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 microl injection volume) for interleukin 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 interleukins 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.

journal_name

J Pharm Biomed Anal

authors

Schenk T,Irth H,Marko-Varga G,Edholm LE,Tjaden UR,van der Greef J

doi

10.1016/s0731-7085(01)00464-2

subject

Has Abstract

pub_date

2001-12-01 00:00:00

pages

975-85

issue

5-6

eissn

0731-7085

issn

1873-264X

pii

S0731708501004642

journal_volume

26

pub_type

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