Further Studies of Specificity of Antibodies Contained in Antiserum against Poliovirus

Abstract

Antigenic components of Mahoney strain (poliovirus type 1) involved in virus neutralization reaction were analyzed with mutant Mahoney strains resistant to inhibitors in equine serum (inhibitorresistant mutants) by means of the kinetic neutralization test. It was shown that absorption of antiMahoney serum with five inhibitor-resistant mutants yielded sera with different antibodies, of which three had distinct specificities and two specificities possibly partly related to one of those three sera. Further, it was found that stepwise selection of Mahoney variants resistant to one, two, three and four different inhibitors resulted in gradual deviation of its antigenic composition from that of the original strain. From these results, the possible presence of three or more distinct antigenic determinant sites on the surface of Mahoney strain was indicated.Although the complexity of poliovirus antigen is suggested by the intratypic serologic differences among strains [3,6,13,18,19,20], the antigenic components constituting poliovirus antigen and the mode in which each of these components participates in virus neutralization reaction are still poorly understood.Previously we reported that by propagating Mahoney strain in cell cultures in the presence of poliovirus inhibitors (i.e., equine serum factors inactivating poliovirus), five different inhibitor-resistant mutants were obtained [17]. Further, antibody absorption tests with these mutants demonstrated the presence in anti-Mahoney serum of at least five antibodies with different specificities. In these experiments, however, antibody activity was measured by the 50% plaque reduction method. In the present study, in contrast, the antigenic constitution of Mahoney strain and the mechanism of virus neutralization with specific antiserum were analyzed by means of the kinetic neutralization test. MATERIALS AND METHODSVirus and cell cultures. Mahoney strain of type 1 poliovirus was used. The virus was purified by three successive plaque isolations and then grown in quantity in monkey kidney stable (MS) cell cultures with Eagle's minimum essential medium (MEM) without animal serum. The infected cell culture fluid was centrifuged at 2000 rpm and the supernatant stored in small aliquots at -20 C.Plaque assay. Monolayers of MS cells in 70-mm petri dishes were inoculated with 0.4 ml per dish of virus. After incubation for lhr at room temperature, the cell layers were overlayed with 8 ml of Eagle's MEM containing 1.1 % Bacto-agar and antibiotics. The dishes were incubated at 37 C in a humidified atmosphere of 5% CO2 and air. A 4 ml of second overlay medium containing 0.01 % neutral red was added 2 days later, and plaques counted after an additional incubation period of 6-8 hr.Anti-Mahoney serum. Hyperimmune serumRequests for reprints should be addressed to Dr.

Reactivity of Neutralizing Antibodies with Different Specificities to H Particles of Poliovirus

“…Absorption of antiMahoney serum with these inhibitor-resistant Mahoney strains yielded five antibodies with different specificities in neutralization reaction (designated as HN31, HN11, H33, HS77, and H520 antibodies) (14). A detailed experiment carried out subsequently (15), however, showed that while three (HN31, HN11, and H33 antibodies) of those five antibodies had mutually distinct specificities, the remaining two had specificities partly related to one of the three (H33 antibody). These results indicate that poliovirus-neutralizing antibody consists of several kinds of antibody having different specificities, and therefore it might be that the surface of the virus particle carries multiple distinct antigenic determinants.…”

Antigenic Variation of Poliovirus Caused by Antibody Components with Different Specificities

“…The method of plaque assay was described elsewhere (17). Briefly, MS cell monolayers in 70 mm petri dishes were inoculated with 0.4 ml of virus, covered with 8 ml of first overlay medium, and then kept at 37 C for 2 days in a CO2 incubator.…”

“…Further, by absorbing anti-Mahoney serum with these mutants, we isolated five kinds of antibodies with different specificities among which, in subsequent experiments (17), three were shown to have distinct specificities and also two specificities partly related to one of the three. This result shows that the an ti-Mahoney serum contains at least three different antibodies having distinct specificities, possibly indicating the presence of at least three different antigenic determinant sites on the virus surface.…”

Antigenic Analysis of Acute Hemorrhagic Conjunctivitis Viruses (Enterovirus Type 70)

“…Urasawa et al (19,20) reported that they succeeded in obtaining various serological variants of poliovirus type 1, Mahoney, using virus inhibitory substances in equine sera and each variant showed resistance to one of several mono-specific antibodies.…”