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Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure
Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594677/ https://www.ncbi.nlm.nih.gov/pubmed/31242246 http://dx.doi.org/10.1371/journal.pone.0218686 |
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author | Carl, Peter Ramos, Inês I. Segundo, Marcela A. Schneider, Rudolf J. |
author_facet | Carl, Peter Ramos, Inês I. Segundo, Marcela A. Schneider, Rudolf J. |
author_sort | Carl, Peter |
collection | PubMed |
description | Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve. |
format | Online Article Text |
id | pubmed-6594677 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-65946772019-07-05 Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure Carl, Peter Ramos, Inês I. Segundo, Marcela A. Schneider, Rudolf J. PLoS One Research Article Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve. Public Library of Science 2019-06-26 /pmc/articles/PMC6594677/ /pubmed/31242246 http://dx.doi.org/10.1371/journal.pone.0218686 Text en © 2019 Carl 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 Carl, Peter Ramos, Inês I. Segundo, Marcela A. Schneider, Rudolf J. Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title | Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title_full | Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title_fullStr | Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title_full_unstemmed | Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title_short | Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure |
title_sort | antibody conjugation to carboxyl-modified microspheres through n-hydroxysuccinimide chemistry for automated immunoassay applications: a general procedure |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594677/ https://www.ncbi.nlm.nih.gov/pubmed/31242246 http://dx.doi.org/10.1371/journal.pone.0218686 |
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