Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.22/16263 |
Resumo: | The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva. |
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Assessment of immunoglobulin capture in immobilized protein A through automatic bead injectionArmoraciaBenzidinesChromatography, AffinityChromogenic CompoundsColorimetryHorseradish PeroxidaseImmobilized ProteinsImmunoglobulin GMicrospheresOxidation-ReductionSalivaSepharoseStaphylococcal Protein AThe repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva.Repositório Científico do Instituto Politécnico do PortoRamos, Inês I.Marques, Sara S.Magalhães, Luís M.Barreiros, LuisaReis, SaletteLima, José L.F. C.Segundo, Marcela A.2020-09-14T13:07:59Z20192019-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.22/16263eng10.1016/j.talanta.2019.06.023metadata only accessinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-03-13T13:02:57Zoai:recipp.ipp.pt:10400.22/16263Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T17:35:57.455131Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
title |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
spellingShingle |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection Ramos, Inês I. Armoracia Benzidines Chromatography, Affinity Chromogenic Compounds Colorimetry Horseradish Peroxidase Immobilized Proteins Immunoglobulin G Microspheres Oxidation-Reduction Saliva Sepharose Staphylococcal Protein A |
title_short |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
title_full |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
title_fullStr |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
title_full_unstemmed |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
title_sort |
Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection |
author |
Ramos, Inês I. |
author_facet |
Ramos, Inês I. Marques, Sara S. Magalhães, Luís M. Barreiros, Luisa Reis, Salette Lima, José L.F. C. Segundo, Marcela A. |
author_role |
author |
author2 |
Marques, Sara S. Magalhães, Luís M. Barreiros, Luisa Reis, Salette Lima, José L.F. C. Segundo, Marcela A. |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Politécnico do Porto |
dc.contributor.author.fl_str_mv |
Ramos, Inês I. Marques, Sara S. Magalhães, Luís M. Barreiros, Luisa Reis, Salette Lima, José L.F. C. Segundo, Marcela A. |
dc.subject.por.fl_str_mv |
Armoracia Benzidines Chromatography, Affinity Chromogenic Compounds Colorimetry Horseradish Peroxidase Immobilized Proteins Immunoglobulin G Microspheres Oxidation-Reduction Saliva Sepharose Staphylococcal Protein A |
topic |
Armoracia Benzidines Chromatography, Affinity Chromogenic Compounds Colorimetry Horseradish Peroxidase Immobilized Proteins Immunoglobulin G Microspheres Oxidation-Reduction Saliva Sepharose Staphylococcal Protein A |
description |
The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019 2019-01-01T00:00:00Z 2020-09-14T13:07:59Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.22/16263 |
url |
http://hdl.handle.net/10400.22/16263 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1016/j.talanta.2019.06.023 |
dc.rights.driver.fl_str_mv |
metadata only access info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
metadata only access |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799131450856964096 |