Flow-injection immunoassay using OPA derivatization Original Research Article

A flow-injection immunoassay (FIIA) using ortho-phthalaldehyde (OPA) derivatization was developed for on-line monitoring of protein products in biotechnological processes. Recombinant tissue-type plasminogen activator (rt-PA) and its antibodies were used in these studies as a model system. The derivatization of rt-PA with OPA led to a lower detection limit (higher fluorescence intensity) and an increase of sensitivity (higher slope of calibration curve) for rt-PA compared with the method without OPA derivatization. The derivatization of rt-PA with OPA was also systematically characterized: influence of pH, derivatization time, OPA concentration and sample volume on fluorescence intensity. Interferences caused by proteins were investigated with different washing 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 range 0–20 μg ml−1. The data obtained showed good agreement with the conventional off-line enzyme linked immunosorbent assay (ELISA) data. The FIIA using o-phthalaldehyde (OPA) derivatization is a valuable tool for on-line monitoring of low concentrated protein products in biotechnical production processes.