We have isolated and identified 22 human lymphoblastoid Interferon-alpha components from a partially purified IFN mixture derived from Sendai virus-induced Namalwa cells by affinity chromatography using 4 monoclonal antibody affinity columns, ultrafiltration, and High Performance Liquid Chromatography. We have characterized the IFN-alpha components using the methods of SDS-PAGE electrophoresis, tryptic digest mapping and amino terminal amino acid sequences. The procedures used to identify the biological properties of these molecules include determining antiviral activities on human, bovine and murine cell lines, their antiproliferative activity on U937 and Daudi cells, their relative affinities for IFN- alpha2b binding sites on U937 and Daudi cells and the evaluation of the IFN-alpha component for in-vitro anti-HIV activity as potential antiviral agents. The chemical characterization involves studying the carbohydrate structure of the IFN-alpha components. Using Anion Exchange Anion Exchange Chromatography and Pulsed Amperometric Detection, we have identified 3 major glycosylated components and 11 minor components with low levels of glycosylation. 6 components appear to contain N- acetylneuraminic acid. Studies are currently being done to determine the oligosaccharide structure and the linkage sites by oligosaccharide mapping and lectin binding. Regulatory activities involve the testing and evaluation of the purity and size of natural and recombinant cytokines that are to be used in patients. This is done by SDS-PAGE to determine the molecular weight, HPLC to assure the purity of the cytokines, and carbohydrate analysis to monitor the recombinant cytokines made in yeast.
