Detection of β-Lactamase Residues in Milk by Sandwich ELISA
Abstract
:1. Introduction
2. Experimental Section
2.1. Chemicals and Materials
2.2. Solutions
2.3. Antibodies and Conjugated Antibodies
2.4. Sandwich ELISA
2.5. Indirect ELISA
2.6. Pairwise Interaction Analysis
2.7. Selection of Optimum mAb Pairs for β-Lactamase Detection
2.8. Assessment of Cross-Reactivity of the Developed Sandwich ELSA
2.9. Sample Preparation
2.10. Analysis of β-Lactamase-Spiked Milk by Sandwich ELISA
3. Results and Discussion
3.1. Production of Anti-β-Lactamase mAbs
3.2. Pairwise Interaction Analysis of mAbs
D mAb | Capture mAb | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | |
1-HRP | 12.29 | 2.07 | 3.48 | 7.30 | 7.77 | 5.72 | 7.25 | 2.00 | 9.85 | 8.29 | 2.59 | 3.56 | 1.84 | 1.18 | |
2-HRP | 11.88 | 2.36 | 15.28 | 6.41 | 5.24 | 6.05 | 5.96 | 4.47 | 3.09 | 3.74 | 3.72 | 4.78 | 3.26 | 1.43 | |
3-HRP | 3.57 | 1.99 | 4.21 | 1.19 | 1.38 | 1.11 | 1.41 | 1.20 | 2.16 | 1.92 | 1.07 | 1.34 | 1.27 | 0.92 | |
4-HRP | 5.95 | 14.70 | 4.51 | 12.56 | 12.81 | 10.60 | 10.73 | 2.78 | 13.09 | 9.11 | 3.84 | 4.69 | 1.93 | 0.91 | |
5-HRP | 9.70 | 4.77 | 1.38 | 11.35 | 3.01 | 1.96 | 2.75 | 1.86 | 5.13 | 2.52 | 1.37 | 2.29 | 2.10 | 1.19 | |
6-HRP | 5.18 | 1.60 | 1.34 | 5.73 | 2.16 | 2.11 | 1.78 | 1.60 | 1.17 | 1.42 | 1.37 | 1.72 | 1.67 | 1.10 | |
7-HRP | 3.01 | 2.26 | 1.00 | 6.20 | 1.29 | 1.59 | 1.76 | 1.45 | 2.75 | 2.09 | 1.06 | 1.25 | 1.33 | 0.89 | |
8-HRP | 3.97 | 1.69 | 1.38 | 7.37 | 2.66 | 2.39 | 2.98 | 0.99 | 1.71 | 2.17 | 1.24 | 2.35 | 1.95 | 1.18 | |
9-HRP | 0.96 | 1.82 | 0.89 | 0.90 | 1.27 | 1.33 | 1.19 | 1.14 | 2.14 | 1.10 | 0.77 | 1.01 | 1.01 | 1.01 | |
10-HRP | 11.41 | 5.16 | 2.41 | 13.10 | 4.93 | 5.23 | 5.80 | 4.95 | 3.65 | 3.97 | 3.80 | 5.06 | 3.71 | 1.79 | |
11-HRP | 10.56 | 5.09 | 2.53 | 15.55 | 6.10 | 4.81 | 6.35 | 5.06 | 3.81 | 2.76 | 4.47 | 4.22 | 3.25 | 1.58 | |
12-HRP | 2.34 | 2.73 | 1.06 | 2.97 | 1.99 | 2.24 | 1.64 | 1.48 | 1.07 | 3.45 | 2.03 | 1.54 | 1.27 | 0.91 | |
13-HRP | 3.13 | 4.64 | 1.21 | 4.66 | 2.25 | 2.57 | 2.00 | 2.78 | 1.47 | 3.93 | 2.88 | 1.77 | 1.49 | 1.31 | |
14-HRP | 3.86 | 9.41 | 2.19 | 3.63 | 4.79 | 6.10 | 5.33 | 5.11 | 1.74 | 10.96 | 7.80 | 2.75 | 1.13 | 0.92 | |
15-HRP | 1.63 | 2.83 | 1.19 | 1.19 | 1.93 | 1.77 | 1.59 | 1.50 | 1.08 | 4.32 | 2.11 | 1.01 | 3.95 | 1.67 |
3.3. Selection of an Optimum mAb Pair for β-Lactamase Detection
3.4. Development of mAb-Based Sandwich ELISA for the Detection of β-Lactamase
3.5. Method Specificity
3.6. Recovery and Validation Tests
Sparked level (ng/mL) | Intra-assay (n = 6) | Inter-assay (n = 6) | ||||
---|---|---|---|---|---|---|
Mean ± SD | Recoveries | CV (%) | Mean ±SD | Recoveries | CV (%) | |
(ng/mL) | (%) | (ng/mL) | (%) | |||
40 | 40.086 ± 2.963 | 100.21 | 7.38 | 38.886 ± 5.035 | 97.22 | 12.96 |
10 | 10.313 ± 0.6422 | 103.13 | 6.21 | 9.682 ± 1.326 | 96.82 | 13.74 |
4. Conclusions
Acknowledgements
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Wang, W.; Liu, L.; Xu, L.; Ma, W.; Kuang, H.; Xu, C. Detection of β-Lactamase Residues in Milk by Sandwich ELISA. Int. J. Environ. Res. Public Health 2013, 10, 2688-2698. https://doi.org/10.3390/ijerph10072688
Wang W, Liu L, Xu L, Ma W, Kuang H, Xu C. Detection of β-Lactamase Residues in Milk by Sandwich ELISA. International Journal of Environmental Research and Public Health. 2013; 10(7):2688-2698. https://doi.org/10.3390/ijerph10072688
Chicago/Turabian StyleWang, Wenbin, Liqiang Liu, Liguang Xu, Wei Ma, Hua Kuang, and Chuanlai Xu. 2013. "Detection of β-Lactamase Residues in Milk by Sandwich ELISA" International Journal of Environmental Research and Public Health 10, no. 7: 2688-2698. https://doi.org/10.3390/ijerph10072688
APA StyleWang, W., Liu, L., Xu, L., Ma, W., Kuang, H., & Xu, C. (2013). Detection of β-Lactamase Residues in Milk by Sandwich ELISA. International Journal of Environmental Research and Public Health, 10(7), 2688-2698. https://doi.org/10.3390/ijerph10072688