Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk
Abstract
:1. Introduction
2. Results and Discussion
2.1. Conjugate Preparation
2.2. Coating Antigen Selection
Plate Coating Antigen | Coating Concentration (ng·mL−1) | Titer | Amax | IC50 (ng·mL−1) | LOD (ng·mL−1) | Working Range (ng·mL−1) |
---|---|---|---|---|---|---|
A | 22.2 | 6 × 104 | 1.601 | 632.8 | 221.0 | ND a |
B | 16.7 | 6 × 103 | 1.87 | 107.5 | 21.0 | 41.5–952.8 |
C | 133.3 | 8 × 103 | 1.42 | 35.4 | 8.9 | 14.9–108.5 |
2.3. Cross-Reactivity
Compound | Structure | Space-filling model | Coating antigen C | Coating antigen B | ||
---|---|---|---|---|---|---|
IC50 (nmol·mL−1) | CR% | IC50 (nmol·mL−1) | CR% | |||
Melamine | 0.28 | 100 | 0.85 | 100.0 | ||
Hapten B | 2.31 | 12.1 | 3.41 | 25.0 | ||
Hapten C | 1.54 | 18.0 | 6.44 | 13.2 | ||
Cyromazine | 1.61 | 17.1 | 4.03 | 21.1 | ||
Hapten A | 2.57 | 10.7 | 8.72 | 9.8 | ||
CAAT | 4.43 | 6.2 | 16.33 | 5.2 | ||
Ammeline | ND a | ≤0.01 | ND | ≤0.01 | ||
Ammelide | ND | ≤0.01 | ND | ≤0.01 | ||
Cyanuric acid | ND | ≤0.01 | ND | ≤0.01 | ||
Cyanuric chloride | ND | ≤0.01 | ND | ≤0.01 | ||
Atrazine | ND | ≤0.01 | ND | ≤0.01 |
2.4. Analysis of Spiked Samples
Sample | Added Levels (ng·mL−1) | Observed Value (ng·mL−1) | Recovery (%, n = 3) | Recovery Mean (%) | CV (%) | CV Mean (%) |
---|---|---|---|---|---|---|
Milk | 120.0 | 116.3 ± 7.4 | 96.9 ± 6.2 | 93.4 | 6.4 | 6.0 |
240.0 | 214.5 ± 5.3 | 89.4 ± 2.2 | 2.5 | |||
360.0 | 340.4 ± 13.8 | 94.6 ± 3.8 | 4.0 | |||
480.0 | 435.0 ± 18.2 | 90.6 ± 3.8 | 4.2 | |||
540.0 | 516.5 ± 66.8 | 95.7 ± 12.4 | 12.9 |
2.5. Validation by HPLC-MS
3. Experimental
3.1. Reagents
3.2. Instrumentation
3.3. Preparation of Hapten-Protein Conjugates
3.4. ELISA Protocol
3.5. Specificity
3.6. Analysis of Spiked Samples
4. Conclusions
Acknowledgements
References
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Lei, H.; Su, R.; Haughey, S.A.; Wang, Q.; Xu, Z.; Yang, J.; Shen, Y.; Wang, H.; Jiang, Y.; Sun, Y. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk. Molecules 2011, 16, 5591-5603. https://doi.org/10.3390/molecules16075591
Lei H, Su R, Haughey SA, Wang Q, Xu Z, Yang J, Shen Y, Wang H, Jiang Y, Sun Y. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk. Molecules. 2011; 16(7):5591-5603. https://doi.org/10.3390/molecules16075591
Chicago/Turabian StyleLei, Hongtao, Rui Su, Simon A. Haughey, Qiang Wang, Zhenlin Xu, Jinyi Yang, Yudong Shen, Hong Wang, Yueming Jiang, and Yuanming Sun. 2011. "Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk" Molecules 16, no. 7: 5591-5603. https://doi.org/10.3390/molecules16075591