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

An on-line liquid chromatography-immunochemical detection (LC-ICD) system f or the quantification of cytokines in cell extracts has been developed usin g a post-column continuous-flow reaction detection system using fluorescenc e labelled antibodies. Cytokines eluting from the micro-HPLC column react w ith antibodies to form fluorescent complexes. In a second step the excess o f free antibody is trapped on a cytokine bound support prior to fluorescenc e detection. The concentration detection limit of the flow injection-ICD sy stem was 50 pM (20 mul injection volume) for interleukine 4 (IL-4). An abso lute detection limit of I fmol was obtained for IL-4. Similar to ICD system s for small non-protein analytes developed earlier, reaction times were in the order of I minute. The immobilised cytokine affinity columns can easily be regenerated and used for months. The present ICD system for interleukin e 4, 6, 8 and 10 was coupled to ion exchange-, size exclusion- and reversed phase chromatography. Important parameters (reaction times, reaction condi tions) were investigated to get a better understanding of post-column ICD s ystems for macromolecules. (C) 2001 Elsevier Science B.V. All rights reserv ed.