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Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection
Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979058/ https://www.ncbi.nlm.nih.gov/pubmed/27600349 http://dx.doi.org/10.3390/microarrays3040282 |
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author | Hospach, Ingeborg Joseph, Yvonne Mai, Michaela Kathrin Krasteva, Nadejda Nelles, Gabriele |
author_facet | Hospach, Ingeborg Joseph, Yvonne Mai, Michaela Kathrin Krasteva, Nadejda Nelles, Gabriele |
author_sort | Hospach, Ingeborg |
collection | PubMed |
description | Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established. |
format | Online Article Text |
id | pubmed-4979058 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-49790582016-09-06 Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection Hospach, Ingeborg Joseph, Yvonne Mai, Michaela Kathrin Krasteva, Nadejda Nelles, Gabriele Microarrays (Basel) Article Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established. MDPI 2014-12-09 /pmc/articles/PMC4979058/ /pubmed/27600349 http://dx.doi.org/10.3390/microarrays3040282 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Hospach, Ingeborg Joseph, Yvonne Mai, Michaela Kathrin Krasteva, Nadejda Nelles, Gabriele Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title | Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title_full | Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title_fullStr | Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title_full_unstemmed | Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title_short | Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection |
title_sort | fabrication of homogeneous high-density antibody microarrays for cytokine detection |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979058/ https://www.ncbi.nlm.nih.gov/pubmed/27600349 http://dx.doi.org/10.3390/microarrays3040282 |
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