Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines

T. Schenk; H. Irth; G. Marko-Varga; L.E. Edholm; U.R. Tjaden; J de Greef

doi:10.1016/S0731-7085(01)00464-2

Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines

T. Schenk, H. Irth, G. Marko-Varga, L.E. Edholm, U.R. Tjaden, J de Greef

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 μl injection volume) for interleukine 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukine 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules. © 2001 Elsevier Science B.V. All rights reserved.

Original language	English
Pages (from-to)	975-85
Journal	Journal of Pharmaceutical and Biomedical Analysis
Volume	26
Issue number	5-6
DOIs	https://doi.org/10.1016/S0731-7085(01)00464-2
Publication status	Published - 2001

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title = "Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines",

abstract = "An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 μl injection volume) for interleukine 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukine 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules. {\textcopyright} 2001 Elsevier Science B.V. All rights reserved.",

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T1 - Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines

AU - Schenk, T.

AU - Irth, H.

AU - Marko-Varga, G.

AU - Edholm, L.E.

AU - Tjaden, U.R.

AU - de Greef, J

PY - 2001

Y1 - 2001

N2 - An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 μl injection volume) for interleukine 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukine 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules. © 2001 Elsevier Science B.V. All rights reserved.

AB - An on-line liquid chromatography-immunochemical detection (LC-ICD) system for the quantification of cytokines in cell extracts has been developed using a post-column continuous-flow reaction detection system using fluorescence labelled antibodies. Cytokines eluting from the micro-HPLC column react with antibodies to form fluorescent complexes. In a second step the excess of free antibody is trapped on a cytokine bound support prior to fluorescence detection. The concentration detection limit of the flow injection-ICD system was 50 pM (20 μl injection volume) for interleukine 4 (IL-4). An absolute detection limit of 1 fmol was obtained for IL-4. Similar to ICD systems for small non-protein analytes developed earlier, reaction times were in the order of 1 minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukine 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction conditions) were investigated to get a better understanding of post-column ICD systems for macromolecules. © 2001 Elsevier Science B.V. All rights reserved.

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DO - 10.1016/S0731-7085(01)00464-2

M3 - Article

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VL - 26

SP - 975

EP - 985

JO - Journal of Pharmaceutical and Biomedical Analysis

JF - Journal of Pharmaceutical and Biomedical Analysis

IS - 5-6

ER -

Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines

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