22 results on '"Spiliotopoulos, Anastasios"'
Search Results
2. Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases
- Author
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Spiliotopoulos, Anastasios, primary, Maurer, Sigrun K., additional, Tsoumpeli, Maria T., additional, Bonfante, Juan A. F., additional, Owen, Jonathan P., additional, Gough, Kevin C., additional, and Dreveny, Ingrid, additional
- Published
- 2022
- Full Text
- View/download PDF
3. Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11
- Author
-
Spiliotopoulos, Anastasios, Blokpoel Ferreras, Lia, Densham, Ruth M., Caulton, Simon G., Maddison, Ben C., Morris, Joanna R., Dixon, James E., Gough, Kevin C., and Dreveny, Ingrid
- Published
- 2019
- Full Text
- View/download PDF
4. Studying social network sites with the combination of traditional social science and computational approaches
- Author
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Spiliotopoulos, Anastasios, Oakley, Ian, and Campos, Pedro
- Subjects
Social network sites ,Facebook API ,Faculdade de Ciências Exatas e da Engenharia ,Ciência social computacional ,Redes sociais ,Computational social science ,Online disclosure ,Engenharia e Tecnologia::Engenharia Eletrotécnica, Eletrónica e Informática [Domínio/Área Científica] ,Uses and gratifications ,Divulgação online ,Usos e gratificações ,Informatics Engineering specialty in Human-Computer Interaction - Abstract
Submitted by António Freitas (amsf@uma.pt) on 2021-02-22T13:53:24Z No. of bitstreams: 1 Spiliotopoulos - PhD thesis - Studying Social Network Sites with the Combination of Traditional Social Science and Comput_1.pdf: 2942408 bytes, checksum: 6b301bb65df306d239f614337b6a43be (MD5) Made available in DSpace on 2021-02-22T13:53:25Z (GMT). No. of bitstreams: 1 Spiliotopoulos - PhD thesis - Studying Social Network Sites with the Combination of Traditional Social Science and Comput_1.pdf: 2942408 bytes, checksum: 6b301bb65df306d239f614337b6a43be (MD5) Previous issue date: 2020-07-03
- Published
- 2020
5. Expression of a cDNA encoding a member of the hexamerin storage proteins from the moth Sesamia nonagrioides (Lef.) during diapause
- Author
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Spiliotopoulos, Anastasios, Gkouvitsas, Theodoros, Fantinou, Argyro, and Kourti, Anna
- Published
- 2007
- Full Text
- View/download PDF
6. Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies
- Author
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Macpherson, Alex, primary, Scott-Tucker, Anthony, additional, Spiliotopoulos, Anastasios, additional, Simpson, Catherine, additional, Staniforth, Justin, additional, Hold, Adam, additional, Snowden, James, additional, Manning, Leah, additional, van den Elsen, Jean, additional, and Lawson, Alastair D. G., additional
- Published
- 2020
- Full Text
- View/download PDF
7. Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets
- Author
-
Spiliotopoulos, Anastasios, Owen, Jonathan.P., Maddison, Ben.C., Dreveny, Ingrid, Rees, Helen.C., and Gough, Kevin.C.
- Published
- 2015
- Full Text
- View/download PDF
8. Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals
- Author
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Naqid, Ibrahim A., primary, Owen, Jonathan P., additional, Maddison, Ben C., additional, Spiliotopoulos, Anastasios, additional, Emes, Richard D., additional, Warry, Andrew, additional, Flynn, Robin J., additional, Martelli, Francesca, additional, Gosling, Rebecca J., additional, Davies, Robert H., additional, La Ragione, Roberto M., additional, and Gough, Kevin C., additional
- Published
- 2016
- Full Text
- View/download PDF
9. Mapping polyclonal antibody responses to bacterial infection using next generation phage display
- Author
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Naqid, Ibrahim A., primary, Owen, Jonathan P., additional, Maddison, Ben C., additional, Spiliotopoulos, Anastasios, additional, Emes, Richard D., additional, Warry, Andrew, additional, Tchórzewska, Monika A., additional, Martelli, Francesca, additional, Gosling, Rebecca J., additional, Davies, Robert H., additional, La Ragione, Roberto M., additional, and Gough, Kevin C., additional
- Published
- 2016
- Full Text
- View/download PDF
10. Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of di erentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identi ed in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and speci city. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.
- Full Text
- View/download PDF
11. Mapping polyclonal antibody responses to bacterial infection using next generation phage display
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy- seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.
- Full Text
- View/download PDF
12. Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets
- Author
-
Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., Gough, Kevin C., Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., and Gough, Kevin C.
- Abstract
Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.
- Full Text
- View/download PDF
13. Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of di erentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identi ed in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and speci city. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.
- Full Text
- View/download PDF
14. Mapping polyclonal antibody responses to bacterial infection using next generation phage display
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy- seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.
- Full Text
- View/download PDF
15. Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets
- Author
-
Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., Gough, Kevin C., Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., and Gough, Kevin C.
- Abstract
Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.
- Full Text
- View/download PDF
16. Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of di erentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identi ed in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and speci city. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.
- Full Text
- View/download PDF
17. Mapping polyclonal antibody responses to bacterial infection using next generation phage display
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy- seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.
- Full Text
- View/download PDF
18. Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets
- Author
-
Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., Gough, Kevin C., Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., and Gough, Kevin C.
- Abstract
Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.
- Full Text
- View/download PDF
19. Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Flynn, Robin J., Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of di erentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identi ed in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and speci city. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.
- Full Text
- View/download PDF
20. Mapping polyclonal antibody responses to bacterial infection using next generation phage display
- Author
-
Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., Gough, Kevin C., Naqid, Ibrahim A., Owen, Jonathan P., Maddison, Ben C., Spiliotopoulos, Anastasios, Emes, Richard D., Warry, Andrew, Tchorzewska, Monika, Martelli, Francesca, Gosling, Rebecca J., Davies, Robert H., La Ragione, Roberto M., and Gough, Kevin C.
- Abstract
Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy- seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.
- Full Text
- View/download PDF
21. Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets
- Author
-
Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., Gough, Kevin C., Spiliotopoulos, Anastasios, Owen, Jonathan P., Maddison, Ben C., Dreveny, Ingrid, Rees, Helen C., and Gough, Kevin C.
- Abstract
Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.
- Full Text
- View/download PDF
22. Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases.
- Author
-
Spiliotopoulos A, Maurer SK, Tsoumpeli MT, Bonfante JAF, Owen JP, Gough KC, and Dreveny I
- Subjects
- Ligands, Peptides, Deubiquitinating Enzymes, Peptide Library, Bacteriophages
- Abstract
Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD) has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
- Full Text
- View/download PDF
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