Cargando…

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability tha...

Descripción completa

Detalles Bibliográficos
Autores principales: O’Brien, Lorna M., Stewart, Linda D., Strain, Sam A. J., Grant, Irene R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729677/
https://www.ncbi.nlm.nih.gov/pubmed/26815790
http://dx.doi.org/10.1371/journal.pone.0147870
_version_ 1782412274669977600
author O’Brien, Lorna M.
Stewart, Linda D.
Strain, Sam A. J.
Grant, Irene R.
author_facet O’Brien, Lorna M.
Stewart, Linda D.
Strain, Sam A. J.
Grant, Irene R.
author_sort O’Brien, Lorna M.
collection PubMed
description The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.
format Online
Article
Text
id pubmed-4729677
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-47296772016-02-04 Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation O’Brien, Lorna M. Stewart, Linda D. Strain, Sam A. J. Grant, Irene R. PLoS One Research Article The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay. Public Library of Science 2016-01-27 /pmc/articles/PMC4729677/ /pubmed/26815790 http://dx.doi.org/10.1371/journal.pone.0147870 Text en © 2016 O’Brien et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
O’Brien, Lorna M.
Stewart, Linda D.
Strain, Sam A. J.
Grant, Irene R.
Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title_full Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title_fullStr Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title_full_unstemmed Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title_short Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation
title_sort novel monoclonal antibody and peptide binders for mycobacterium avium subsp. paratuberculosis and their application for magnetic separation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729677/
https://www.ncbi.nlm.nih.gov/pubmed/26815790
http://dx.doi.org/10.1371/journal.pone.0147870
work_keys_str_mv AT obrienlornam novelmonoclonalantibodyandpeptidebindersformycobacteriumaviumsubspparatuberculosisandtheirapplicationformagneticseparation
AT stewartlindad novelmonoclonalantibodyandpeptidebindersformycobacteriumaviumsubspparatuberculosisandtheirapplicationformagneticseparation
AT strainsamaj novelmonoclonalantibodyandpeptidebindersformycobacteriumaviumsubspparatuberculosisandtheirapplicationformagneticseparation
AT grantirener novelmonoclonalantibodyandpeptidebindersformycobacteriumaviumsubspparatuberculosisandtheirapplicationformagneticseparation