Rational design of an immunochemical assay for quantifying several therapeutic forms of recombinant human interferon alpha2b

NATALIA CEAGLIO; MARCOS OGGERO; RICARDO KRATJE; MARINA ETCHEVERRIGARAY

Pinamar. Pcia. Buenos Aires. Argentina

Congreso; X Congreso de la Panamerican Association of Biochemistry and Molecular Biology (PABMB) – XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) – XX Reunión Anual de la Sociedad Argentina de Neuroquímica.; 2005

Panamerican Association of Biochemistry and Molecular Biology; Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; Sociedad Argentina de Neuroquímica

Recombinant hIFN-alpha2a, hIFN-alpha2b and the corresponding long lasting forms of pegylated IFN-alpha2 have undergone extensive clinical investigations and have become useful drugs for viral and oncological indications. Therefore, methods for quantifying them are relevant in order to evaluate different stages of their production processes and their final formulation, where the molecule is not pure and conventional methods cannot be applied. Three anti-IFN-alpha2b monoclonal antibodies (mAbs CA5E6, CB15D7 and CA1A3), mapping three distinct epitopes, were selected for immunochemical procedures. A sandwich mAb-polyclonal antibody ELISA and a competitive ELISA with each mAb were carried out. mAb CB15D7 was suitable to quantify rhIFN-alpha2b and 12-kDa monomethoxy polyethylene glycol (PEG)-conjugated rhIFN-alpha2b in both ELISA versions. Nevertheless, the 40-kDa PEG-rhIFN-alpha2a derivative was only accurately quantified by the competitive assay using mAb CB15D7 or CA5E6. The sandwich ELISA comprises some restraints for the native cytokine recognition that were clearly evidenced with the highest pegylated form of the IFN. The competitive assay is appropriated for all therapeutic forms of rhIFN-alpha2 independently of the solution to be tested.