140 ng / mL, the fluorescence intensity at 426 nm shows no significant change. Therefore, based on the principle of reagent conservation, 140 ng / mL is selected as the optimal working concentration of HRP-IgG.

[0074] (4) Optimization of substrate incubation time.

[0075] Based on the optimization conditions of the above three steps, the optimal substrate interaction time is further optimized.

[0076] Six different substrate incubation times were set: 5, 10, 15, 20, 25, and 30 min. Three replicate wells and one negative control well were grouped together. The remaining steps were the same as the detection steps described above. The time corresponding to the minimum fluorescence intensity and the maximum P / N value was determined as the optimal substrate incubation time.

[0077] The results are as follows Figure 8As shown, when the substrate incubation time is 5–20 min, the P / N ratio gradually increases with increasing incubation time. However, when the incubation time exceeds 20 min, the P / N ratio shows no significant change with further increases in incubation time. Similarly, when the substrate incubation time is 5–20 min, the fluorescence intensity at 426 nm gradually decreases with increasing incubation time. However, when the incubation time exceeds 20 min, the fluorescence intensity at 426 nm shows no significant change with further increases in incubation time. Therefore, based on the principle of saving time, 20 min is selected as the optimal substrate incubation time.

[0078] (5) The optimized ZEN detection process is as follows:

[0079] ① Dilute ZEN-BSA with CBS buffer to 250 ng / mL, coat it in a 96-well microplate, incubate at room temperature for 60 min, wash three times with PBST buffer and pat dry;

[0080] ② The sample to be tested was reacted with Ab1 (250 ng / mL) diluted with PBS buffer. The resulting reaction was also added to the microplate and incubated at room temperature for 1 h. After washing with PBST buffer, the plate was patted dry.

[0081] ③ Add 100 μL of HRP-IgG diluted in PBST buffer (140 ng / mL) to each well, incubate at room temperature for 1 h, then wash and pat dry;

[0082] ④ Add 150 μL of substrate solution (including 10 mM PBS buffer, 50 mM PPD, 2 μM PTA-NH2 and 4 mM H2O2) and incubate at room temperature for 20 min;

[0083] ⑤ Using an ELISA reader, record the fluorescence intensity (F) at 426 nm under 360 nm excitation light. 426 );

[0084] ⑥ Add 50 μL of 2M H2SO4 to terminate the reaction, and record the absorbance (OD) at 415 nm using a microplate reader. 415 ).

[0085] (6) Confirmation of sensitivity and linear detection range.

[0086] Following step (5), the sensitivity and linear detection range of the ratiometric immunoassay were determined by establishing a standard curve. ZEN standard was diluted 2-fold from 200 ng / mL in PBS buffer to 17 concentration gradients, and a blank control group was set up. The F values ​​at different concentrations of ZEN standard were obtained. 426 The inhibition rate and the fluorescence and colorimetric standard curves for detecting ZEN were plotted through data analysis and processing.

[0087] ① Establish a standard curve equation for fluorescence method with the logarithm of ZEN concentration on the x-axis and F / F0 on the y-axis. F is the concentration of ZEN in the presence of ZEN. 426 F0 is F without ZEN. 426 .like Figure 9 As shown, the standard curve equation for the linear fit is: Y = 1.310 + 0.135X, R0 2 =0.996. The limit of detection is 0.012 ng / mL, and the detection range is 0.012 ng / mL to 3.125 ng / mL.

[0088] ② Establish a colorimetric standard curve equation with the logarithm of ZEN concentration on the x-axis and the inhibition rate on the y-axis. Inhibition rate (%) = [1 - (OD)] A -OD 对照 ) / OD A ]×100%, of which OD 对照 Zero standard sample well OD 415 Value, OD A OD of standards at different concentrations 415 Value. For example... Figure 10 As shown, the standard curve equation for the linear fit is: Y = 49.517 + 32.884X, R0 2 =0.995. The limit of detection is 0.048 ng / mL, and the detection range is 0.048 ng / mL to 3.125 ng / mL.

[0089] Example 3

[0090] Based on the detection method, optimal detection conditions, and standard curve constructed in Example 2, this example further analyzes the selectivity of the immunization method.

[0091] Five different mycotoxins at concentrations of 200 ng / mL—aflatoxin B1 (AFB1), vomitoxin (DON), fumonisin B1 (FB1), ochratoxin A (OTA), and T-2 toxin (T-2)—were used for specificity analysis. A dual-mode immunoassay was performed using six mycotoxins instead of 10 ng / mL ZEN.

[0092] The selectivity of the dual-modality immunoassay was evaluated by detecting cross-reactivity with five fungal toxins: AFB1, AFM1, FB1, T-2, OTA, and ZEN. Results are as follows: Figure 11 As shown, only ZEN elicited a significant dual-signal change, while other fungal toxins remained at a blank level (F / F0 = 1, P / N = 1). The non-specific binding-induced response was significantly lower than the ZEN-induced response. These results indicate that the established dual-mode immunoassay has good selectivity for the detection of ZEN.

[0093] Example 4

[0094] This example uses an actual cornmeal sample for detection and analysis, based on the detection method, optimal detection conditions, and standard curve constructed in Example 2. The experimental process is as follows:

[0095] (1) Add ZEN standard to the ground and crushed corn flour sample and store at room temperature overnight. The final concentrations of ZEN are 0, 0.1, 1 and 2 μg / kg.

[0096] (2) Add 70% (v / v) methanol-water solution, sonicate for 10 min, and centrifuge at 25℃ and 4000g for 10 min.

[0097] (3) Take the supernatant and dilute it 20 times with PBS buffer. Then, follow the steps (5) of Example 2 to recover and analyze the spiked samples.

[0098] (4) Calculate the recovery rate (ZEN concentration determined / ZEN concentration added × 100%) and the coefficient of variation (CV, standard deviation / mean × 100%).

[0099] The applicability of ZEN detection in real maize samples is shown in Table 3. The recovery rate of the fluorescence method was 95.5%–102.0%, with a coefficient of variation of less than 8%; the recovery rate of the colorimetric method was 94.0%–104.0%, with a coefficient of variation of less than 6%. This meets the requirements of the American Association of Analytical Chemists (AOAC) for the effective range of recovery rates. This indicates that the proposed dual-mode immunoassay method has good accuracy and precision. These results demonstrate that the dual-mode immunoassay method proposed in this invention can be used for the detection of ZEN in maize samples.

[0100] Table 3. Recovery rate of ZEN in cornmeal samples

[0101]

[0102]

[0103] In summary, the detection kit and method provided by this invention can achieve dual-signal detection of ZEN, and are simple to operate. They exhibit high sensitivity and specificity, and can be used for the detection of ZEN in grains and their processed products, providing a new method for detecting ZEN residues in grains.

[0104] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A colorimetric / fluorescent dual-mode immunoassay kit for zearalenone, characterized in that, The product includes an ELISA plate, zearalenone antigen, zearalenone monoclonal antibody, horseradish peroxidase-labeled IgG antibody, substrate solution, and H2SO4, wherein the substrate solution includes PPD, PTANH2, H2O2, and PBS buffer. The enzyme-labeled plate is used to immobilize the zearalenone antigen, and the zearalenone antigen is zearalenone conjugated with bovine serum albumin. The H2SO4 is used to oxidize PPD to obtain PPDox and generate PBQ.

2. A colorimetric / fluorescence dual-mode immunoassay method for zearalenone, characterized in that, The detection using the test kit described in claim 1 specifically includes the following steps: S1. Coat the zearalenone antigen onto an ELISA plate; S2. Add the sample to be tested and the zearalenone monoclonal antibody to the enzyme-labeled plate prepared in S1, incubate, wash and pat dry. S3. Add horseradish peroxidase-labeled IgG antibody, incubate, wash and pat dry; S4. After adding the substrate solution and incubating, record the fluorescence intensity at 426 nm under 360 nm excitation light, then add H2SO4 and record the absorbance at 415 nm. S5. Calculate the concentration of zearalenone in the sample based on fluorescence intensity, absorbance, and standard curve.

3. The colorimetric / fluorescence dual-mode immunoassay method according to claim 2, characterized in that, The standard curve includes: The standard curve of fluorescence method is plotted with the logarithm of zearalenone concentration on the x-axis and F / F0 on the y-axis, where F is the fluorescence intensity value in the presence of different concentrations of zearalenone and F0 is the fluorescence intensity value in the absence of zearalenone. A colorimetric standard curve was constructed with the logarithm of zearalenone concentration on the x-axis and the inhibition rate on the y-axis. The inhibition rate (%) was calculated as [1 - (ODA - OD control) / ODA] × 100%, where OD control was the OD value in the absence of zearalenone. 415 Values, where ODA is the OD value in the presence of different concentrations of zearalenone. 415 value.

4. The colorimetric / fluorescence dual-mode immunoassay method according to claim 2, characterized in that, The process of coating zearalenone antigen onto an ELISA plate is as follows: dilute the zearalenone antigen with CBS buffer, add it to the ELISA plate for incubation, wash with PBST buffer, and then pat dry.

5. The colorimetric / fluorescence dual-mode immunoassay method according to claim 4, characterized in that, The concentration of the zearalenone antigen in CBS buffer is 250 ng / mL, and its coating incubation time is 60–80 min.

6. The colorimetric / fluorescence dual-mode immunoassay method according to claim 2, characterized in that, The incubation time of the substrate solution in step S4 is 20 to 30 minutes.

7. The colorimetric / fluorescence dual-mode immunoassay method according to claim 2, characterized in that, The method for preparing the test sample is as follows: add methanol aqueous solution to the ZEN contaminated sample, extract by ultrasonication, and then centrifuge to obtain the supernatant.

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