FLOW-INJECTION IMMUNOASSAY USING OPA DERIVATIZATION

A flow-injection immunoassay (FIIA) using ortho-phthalaldehyde (OPA) d erivatization was developed for on-line monitoring of protein products in biotechnological processes. Recombinant tissue-type plasminogen ac tivator (rt-PA) and its antibodies were used in these studies as a mod el system. The derivatization of rt-PA with OPA led to a lower detecti on limit (higher fluorescence intensity) and an increase of sensitivit y (higher slope of calibration curve) for rt-PA compared with the meth od without OPA derivatization. The derivatization of rt-PA with OPA wa s also systematically characterized: influence of pH, derivatization t ime, OPA concentration and sample volume on fluorescence intensity. In terferences caused by proteins were investigated with different washin g buffer solutions. A 0.1 M potassium phosphate buffer (PPB) solution containing 1 g l(-1) polyethylene glycol (PEG) was found to be optimal for minimizing the interference with BSA in the measurement. On-line monitoring for rt-PA is carried out on a simulated bioprocess in the r ange 0-20 mu g ml(-1). The data obtained showed good agreement with th e conventional off-line enzyme linked immunosorbent assay (ELISA) data . The FIIA:using ophthalaldehyde (OPA) derivatization is a valuable to ol for on-line monitoring of low concentrated protein products in biot echnical production processes. (C) 1998 Elsevier Science B.V. All righ ts reserved.