Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Bactrian Camel Immunization
2.3. Constructing the Phage Display Nb Library
2.4. Biopanning of the Phage Display Anti-SEC Nbs
2.5. Preparation of the Phage Display Nbs
2.6. The Purification and Expression of the Anti-SEC Nbs
2.7. Pairwise Selection for Sandwich Nbs-ELISA
2.8. Developing a Sandwich Nbs-ELISA for SEC Detection
2.9. Cross-Reactivity Examination
2.10. Analysis of the Spiked Dairy Product
3. Results
3.1. Anti-SEC Nb Library Construction
3.2. Biopanning of the Anti-SEC Nbs
3.3. Characterization of the Binding Properties of Anti-SEC Nbs
3.4. Pairwise Interaction Analysis
3.5. Sandwich Nbs-ELISA for SEC Detection
3.6. Cross-Reactivity Assay
3.7. Spiked Sample Analysis
4. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Phage-Displayed Nbs | Capture Nbs (POD450/NOD450) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
C2 | C3 | C6 | C7 | C11 | C15 | C19 | C49 | C61 | C64 | C91 | |
C2 | 1.14 | 2.03 | 1.52 | 1.12 | 2.08 | 1.08 | 1.81 | 2.07 | 1.29 | 1.06 | 0.81 |
C3 | 1.80 | 1.75 | 3.29 | 1.64 | 1.95 | 1.59 | 3.44 | 2.08 | 2.27 | 1.70 | 0.86 |
C6 | 1.00 | 1.83 | 1.21 | 0.95 | 1.87 | 0.92 | 1.82 | 1.98 | 1.21 | 1.24 | 0.78 |
C7 | 1.05 | 4.12 | 1.87 | 1.71 | 3.34 | 1.32 | 1.58 | 3.68 | 1.54 | 1.77 | 1.16 |
C11 | 2.74 | 2.32 | 8.08 | 3.53 | 2.15 | 3.34 | 5.61 | 2.42 | 4.00 | 4.43 | 0.94 |
C15 | 1.43 | 4.25 | 2.78 | 1.31 | 4.33 | 1.21 | 2.51 | 4.37 | 1.44 | 1.52 | 0.88 |
C19 | 0.96 | 3.24 | 1.43 | 0.96 | 2.98 | 1.26 | 2.45 | 2.75 | 1.84 | 1.15 | 0.84 |
C49 | 1.57 | 6.07 | 4.09 | 1.94 | 5.13 | 1.10 | 2.64 | 4.35 | 2.02 | 2.81 | 1.80 |
C61 | 1.71 | 2.45 | 5.57 | 3.84 | 2.61 | 3.25 | 3.35 | 2.43 | 3.00 | 5.22 | 0.90 |
C64 | 1.05 | 2.36 | 1.86 | 1.37 | 2.09 | 1.08 | 1.41 | 2.18 | 1.30 | 1.58 | 0.94 |
C91 | 1.72 | 7.51 | 5.30 | 2.89 | 6.48 | 1.95 | 3.19 | 7.23 | 2.49 | 3.03 | 0.81 |
Sample | Spiked Levels (ng/mL) | Detection Level (ng/mL) (ng/mL) (n = 3 a) | Recovery (%) |
---|---|---|---|
Fresh milk | 250 | 255.22 ± 13.76 | 102.09 ± 5.51 |
500 | 497.42 ± 42.47 | 99.48 ± 8.49 | |
1000 | 1000.05 ± 90.34 | 100.05 ± 9.03 | |
Yogurt in shelf-life | 250 | 233.79 ± 21.88 | 93.52 ± 8.75 |
500 | 540.32 ± 36.27 | 108.06 ± 7.25 | |
1000 | 950.42 ± 119.42 | 95.04 ± 11.94 | |
Cheese | 250 | 222.81 ± 9.92 | 89.12 ± 3.97 |
500 | 422.62 ± 12.62 | 84.52 ± 2.52 | |
1000 | 1020.48 ± 90.03 | 102.05 ± 9.00 |
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Ji, Y.; Chen, L.; Wang, Y.; Zhang, K.; Wu, H.; Liu, Y.; Wang, Y.; Wang, J. Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products. Foods 2021, 10, 2426. https://doi.org/10.3390/foods10102426
Ji Y, Chen L, Wang Y, Zhang K, Wu H, Liu Y, Wang Y, Wang J. Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products. Foods. 2021; 10(10):2426. https://doi.org/10.3390/foods10102426
Chicago/Turabian StyleJi, Yanwei, Lili Chen, Yingying Wang, Kaihui Zhang, Haofen Wu, Yuan Liu, Yanru Wang, and Jianlong Wang. 2021. "Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products" Foods 10, no. 10: 2426. https://doi.org/10.3390/foods10102426