Cargando…
Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies
The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker...
Autores principales: | , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498459/ https://www.ncbi.nlm.nih.gov/pubmed/32943681 http://dx.doi.org/10.1038/s41598-020-72159-4 |
_version_ | 1783583514906591232 |
---|---|
author | Moreira, Gustavo Marçal Schmidt Garcia Köllner, Sarah Mara Stella Helmsing, Saskia Jänsch, Lothar Meier, Anja Gronow, Sabine Boedeker, Christian Dübel, Stefan Mendonça, Marcelo Moreira, Ângela Nunes Conceição, Fabricio Rochedo Hust, Michael |
author_facet | Moreira, Gustavo Marçal Schmidt Garcia Köllner, Sarah Mara Stella Helmsing, Saskia Jänsch, Lothar Meier, Anja Gronow, Sabine Boedeker, Christian Dübel, Stefan Mendonça, Marcelo Moreira, Ângela Nunes Conceição, Fabricio Rochedo Hust, Michael |
author_sort | Moreira, Gustavo Marçal Schmidt Garcia |
collection | PubMed |
description | The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex—enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35–100.0%) and specificity (CI 78.20–100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria. |
format | Online Article Text |
id | pubmed-7498459 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-74984592020-09-18 Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies Moreira, Gustavo Marçal Schmidt Garcia Köllner, Sarah Mara Stella Helmsing, Saskia Jänsch, Lothar Meier, Anja Gronow, Sabine Boedeker, Christian Dübel, Stefan Mendonça, Marcelo Moreira, Ângela Nunes Conceição, Fabricio Rochedo Hust, Michael Sci Rep Article The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex—enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35–100.0%) and specificity (CI 78.20–100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria. Nature Publishing Group UK 2020-09-17 /pmc/articles/PMC7498459/ /pubmed/32943681 http://dx.doi.org/10.1038/s41598-020-72159-4 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Moreira, Gustavo Marçal Schmidt Garcia Köllner, Sarah Mara Stella Helmsing, Saskia Jänsch, Lothar Meier, Anja Gronow, Sabine Boedeker, Christian Dübel, Stefan Mendonça, Marcelo Moreira, Ângela Nunes Conceição, Fabricio Rochedo Hust, Michael Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title | Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title_full | Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title_fullStr | Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title_full_unstemmed | Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title_short | Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies |
title_sort | pyruvate dehydrogenase complex—enzyme 2, a new target for listeria spp. detection identified using combined phage display technologies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498459/ https://www.ncbi.nlm.nih.gov/pubmed/32943681 http://dx.doi.org/10.1038/s41598-020-72159-4 |
work_keys_str_mv | AT moreiragustavomarcalschmidtgarcia pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT kollnersarahmarastella pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT helmsingsaskia pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT janschlothar pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT meieranja pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT gronowsabine pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT boedekerchristian pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT dubelstefan pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT mendoncamarcelo pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT moreiraangelanunes pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT conceicaofabriciorochedo pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies AT hustmichael pyruvatedehydrogenasecomplexenzyme2anewtargetforlisteriasppdetectionidentifiedusingcombinedphagedisplaytechnologies |