Isolation and Analysis of Adenovirus 12 Sub-units from Adenovirus Type 12-infected Cells

Abstract

THE isolation of adenovirus 12 group-specific (“A”) and type-specific (“C”) antigens has been described by Huebner et al.¹ The method used for isolation of the sub-units was step by step elution from columns of diethylaminoethyl cellulose (DEAE). Because the amount of material available at a given time varied, reabsorption was less important in this procedure than with gradient elution procedures. Eluent changes were regulated by a series of tests using cell protein alone in order to eliminate the possibility of widening or tailing of bands. Virus-antigen mixtures were equilibrated with 0.01 M phosphate buffer at 6.8 pH. before application to the column of DEAE, which was prepared in 0.01 M phosphate buffer at pH of 7.2. All operations were carried out in a cold room. Sodium chloride solutions were prepared in 0.01 M phosphate buffer at 6.8 pH. so that there was a constant pH but an increase in salt concentration during elution. An investigation of the effectiveness of changes in salt concentration versus changes in pH with cell protein showed that a constant pH was desirable and that changes in salt fronts were predictable. It was necessary to re-cycle eluates once or twice over fresh columns in order to obtain purified material in single peaks. A critical aspect of the procedure was the resin exchange capacity of 0.56 m. equiv./g.