A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum
Abstract
:1. Introduction
2. Results
2.1. Expression, Purification and Identification of IBV-N Protein
2.2. Construction of a Phage Display VHH Library
2.3. Screening and Sequencing of Nanobodies against IBV-N Protein
2.4. Expression of RANbodies against IBV-N Protein
2.5. Development the cELISA for Detecting IBV Antibody
2.6. Cut-Off Values for the cELISA
2.7. Specificity, Sensitivity and Reproducibility of the cELISA
2.8. Agreements of the cELISA with a Commercial ELISA Kit
3. Discussion
4. Materials and Methods
4.1. Serum Samples, Cells, and Vectors
4.2. Experiment Animals
4.3. Expression, Purification and Identification of IBV-N Protein
4.4. Immunization of Camel
4.5. Generation of VHH Library
4.6. Screening of Specific VHH against IBV-N Protein
4.7. Production of Nanobody-Horseradish Peroxidase Fusion against IBV-N Protein
4.8. Development of the Competitive ELISA using RANbodies
4.9. Cut-off Value
4.10. Sensitivity, Specificity and Repeatability of cELISA
4.11. Comparisons of the cELISA with the Commercial ELISA Kit
4.12. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Round of Screening | Input (Pfu/Well) | P Output (Pfu/Well) | N Output (Pfu/Well) | Recovery (P/Input) | P/N |
---|---|---|---|---|---|
1st Round | 5.0 × 1010 | 3.0 × 104 | 3.06 × 103 | 6.0 × 10−5 | 9.8 |
2nd Round | 5.0 × 1010 | 6.0 × 105 | 2.7 × 104 | 1.2 × 10−5 | 22.2 |
3rd Round | 5.0 × 1010 | 3.6 × 106 | 1.5 × 105 | 1.8 × 10−4 | 2.4 × 103 |
Coated IBV-N Protein | Different Dilution of the IBV-N-Nb66-vHRP | |||||||
---|---|---|---|---|---|---|---|---|
1:2 | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | 1:128 | 1:256 | |
1600 ng/well | 1.544 ± 0.020 | 1.330 ± 0.008 | 1.207 ± 0.070 | 1.157 ± 0.106 | 0.833 ± 0.103 | 0.678 ± 0.002 | 0.421 ± 0.020 | 0.287 ± 0.002 |
800 ng/well | 1.513 ± 0.020 | 1.313 ± 0.014 | 1.136 ± 0.069 | 1.086 ± 0.053 | 0.742 ± 0.027 | 0.653 ± 0.005 | 0.313 ± 0.115 | 0.291 ± 0.001 |
400 ng/well | 0.952 ± 0.005 | 0.834 ± 0.001 | 0.831 ± 0.010 | 0.779 ± 0.020 | 0.674 ± 0.050 | 0.479 ± 0.012 | 0.308 ± 0.030 | 0.210 ± 0.020 |
200 ng/well | 0.543 ± 0.143 | 0.437 ± 0.061 | 0.387 ± 0.018 | 0.366 ± 0.001 | 0.321 ± 0.005 | 0.223 ± 0.020 | 0.141 ± 0.010 | 0.130 ± 0.017 |
Item | Range | Mid-Value |
---|---|---|
Coefficient of variation in intra-plates | 0.55–1.65% | 1.10% |
Coefficient of variation between inter-plates | 2.58–6.03% | 4.17% |
Samples | Number | The Developed cELISA | IDEXX ELISA Kit | Agreement (%) | Kappa Value | |
---|---|---|---|---|---|---|
+ | - | |||||
Chicken serum samples with unknown exposure to the IBV virus or vaccine immunization | 83 | + | 82 | 1 | 98.0% (343/350) | 0.94 |
67 | - | 2 | 65 | |||
Chicken serum samples with a live attenuated vaccine strain of H120 strain | 195 | + | 191 | 4 | ||
5 | - | 0 | 5 |
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Gu, K.; Song, Z.; Ma, P.; Liao, Z.; Yang, M.; Zhou, C.; Li, C.; Zhao, Y.; Li, H.; Yang, X.; et al. A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum. Int. J. Mol. Sci. 2022, 23, 7589. https://doi.org/10.3390/ijms23147589
Gu K, Song Z, Ma P, Liao Z, Yang M, Zhou C, Li C, Zhao Y, Li H, Yang X, et al. A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum. International Journal of Molecular Sciences. 2022; 23(14):7589. https://doi.org/10.3390/ijms23147589
Chicago/Turabian StyleGu, Kui, Zengxu Song, Peng Ma, Ziwei Liao, Ming Yang, Changyu Zhou, Chao Li, Yu Zhao, Hao Li, Xin Yang, and et al. 2022. "A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum" International Journal of Molecular Sciences 23, no. 14: 7589. https://doi.org/10.3390/ijms23147589