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 (lambda(ex)=315 nm and lambda(em)=425 nm). The method for the determination of monoclonal mouse -IgG shows a linear range between 1 and 200 ng ml(-1) (r(2)=0.9997, n=10) with an RSD less than or equal to 3% at 10 and 100 ng ml(-1) (n=33). The 3 sigma detection limit is 0.1 ng ml(-1). The approach should be generally applicable for the determination of monoclonal antibodies.