Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections
Abstract
:1. Introduction
2. Results
2.1. Antigen Design and Screening Approach to Generate mAbs Specific for NS1 from Rodent PVs, Human CuV, and b19v
2.2. Identification of Hybridoma Producing mAbs for NS1 Detection
2.2.1. H-1PV NS1
2.2.2. CuV NS1
2.2.3. B19V NS1
2.3. Assessment of Antibody Cross-Reactivity between Rodent and Human Parvoviruses
2.4. Monoclonal Antibodies Detecting the C-Terminal Part of NS1
2.5. Monoclonal Antibodies Detecting H-1PV in Paraffin-Embedded Samples
3. Discussion
4. Material and Methods
4.1. Animal Ethics Statement
4.2. DNA Constructs
4.3. Reagents
4.4. Cells and Viruses
4.5. Bacterial Expression and Purification of Immunogens
4.6. Immunization, Cell Fusion, and the Isolation of Monoclonal Antibodies
4.7. Immunofluorescence Microscopy
4.8. Western Blot Analysis
4.9. Paraffin Embedment of Cultured Cells and Detection of Target Proteins
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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H-1PV NS1 Screening | |||||
IF-positive | WB-positive | ||||
253 | 225 | ||||
Excellent 1 | Intermediate 2 | Weak 3 | Excellent | Intermediate | Poor |
90 | 101 | 62 | 32 | 50 | 94 |
CuV NS1 Screening | |||||
IF-positive | WB-positive | ||||
16 | 3 | ||||
B19V NS1 Screening | |||||
IF-positive | WB-positive | ||||
10 | 5 |
Name | neg | vTF7-3 | H1 | Cuta | Bufa | B19 | KRV | TVX | LuIII | MVM | Hg#1 | Hg#13 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
H-1PV-NS1-specific | ||||||||||||
H1#742/1 | - | - | +++ | - | - | - | +++ | - | - | +++ | +++ | - |
H1#1420/5 | - | - | +++ | - | - | - | +++ | - | - | +++ | +++ | - |
H1#700/6 | - | ++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
H1#818/3 | - | - | +++ | - | - | - | +++ | - | +++ | +++ | +++ | +++ |
H1#1539/2 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | - | +++ | +++ | - |
H1#705 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | +++ | ++ | - | - | +++ |
H1#721 | - | n.d. | +++ | n.d. | n.d. | n.d. | + | - | ++ | ++ | +++ | +++ |
H1#737 | - | n.d. | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | +++ | +++ |
H1#756 | - | n.d. | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | +++ | +++ |
H1#853 | - | n.d. | +++ | n.d. | n.d. | n.d. | ++ | - | + | ++ | +++ | +++ |
H1#867 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | +++ | +++ | +++ | +++ |
H1#888/1 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | +++ | +++ | +++ | +++ |
H1#892/5 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | - | +++ | +++ | - |
H1#905 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | - | +++ | +++ | - |
H1#918 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | +++ | +++ | +++ | +++ | +++ |
H1#1252 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | - | ++ | +++ | - |
H1#1320 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | ++ | +++ | +++ | +++ |
H1#1428/5 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | - | - | (+) | +++ | - |
H1#1509/3 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | +++ | +++ | +++ | +++ | +++ |
H1#1581/3 | - | n.d. | +++ | n.d. | n.d. | n.d. | +++ | +++ | - | +++ | +++ | +++ |
B19-NS1-specific | ||||||||||||
B19#892/5 | - | - | - | - | - | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
B19#757/10 | - | - | - | - | - | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
B19#5/12/3 | - | - | (+) | (+) | (+) | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
B19#278/3 | - | +++ | +++ | +++ | +++ | +++ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
Cuta-NS1-specific | ||||||||||||
C#177/2 | - | - | - | +++ | - | - | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
C#98/1 | - | - | - | +++ | + | - | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
C#93/8 | - | + | + | +++ | +++ | +++ | +++ | +++ | +++ | +++ | n.d. | n.d. |
C#237/1 | - | - | +++ | +++ | +++ | ++ | +++ | +++ | +++ | +++ | +++ | +++ |
Others | ||||||||||||
pNS1H | - | - | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | n.d. | n.d. |
pNS1-MK/SP8 | - | - | +++ | ++ | - | - | +++ | +++ | +++ | +++ | n.d. | n.d. |
NS1-3D9 | - | - | +++ | - | - | - | +++ | - | +++ | +++ | n.d. | n.d. |
m#236/10 | - | - | +++ | - | - | - | +++ | - | +++ | +++ | +++ | - |
Forward Primer | Backward Primer | |
---|---|---|
Immunogens | ||
H1-NS1Heli | ggatccgcatggctagcaccagaacctgtagaatctttgc | gcggccgcttaaataactggtgttggttcaatctgtttgc |
H1-NS1TA | ggatccgcatggcttgttactgtgctaaatggggcaaagt | gcggccgcttagtccaaggtcagctcctcgttgaagtcgc |
B19-NS1Heli | gaaatttcctatgtgctgcttaaacaaacaaatgggaaaaag | gcggccgctattcgttggttgtcattatgactggtgttgg |
B19-NS1TA | gtttaaacctatgggcgcctggaacactgaaaccccgcgctc | gcggccgcttactcataatctacaaagctttgcaatcc |
Cuta-NS1Im | gaaatttcctatgtgctgcttaaacaaacaaatgggaaaaag | aagcttgcggccgcctaatagctggcattcacatccgttccggatatc. |
Recombinant Viruses | ||
FL-H1-NS1 | gtttaaacatggctgactacaaggacgacgatggctggaaacgcttactc | gcggccgcttactcataatctacaaagctttgcaatcc |
Y-CutaNS1 | gtttaaacatgagaccagagatcacgtggttgatggctctctcagcaaagagatg | aagcttgcggccgcctaatagctggcattcacatccgttccggatatc |
FL-CutaNS1 | gtttaaacatggctgactacaaggacgacgatggctctcagcaaagagatg | aagcttgcggccgcctaatagctggcattcacatccgttccggatatc |
FL-B19NS1 | gtttaaacatggctgactacaaggacgacgatggagctatttagaggggtg | gcggccgcttactcataatctacaaagctttgcaatcc |
HisB19NS1 | gtttaaacatgcactaccaccactaccac atggagctatttagaggggtg | gcggccgcttactcataatctacaaagctttgcaatcc |
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Tessmer, C.; Plotzky, C.; Fees, J.; Welsch, H.; Eudenbach, R.; Faber, M.; Simón, A.; Angelova, A.; Rommelaere, J.; Hofmann, I.; et al. Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections. Pathogens 2022, 11, 208. https://doi.org/10.3390/pathogens11020208
Tessmer C, Plotzky C, Fees J, Welsch H, Eudenbach R, Faber M, Simón A, Angelova A, Rommelaere J, Hofmann I, et al. Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections. Pathogens. 2022; 11(2):208. https://doi.org/10.3390/pathogens11020208
Chicago/Turabian StyleTessmer, Claudia, Claudia Plotzky, Jana Fees, Hendrik Welsch, Rebecca Eudenbach, Martin Faber, Alicia Simón, Assia Angelova, Jean Rommelaere, Ilse Hofmann, and et al. 2022. "Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections" Pathogens 11, no. 2: 208. https://doi.org/10.3390/pathogens11020208
APA StyleTessmer, C., Plotzky, C., Fees, J., Welsch, H., Eudenbach, R., Faber, M., Simón, A., Angelova, A., Rommelaere, J., Hofmann, I., & Nüesch, J. P. F. (2022). Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections. Pathogens, 11(2), 208. https://doi.org/10.3390/pathogens11020208