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 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.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: Schenk
authors.forename: H
authors.initials: H
authors.lastname: Irth
authors.forename: G
authors.initials: G
authors.lastname: Marko-Varga
authors.forename: L E
authors.initials: LE
authors.lastname: Edholm
authors.forename: U R
authors.initials: UR
authors.lastname: Tjaden
authors.forename: J
authors.initials: J
authors.lastname: van der Greef
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D016207:Cytokines / Q000032:analysis*; D017022:Flow Injection Analysis; D005455:Fluorescent Antibody Technique; D007120:Immunochemistry; D009862:Online Systems; D011994:Recombinant Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D016207:Cytokines; D011994:Recombinant Proteins