On-line competitive immunoassay for insulin based on capillary electrophoresis with laser-induced fluorescence detection.

An on-line competitive immunoassay for insulin has been developed and applied to monitoring insulin concentration in a flowing stream. In the assay, solutions of fluorescein-labeled insulin (FITC-insulin), monoclonal anti-insulin, and sample containing insulin are pumped into a cross where they begin to mix. The mixture flows through a fused silica reactor capillary to a flow-gated interface. During transfer to the interface, insulin and FITC-insulin compete to form a complex with the antibody. At the interface, a plug of the mixture is injected into a separation capillary, where the bound and free FITC-insulin are separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The amount of bound FITC-insulin, amount of free FITC-insulin, or bound/free ratio can be used to quantify insulin concentration. Typical relative standard deviations of bound over free ratio are 5%. The detection limit of the immunoassay in the on-line mode is < 0.3 nM. Each separation requires as little as 3 s, and over 1600 consecutive assays can be acquired with no need to rinse the separation capillary. Thus, the system can be used to monitor insulin in a flowing stream for flow injection analysis or for sensor-like monitoring. Dilution and zone broadening during transfer of sample to the interface limit the response time of the on-line system to about 25 s. As a demonstration of the on-line immunoassay, the insulin content of single islets of Langerhans was determined by flow injection analysis.