Flow-injection sandwich ELISA for bioprocess monitoring

Abstract

A fully automated flow-injection immunoassay based on sandwich enzyme-linked immunosorbent assay (ELISA) is described for the model system: protein G-sepharose, rabbit IgG and horseradish peroxidase (HRP)-labelled protein A. After injecting rabbit IgG and HRP-labelled protein A into a cartridge containing protein G-sepharose sequentially, a mixture of hydrogen peroxide and the redox indicator, 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) is passed through the cartridge. The HRP-labelled protein A bound in the cartridge is directly proportional to the concentration of rabbit IgG. The colour variation of ABTS caused during the reaction between HRP and H2O2 in the cartridge is detected photometrically. The whole assay procedure is controlled and evaluated by a computer. Rabbit Ige and HRP-labelled protein A are also detected by a fluorometer, which is introduced into the flow system. In the flow-injection sandwich ELISA, the slope of the calibration curve is 0.4491 in the range of 0 and 300 mu g ml(-1) rabbit IgG, while it is 0.1274 in the heterogeneous immunoassay. So the flow-injection sandwich ELISA system is found to be more sensitive than a heterogeneous immunoassay for the monitoring of the model protein.