Sequential injection-ELISA based system for online determination of hyaluronan

Sequential injection-bead-based immunoassay system has been developed. The main purpose is to make immunoassay process more automated by manipulating the precise delivery of micro-volumes of reagents and the precise timing of incubation and washing steps with a computer program that controls the bi-...

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Main Authors: Supaporn Kradtap Hartwell, Boonraksa Srisawang, Prachya Kongtawelert, Jaroon Jakmunee, Kate Grudpan
Format: Journal
Published: 2018
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http://cmuir.cmu.ac.th/jspui/handle/6653943832/62148
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Institution: Chiang Mai University
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spelling th-cmuir.6653943832-621482018-09-11T09:22:41Z Sequential injection-ELISA based system for online determination of hyaluronan Supaporn Kradtap Hartwell Boonraksa Srisawang Prachya Kongtawelert Jaroon Jakmunee Kate Grudpan Chemistry Sequential injection-bead-based immunoassay system has been developed. The main purpose is to make immunoassay process more automated by manipulating the precise delivery of micro-volumes of reagents and the precise timing of incubation and washing steps with a computer program that controls the bi-directional syringe pump. The manifold was designed with the aims of reducing back pressure from beads that act as solid surfaces for immobilization of the target substance, reducing dispersion and dilution of the reagent during incubation, and maximizing signal while minimizing incubation time. This was done by introducing air segment to separate the reagent zone from the carrier stream and by using a suitable sensitive detector which, in this case, was an amperometer. In this study, hyaluronan (HA) was used as a target analyte because of its clinical significance as a potential biomarker for liver, bone and cancer diseases. Amount of hyaluronan was determined using competitive enzyme linked immuno sorbent assay (ELISA) based technique where immobilized HA and HA in solution compete to bind with a fixed amount of biotinylated HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs whose degree of binding is indirectly proportional to the amount of HA in solution. A calibration curve was constructed from a series of concentrations of HA standards. Lowest detectable concentration was found to be 1 ng/mL with the dynamic working range of 1-5000 ng/mL and R.S.D. of intra-assay (n = 7) and inter-assay (n = 3) of various HA concentrations were 4-10% and 9-12%, respectively. Used beads could be reused by washing with 2 M guanidine. Total analysis time for this automatic assay was about 30 min as compared to the 5-8 h used in conventional batch well ELISA. The system could be applied to assay HA in human serum. © 2004 Elsevier B.V. All rights reserved. 2018-09-11T09:22:41Z 2018-09-11T09:22:41Z 2005-04-15 Journal 00399140 2-s2.0-17744392628 10.1016/j.talanta.2004.11.032 https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=17744392628&origin=inward http://cmuir.cmu.ac.th/jspui/handle/6653943832/62148
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Chemistry
spellingShingle Chemistry
Supaporn Kradtap Hartwell
Boonraksa Srisawang
Prachya Kongtawelert
Jaroon Jakmunee
Kate Grudpan
Sequential injection-ELISA based system for online determination of hyaluronan
description Sequential injection-bead-based immunoassay system has been developed. The main purpose is to make immunoassay process more automated by manipulating the precise delivery of micro-volumes of reagents and the precise timing of incubation and washing steps with a computer program that controls the bi-directional syringe pump. The manifold was designed with the aims of reducing back pressure from beads that act as solid surfaces for immobilization of the target substance, reducing dispersion and dilution of the reagent during incubation, and maximizing signal while minimizing incubation time. This was done by introducing air segment to separate the reagent zone from the carrier stream and by using a suitable sensitive detector which, in this case, was an amperometer. In this study, hyaluronan (HA) was used as a target analyte because of its clinical significance as a potential biomarker for liver, bone and cancer diseases. Amount of hyaluronan was determined using competitive enzyme linked immuno sorbent assay (ELISA) based technique where immobilized HA and HA in solution compete to bind with a fixed amount of biotinylated HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs whose degree of binding is indirectly proportional to the amount of HA in solution. A calibration curve was constructed from a series of concentrations of HA standards. Lowest detectable concentration was found to be 1 ng/mL with the dynamic working range of 1-5000 ng/mL and R.S.D. of intra-assay (n = 7) and inter-assay (n = 3) of various HA concentrations were 4-10% and 9-12%, respectively. Used beads could be reused by washing with 2 M guanidine. Total analysis time for this automatic assay was about 30 min as compared to the 5-8 h used in conventional batch well ELISA. The system could be applied to assay HA in human serum. © 2004 Elsevier B.V. All rights reserved.
format Journal
author Supaporn Kradtap Hartwell
Boonraksa Srisawang
Prachya Kongtawelert
Jaroon Jakmunee
Kate Grudpan
author_facet Supaporn Kradtap Hartwell
Boonraksa Srisawang
Prachya Kongtawelert
Jaroon Jakmunee
Kate Grudpan
author_sort Supaporn Kradtap Hartwell
title Sequential injection-ELISA based system for online determination of hyaluronan
title_short Sequential injection-ELISA based system for online determination of hyaluronan
title_full Sequential injection-ELISA based system for online determination of hyaluronan
title_fullStr Sequential injection-ELISA based system for online determination of hyaluronan
title_full_unstemmed Sequential injection-ELISA based system for online determination of hyaluronan
title_sort sequential injection-elisa based system for online determination of hyaluronan
publishDate 2018
url https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=17744392628&origin=inward
http://cmuir.cmu.ac.th/jspui/handle/6653943832/62148
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