Homogeneous preparation of fluorescent-derivatized insulin and its application to competitive chromatographic immunoassays.

ABSTRACT

A homogeneously labeled insulin sample was prepared using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as the fluorescent-labeling reagent, and this was successfully applied to a chromatographic immunoassay. This labeled insulin was prepared by tagging all the three amino groups with AQC. Both CE and chromatographic immunoassay experiments indicated that the prepared insulin still kept its immunoaffinity to its antibody. It was observed that appropriate concentrations of acetonitrile (ACN) were efficient in lowering the quenching of the fluorescent signal of tagged insulin, in keeping the dilute, tagged insulin in solution, and in improving its peak shape during a chromatographic immunoassay. The tagged insulin was found to be 20-400 times more sensitive than native insulin detected under ultraviolet detection conditions. A competitive chromatographic immunoassay system was set up and calibrated. The system was used for analyses of an insulin-spiked urine sample, with a 96% recovery obtained.