Antigenic Analysis of Polioviruses Employing the Disk Neutralization Test

Abstract

A disk neutralization test is described for measuring intratypic antigenic differences that exist among the polioviruses. Varying dilutions of strain specific antisera are incorporated into the agar overlaid on cell culture monolayers. Paper disks containing uniform amounts of homologous or heterologous virus are then placed on the serum-agar surface. If the test virus is not neutralized, a zone of degeneration develops below the disk. Five strains may be tested against one serum dilution on the monolayer of a single 60-mm Petri dish. Serum titers were reproducible from test to test within a 2-fold range. Thus, test strains which yielded serum titers within 50% of that yielded with the homologous strain can be regarded as antigenically the same. The smaller the titer relative to the titer with the homologous strain, the greater the antigenic differences between the strains. Early immune sera obtained in monkeys, rabbits, or guinea pigs were more discriminating in being able to detect differences than late immune sera even though the late sera yielded higher neutralization titers. The disk antigenic test, like the neutralization kinetic test, is dependent directly on the antigen-antibody reaction and is not affected by the growth rate of the virus and other variables. Comparative tests were carried out on the same strains and both methods yielded essentially the same results. The disk method was applied epidemiologically to follow Sabin's type 3 and Lederle's type 1 vaccines after multiplication in vaccinated persons. Even though the vaccinated persons or contacts may excrete strains with the properties different from those of the vaccine virus, the disk antigenic tests showed that the new virus particles with the altered properties were progeny of the vaccine viruses.