A flow-injection continuous filtration approach for the automatic determination of monoclonal antibodies

An unsegmented continuous manifold for development of a heterogeneous immunoassay has been developed in which a microfilter located prior to the detector retains the precipitate formed in the immunochemical reaction. The system used to check the performance of the dynamic approach is monoclonal mouse immunoglobulin G (IgG) as analyte and its corresponding antimouse immunoglobulin G labelled with peroxidase. The derivatizing reaction used to monitor the immunoreaction is based on the oxidation of 4-hydroxyphenylacetic acid with hydrogen peroxide in the presence of the peroxidase bound to the antimouse IgG, which was monitored fluorimetrically (λex = 315 nm and λem = 425 nm). The method for the determination of monoclonal mouse -IgG shows a linear range between 1 and 200 ng ml−1 (r2 = 0.9997, n = 10) with an RSD ≤ 3% at 10 and 100 ng ml−1 (n = 33). The 3σ detection limit is 0.1 ng ml−1. The approach should be generally applicable for the determination of monoclonal antibodies.