The Purification of Poliomyelitis Virus as Studied by Complement Fixation

Abstract

Summary and Conclusions Complement fixation (CF) assays for poliomyelitis virus and for monkey kidney antigens have been found useful in guiding and controlling the processes of concentration and fractionation required for the purification of poliomyelitis virus. In conjunction with infectivity measurements, as well as nitrogen analyses, CF titers furnish information on yield and purity. Three variations of the basic purification procedure have been studied with respect to yield and purity gain. Usually, the yield of purified virus was equal to or better than 50%, with purity gains up to about 1000-fold relative to the starting material. The infected tissue culture fluids (ITCF) used as starting material were found to contain about 0.01 to 0.2 μg of virus nitrogen/ml. ITCF from minced tissue cultures contained about 20 μg of total non-dialyzable N per ml, while ITCF from cultures of the trypsinized type contained only about 3–5 μg of total non-dialyzable N. Concomitantly, the former type of ITCF showed monkey kidney antigen titers by CF about 40 times greater than those of the latter type. Tentative estimates indicate that the most highly purified fractions were composed primarily of viral substance. Those which were derived from ITCF of the minced tissue type still contained appreciable amounts of monkey kidney substance, perhaps 25%. On the other hand, purified products from ITCF of the trypsinized type were free of detectable monkey kidney antigens, but the presence of non-antigenic contaminants could not be excluded. However, only a small proportion, somewhat less than 0.1%, of the actual mass was infectious virus, as detected on monkey kidney cultures. The remainder, presumably represents non-infectious virus, or viral products, which are immunologically reactive but which do not form plaques on tissue cultures. Fractionation of purified products by ultracentrifugation in a sucrose density gradient (SDG) led to a separation of fast sedimenting infectious units from slower non-infectious components. Both were reactive in CF tests with polio antisera. It is shown that infectivity titers should not be used as a measure of viral substance, but, instead, CF titers should be employed.