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Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans
The serum level of trimethylamine N-oxide (TMAO), a gut microbiota metabolite associated with diabetes, cancer, inflammatory and neurological diseases, can be determined by the micro-enzyme-linked immunosorbent assay (ELISA) method. However, we had problems obtaining accurate standard curves with th...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Leibniz Research Centre for Working Environment and Human Factors
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10415589/ https://www.ncbi.nlm.nih.gov/pubmed/37575362 http://dx.doi.org/10.17179/excli2022-5617 |
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author | Aksoyalp, Zinnet Sevval Erdogan, Betül Rabia Aksun, Saliha |
author_facet | Aksoyalp, Zinnet Sevval Erdogan, Betül Rabia Aksun, Saliha |
author_sort | Aksoyalp, Zinnet Sevval |
collection | PubMed |
description | The serum level of trimethylamine N-oxide (TMAO), a gut microbiota metabolite associated with diabetes, cancer, inflammatory and neurological diseases, can be determined by the micro-enzyme-linked immunosorbent assay (ELISA) method. However, we had problems obtaining accurate standard curves with the original kit protocol from Bioassay Technology Laboratory. We aimed to acquire proper standard curves by modifying the kit protocol in this study. First, we evaluated the human TMAO ELISA kit protocols and other human ELISA kits. We maintained the incubation times longer and increased the wash cycle. Moreover, we incubated the standards containing biotinylated antibody in the wells alone. Then we washed the wells and added streptavidin-HRP for the second incubation step. The data of original and modified ELISA kit protocol were analyzed with Student's t-test. We measured higher absorbance with lower standard solution concentration in experiments that followed the original kit protocol. After investigating other human TMAO ELISA kits, we noticed that the SunRed Biotechnology Company and MyBioSource companies suggested similar protocols to the Bioassay Technology Laboratory company. The ELK Biotechnology ELISA protocol was different from others. However, since there is no biotinylated antibody in the standard solution in the ELK biotechnology kit, we changed some steps by examining other human ELISA protocols from different companies. After performing the modified protocol, we found that the absorbances of the standard solutions were consistent with their concentrations, and we obtained an accurate standard curve. Higher R(2 )values and lower absolute difference of standard concentrations were found in the modified kit protocol. The human TMAO ELISA protocol, which we modified in this study, will enable researchers to obtain more reliable results and prevent them from failing time and resources. |
format | Online Article Text |
id | pubmed-10415589 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Leibniz Research Centre for Working Environment and Human Factors |
record_format | MEDLINE/PubMed |
spelling | pubmed-104155892023-08-12 Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans Aksoyalp, Zinnet Sevval Erdogan, Betül Rabia Aksun, Saliha EXCLI J Methods The serum level of trimethylamine N-oxide (TMAO), a gut microbiota metabolite associated with diabetes, cancer, inflammatory and neurological diseases, can be determined by the micro-enzyme-linked immunosorbent assay (ELISA) method. However, we had problems obtaining accurate standard curves with the original kit protocol from Bioassay Technology Laboratory. We aimed to acquire proper standard curves by modifying the kit protocol in this study. First, we evaluated the human TMAO ELISA kit protocols and other human ELISA kits. We maintained the incubation times longer and increased the wash cycle. Moreover, we incubated the standards containing biotinylated antibody in the wells alone. Then we washed the wells and added streptavidin-HRP for the second incubation step. The data of original and modified ELISA kit protocol were analyzed with Student's t-test. We measured higher absorbance with lower standard solution concentration in experiments that followed the original kit protocol. After investigating other human TMAO ELISA kits, we noticed that the SunRed Biotechnology Company and MyBioSource companies suggested similar protocols to the Bioassay Technology Laboratory company. The ELK Biotechnology ELISA protocol was different from others. However, since there is no biotinylated antibody in the standard solution in the ELK biotechnology kit, we changed some steps by examining other human ELISA protocols from different companies. After performing the modified protocol, we found that the absorbances of the standard solutions were consistent with their concentrations, and we obtained an accurate standard curve. Higher R(2 )values and lower absolute difference of standard concentrations were found in the modified kit protocol. The human TMAO ELISA protocol, which we modified in this study, will enable researchers to obtain more reliable results and prevent them from failing time and resources. Leibniz Research Centre for Working Environment and Human Factors 2023-02-20 /pmc/articles/PMC10415589/ /pubmed/37575362 http://dx.doi.org/10.17179/excli2022-5617 Text en Copyright © 2023 Aksoyalp et al. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ) You are free to copy, distribute and transmit the work, provided the original author and source are credited. |
spellingShingle | Methods Aksoyalp, Zinnet Sevval Erdogan, Betül Rabia Aksun, Saliha Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title | Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title_full | Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title_fullStr | Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title_full_unstemmed | Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title_short | Optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine N-oxide levels in humans |
title_sort | optimization of enzyme-linked immunosorbent assay kit protocol to detect trimethylamine n-oxide levels in humans |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10415589/ https://www.ncbi.nlm.nih.gov/pubmed/37575362 http://dx.doi.org/10.17179/excli2022-5617 |
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