SUMMARYNeutralizing monoclonal antibodies incorporated into plaque assay overlay medium were used to select antibody-resistant (Ab ~) mutants of both the Herts (using antifusion protein monoclonal 481) and Beaudette C (using anti-haemagglutininneuraminidase protein monoclonal 445) strains of Newcastle disease virus at the permissive temperature of 34 °C. Certain of the Herts, but none of the Beaudette C, Ab R mutants were also temperature-sensitive (ts-) and failed to form plaques at the non-permissive temperature of 41.5 °C. pS]Methionine-labelled proteins from chick embryo fibroblasts infected with wild-type, ts ÷ Ab R and ts-Ab R virus when separated by two-dimensional polyacrylamide gel electrophoresis revealed a variety of changes in the isoelectric point of the fusion protein F (using monoclonal 481) and the haemagglutinin-neuraminidase protein HN (using monoclonal 445). The ts ÷ 'revertants' of ts-Ab R mutants remained Ab R and also showed changed isoelectric points in the F protein.Monoclonal antibodies raised against viruses are of value in identifying virus antigens in virus-infected cells, in aiding purification of virus proteins by affinity chromatography (Varsanyi et al., 1984), and in investigating site(s) on virus surface antigens that are important in virus neutralization (Wiley et aL, 1981; Emini et al., 1983;Dimmock, 1984), This study investigates the utility of neutralizing monoclonal antibodies directed against the fusion (F) and haemagglutinin-neuraminidase (HN) surface glycoproteins of Newcastle disease virus (NDV) to select temperature-sensitive (is-) antibody-resistant (Ab R) mutants. Such mutants would be expected to have lesion(s) within one or the other glycoprotein and ts ÷ 'revertants' of these mutants should shed light on interactions of the antibody-binding sites with other sites in the same or indeed other virus proteins.The monoclonal antibodies 481 (Abe81, anti-F) and 445 (Ab~5, anti-HN-2) used in this study, and isolated and characterized earlier by one of us (Russell et al., 1983), were raised against the Ulster strain of NDV. This strain requires the presence of trypsin in the overlay medium to allow plaque formation; therefore, for convenience the Herts (sensitive to Ab4sl) and Beaudette C (sensitive to Ab44s) strains of NDV were used in this study, as these strains do not require the addition of trypsin in the plaque overlay medium.Serial dilutions of chorioallantoic fluids from eggs infected with plaque-purified Herts and Beaudette C virus were plated on chick embryo fibroblast (CEF) secondary monolayers in 50 mm diam. Petri dishes (Flow Laboratories) and overlaid with 5 ml HEPES-buffered Medium 199 containing 5~ calf serum and t ~ agar to which was added unheated ascites fluid (5 ~tI per monolayer) prepared from the monoclonal antibody-producing cells. Selection and control plates were incubated at 34 + 0-1 °C in sealable plastic boxes which were completely immersed in a water-bath for 4 days. Plaques formed at 34 °C in the presence of monoclonal antibody were picked usin...