Potential of on-line micro-LC immunochemical detection in the bioanalysis of cytokines.
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.
Schenk
T
T
Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
Irth
H
H
Marko-Varga
G
G
Edholm
L E
LE
Tjaden
U R
UR
van der Greef
J
J
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
D016207:Cytokines; D011994:Recombinant Proteins