Purification of a protein (60K/58K) associated with the Epstein-Barr virus-induced early antigen complex in Raji cells

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Abstract
A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) has been established for the detection and quantitation of EBV-associated early antigens (EA) in 5-iodo-2-deoxyuridine-induced human lymphoblastoid Raji cells. The EA complex extracted from Raji cells could be separated by ion exchange chromatography and isoelectric focusing into several components. One EA-associated subspecificity has been purified by DEAE-, CM- and Blue-Sepharose chromatography followed by isoelectric focusing. The isolated protein has an apparent MW of 240,000 .+-. 20,000 daltons under nondissociating conditions on Sephacryl S-300, an isoelectric point of 4.5, and seems to be composed of 2 polypeptides of 60,000 and 58,000 daltons as shown by sodium dodecyl sulfate-gel electrophoresis and 2-dimensional gel electrophoresis. Preliminary data indicate that the 58,000 polypeptide is generated by limited proteolysis of the 60,000 polypeptide. The EA activity of the isolated protein has been confirmed by the double antibody sandwich ELISA and its reactivity with anti-EA-positive sera in an ELISA for the detection of anti-EA antibodies.

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