In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16
Abstract
:1. Introduction
2. Results
2.1. Characterization of Polyclonal Immunoglobulin Yolk (IgY)-Binding Protein Antibodies
2.2. Cloning of Single-Chain Variable Fragment (scFv) Gene
2.3. Characterization of Phage-Displaying scFv Libraries and Selection of scFv-Binding Protein Clones
2.4. Characterization of scFv Antibody after Fifth Cycle of Bio-Panning
2.5. Neutralization Effect of IgY- and scFv-Binding Protein Antibodies
2.6. Determination of Cross-Reactivity of scFv-Binding Protein S1 and IgY against CA16- and EV71-Infected Lysate and BSA
3. Discussion
4. Materials and Methods
4.1. Ethics Declaration
4.2. Cells, Viruses, and Bacteria
4.3. Preparation of CA16-Infected Lysate
4.4. Hen Vaccination
4.5. Hen IgY Purification from Egg Yolk
4.6. Titration of IgY Antibodies
4.7. RNA Extraction and cDNA Synthesis
4.8. Preparation of Hen scFv Gene
4.9. Cloning of Hen scFv Gene into pComb3X DNA Vector
4.10. Construction of Phage-Displaying scFv Libraries
4.11. Bio-Panning of scFv-Binding Protein Antibodies and Selection
4.12. SDS-PAGE and Immunoblotting Techniques
4.13. Enzyme-Linked Immunosorbent Assay (ELISA)
4.14. In Vitro scFv-Binding Protein Neutralization Assay
4.15. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
SCFV | Single-chain variable fragment |
TMB | 3,3′, 5,5′-tetramethylbenzidene |
PFU | Plaque-forming unit |
SD | Standard deviation |
OD | Optical density |
µg/mL | Microgram per milliliter |
% | Percentage |
M | Molar |
MgCl2 | Magnesium chloride |
mg/mL | Milligram per milliliter |
w/v | Weight per volume |
pmole | Picomole |
µL | Microliter |
ng | Nanogram |
°C | Degree Celsius |
PVDF | Polyvinylidene fluoride |
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Amount of Antigen (μg, CA16-Infected Lysate) | Volume of 1× PBS (μL) | Adjuvants (500 μL) | |
---|---|---|---|
1st-vaccination | 100 | 500 | Complete freund’s adjuvant |
2nd-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
3rd-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
4th-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
5th-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
6th-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
7th-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
8th-vaccination | 100 | 500 | Incomplete freund’s adjuvant |
ScFv Clone | Region | Diff/totl (%) * | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
FR1 | FR2 | FR3 | FR4 | Total FRs | CDR1 | CDR2 | CDR3 | Total CDRs | ||
ScFv-binding protein S1 | VL | 1/20 (5) | 1/16 (6.25) | 6/32 (18.75) | 0/10 (0) | 8/78 (10.25) | 7/13 (53.85) a | 2/7 (28.57) | 4/11 (36.36) | 13/31 (41.94) a |
VH | 3/30 (10) | 0/14 (0) | 3/32 (9.38) | 0/11 (0) | 6/87 (6.90) | 2/5(40) | 8/17 (47.06) | 12/15 (80) a | 22/37 (59.46) a |
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Mwale, P.F.; Lee, C.-H.; Huang, P.-N.; Tseng, S.-N.; Shih, S.-R.; Huang, H.-Y.; Leu, S.-J.; Huang, Y.-J.; Chiang, L.-C.; Mao, Y.-C.; et al. In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16. Int. J. Mol. Sci. 2021, 22, 4146. https://doi.org/10.3390/ijms22084146
Mwale PF, Lee C-H, Huang P-N, Tseng S-N, Shih S-R, Huang H-Y, Leu S-J, Huang Y-J, Chiang L-C, Mao Y-C, et al. In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16. International Journal of Molecular Sciences. 2021; 22(8):4146. https://doi.org/10.3390/ijms22084146
Chicago/Turabian StyleMwale, Pharaoh Fellow, Chi-Hsin Lee, Peng-Nien Huang, Sung-Nien Tseng, Shin-Ru Shih, Hsin-Yuan Huang, Sy-Jye Leu, Yun-Ju Huang, Liao-Chun Chiang, Yan-Chiao Mao, and et al. 2021. "In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16" International Journal of Molecular Sciences 22, no. 8: 4146. https://doi.org/10.3390/ijms22084146