Reactivation Of Neutralized Virus By Fluorocarbon - Mechanism Of Action And Demonstration Of Reduced Reactivability With Time Of Virus-Antibody Interaction

The method of fluorocarbon reactivation of neutralized virus (poliovirus and an enteric bovine orphan virus) was used to determine whether a change in reactivability occurred with time (duration) of virus-antibody interaction. The mechanism of fluorocarbon reactivation was also studied. The effect of fluorocarbon treatment on free neutralizing antibody (rabbit) was first examined. Such antibody partitioned rather evenly in the buffer-fluorocarbon system, with a small preference for the organic phase ( 2 3 of the antibody) at equilibrium. All antibody that had diffused from aqueous phase to organic phase could be recovered in the latter phase, a result indicating that no denaturation of antibody occurred. Only when antibody in the organic phase was allowed to diffuse back into fresh aqueous phase did it exhibit slightly reduced stability. Neutralized virus was quantitatively reactivated by fluorocarbon treatment, even after prolonged virus-antibody interaction at 37°C, when the aqueous: organic phase ratio was 2. In contrast, with a ratio of 9, which offers a more sensitive method for detecting changes in virus-antibody bond strength, a progressive decrease in the reactivability of neutralized virus occurred. Three different mechanisms of action of fluorocarbon reactivation were considered: (1) denaturation of antibody; (2) selective removal of free antibody from the aqueous phase resulting in reequilibration of a reversible virus-antibody reaction, and (3) dissociation of virus-antibody complexes. The data indicated that virus reactivation was most likely the result of dissociation of virus-antibody complexes.