Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples
Abstract
:1. Introduction
2. Results and Discussion
2.1. Development of Concept for Multiplex Antibody Microarray Immunoassay
2.2. Preparation of the Hardware Prototype for Antibody Microarray Reaction
2.3. Evaluation of Specificity Among Ag-Ab Pairs for the Antibody Microarray
2.4. Calibration Curves for AFB1, OTA, ZEN, and FB1 Detection
2.5. Recovery Studies
2.6. Commercial Samples Analysis
3. Conclusions
4. Materials and Methods
4.1. Reagents
4.2. Apparatus
4.3. Biotinylation and Identification of Mycotoxin-Protein Conjugates
4.4. Antibody Microarray and Reaction Device Design
4.5. Preparation of the Antibody Microarray
4.6. Detection Procedure of the Antibody Microarray
4.7. Signal Acquisition and Data Analysis
4.8. Specificity Among the Four Antibodies and Biotinylated Conjugates
4.9. Recovery Studies
4.10. Detection of Commercial Samples by Antibody Microarray and LC-MS/MS.
Author Contributions
Funding
Conflicts of Interest
References
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Mycotoxins | Concentration of the Antibody (μg/mL) | Concentration of the Biotinylated Antigen (μg/mL) |
---|---|---|
AFB1 | 25 | 0.2 |
OTA | 25 | 0.1 |
ZEN | 50 | 0.4 |
FB1 | 12.5 | 0.4 |
Mycotoxins | LOD (ng/mL) | IC50 (ng/mL) | Detection Range (IC20-IC80, ng/mL) | Regression Equation |
---|---|---|---|---|
AFB1 | 0.21 | 5.12 | 0.47–55.69 | y = 0.2895x + 0.2946 (R2 = 0.9806) |
OTA | 0.19 | 7.82 | 0.48–127.11 | y = 0.2477x + 0.2788 (R2 = 0.9887) |
ZEN | 0.09 | 2.62 | 0.22–31.36 | y = 0.2782x + 0.3837 (R2 = 0.9810) |
FB1 | 0.24 | 7.23 | 0.56–92.57 | y = 0.2709x + 0.2673 (R2 = 0.9936) |
Samples | Concentrations (μg/kg) | Recovery Rate (%) (Mean ± SD a) | CV b (%) | |
---|---|---|---|---|
Spiked | Detected | |||
AFB1 | 20 | 17.27 | 86.3 ± 6.4 | 7.4 |
40 | 31.68 | 79.2 ± 4.7 | 5.9 | |
60 | 54.87 | 91.5 ± 7.1 | 7.8 | |
OTA | 20 | 16.71 | 83.6 ± 8.1 | 9.7 |
40 | 34.87 | 87.2 ± 7.6 | 8.7 | |
60 | 49.73 | 82.9 ± 6.8 | 8.2 | |
ZEN | 50 | 45.86 | 91.7 ± 8.9 | 9.7 |
100 | 89.13 | 89.1 ± 5.7 | 6.4 | |
200 | 213.07 | 106.5 ± 7.3 | 6.9 | |
FB1 | 200 | 189.27 | 94.6 ± 9.1 | 9.6 |
400 | 427.62 | 106.9 ± 7.8 | 7.3 | |
800 | 907.21 | 113.4 ± 8.6 | 7.6 |
Samples | Antibody Microarray (μg/kg, Mean ± SD a) | LC-MS/MS (μg/kg, Mean ± SD) | ||||||
---|---|---|---|---|---|---|---|---|
AFB1 | OTA | ZEN | FB1 | AFB1 | OTA | ZEN | FB1 | |
Corn 1 | - b | 12.31 ± 2.13 | - | - | - | 14.87 ± 1.21 | - | - |
Corn 2 | - | - | 95.37 ± 8.73 | 724.53 ± 57.29 | - | - | 109.14 ± 8.16 | 688.47 ± 32.07 |
Corn 3 | - | - | 72.85 ± 5.26 | 920.76 ± 64.79 | - | - | 85.03 ± 7.39 | 846.59 ± 50.61 |
Corn 4 | - | 27.64 ± 2.49 | - | - | - | 32.36 ± 2.14 | - | - |
Corn 7 | - | 20.86 ± 2.43 | - | 593.71 ± 41.15 | - | 25.57 ± 2.71 | - | 542.04 ± 38.32 |
Corn 11 | - | 22.59 ± 2.04 | - | - | - | 26.71 ± 2.13 | - | - |
Wheat 1 | - | 16.76 ± 1.36 | - | - | - | 20.99 ± 2.71 | - | - |
Wheat 6 | - | - | 29.78 ± 2.54 | 811.23 ± 62.15 | - | - | 34.21 ± 2.33 | 741.48 ± 40.32 |
Wheat 7 | - | 42.33 ± 3.89 | - | - | - | 49.55 ± 3.23 | - | - |
Wheat 11 | 8.79 ± 1.05 | - | 13.96 ± 1.19 | - | 10.11 ± 1.62 | - | 16.67 ± 1.35 | - |
Feed 2 | 23.85 ± 2.68 | - | 15.97 ± 1.34 | 398.92 ± 35.67 | 27.52 ± 1.84 | - | 19.35 ± 2.17 | 347.54 ± 26.38 |
Feed 3 | - | - | 14.69 ± 1.37 | 321.27 ± 28.94 | - | - | 17.52 ± 1.34 | 299.93 ± 21.15 |
Feed 6 | - | - | 11.38 ± 1.67 | - | - | - | 14.84 ± 1.89 | - |
Feed 7 | - | 39.41 ± 2.97 | - | 713.25 ± 61.68 | - | 45.62 ± 3.12 | - | 667.06 ± 48.81 |
Feed 11 | - | - | 26.72 ± 2.51 | 684.82 ± 46.39 | - | - | 30.34 ± 2.19 | 631.28 ± 37.43 |
Feed 12 | - | - | 21.85 ± 2.62 | 461.56 ± 35.73 | - | - | 27.03 ± 2.31 | 420.79 ± 22.27 |
Method | Target Analyte | LOD (ng/mL) | Ref. |
---|---|---|---|
Fluorescent sensor | AFB1 | 14 | [27] |
Aptamer-based fluorescent assay | AFB1 | 1.6 | [28] |
FRET based fluorescence | OTA | 3.5 | [29] |
Lateral flow strip based aptasensor | OTA | 0.40 | [30] |
Novel biosensor platform | OTA | 2.57 | [31] |
ic-ELISA | ZEN | 0.114 | [32] |
Aptamer-based fluorescence assay | ZEN | 0.5 | [34] |
Microarray-Based Immunoassay | FB1 | 11.1 | [35] |
Ic-ELISA | FB1 | 1.15 | [36] |
Cell fluorescence biosensor | ZEN and DON | ZEN: 3.2 | [33] |
Lateral Flow Dual Immunoassay | ZEN and FB1 | ZEN: 0.35, FB1: 5.23 | [38] |
Multicolor ICGA | AFB1, ZEN, and T-2 | AFB1: 0.5, ZEN: 2 | [37] |
Suspension Array | AFB1, ZEN, DON, and FB1 | AFB1: 0.56, ZEN: 0.51, FB1: 6.0 | [14] |
Microarray immunoassay | AFB1, OTA, ZEN, and FB1 | AFB1: 0.21, OTA: 0.19, ZEN: 0.09, FB1: 0.24 | This work |
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Zhang, X.; Wang, Z.; Fang, Y.; Sun, R.; Cao, T.; Paudyal, N.; Fang, W.; Song, H. Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples. Toxins 2018, 10, 415. https://doi.org/10.3390/toxins10100415
Zhang X, Wang Z, Fang Y, Sun R, Cao T, Paudyal N, Fang W, Song H. Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples. Toxins. 2018; 10(10):415. https://doi.org/10.3390/toxins10100415
Chicago/Turabian StyleZhang, Xian, Zuohuan Wang, Yun Fang, Renjie Sun, Tong Cao, Narayan Paudyal, Weihuan Fang, and Houhui Song. 2018. "Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples" Toxins 10, no. 10: 415. https://doi.org/10.3390/toxins10100415