Anomalous Behaviour of Newcastle Disease Virus Haemagglutinin-Neuraminidase Protein in Western Blotting Analysis of Monoclonal Antibody Binding Sites

Abstract

Previous studies with a particular monoclonal antibody (MAb 445) raised against the Ulster strain of Newcastle disease virus (NDV) have shown that this MAb immune-precipitates only the 74000 mol. wt. (74K) haemagglutinin-neuraminidase protein (HN) of both Ulster and Beaudette C strains of NDV. Using the technique of Western blotting, it is shown that under certain denaturing conditions virion proteins co-migrating in SDS-polyacrylamide gels with the faster migrating 67K fusion protein and 53K nucleocapsid/nucleocapsid-associated protein, as well as the 74K HN, all reacted strongly with MAb 445 and with MAb 14 which is also directed against the HN protein. Analysis of this anomalous behaviour using SDS-polyacrylamide diagonal gel electrophoresis has ted to the unexpected conclusion that the 74K HN can electrophorese as three immunoreactive species of apparent mol. wt. 74K, 67K and 53K. If the Western blotting technique is to be applied to proteins which have not been sufficiently denatured (in an attempt to preserve epitope integrity) it is important to establish that no additional proteins remain associated with the protein bands that react with the MAb. Monoclonal antibodies (MAbs) are particularly useful in detecting and purifying individual virus proteins because a given MAb should only react with an epitope unique to a particular protein. Various methods can be used to determine to which protein a given MAb is directed; immunoprecipitation and Western blotting (Burnette, 1981) are the most commonly used techniques. MAbs 445 and 14, raised against Newcastle disease virus (NDV) (Russell et al., 1983a, b), immune-precipitate only the 74000 mol. wt. (74K) haemagglutinin-neuraminidase protein (HN) from NDV strains (Russell et al., 1983b) and have been used to select independent mutants of the Beaudette C strain resistant to neutralization by MAb 445 and MAb 14. All of these mutants have an altered HN (revealed by isoelectric focusing gel electrophoresis) (Samson et al., 1985; and unpublished data). In an attempt to use the technique of Western blotting to investigate further the properties of a temperature-sensitive (but still MAb 445-and MAb 14-sensitive) Beaudette C mutant ts53 [which had been shown earlier to possess a thermolabile HN (Harper et at., 1983)], it was found that three proteins from wild-type virions reacted with either MAi~ 445 or 14 but only the expected HN reacted from the ts53 virions. The original anomalous behaviour of wild-type HN was noticed in an experiment designed to see whether the known thermolability of HN in ts53 virions [measured by both haemagglutina-tion and neuraminidase activity of intact virions (Harper et al., 1983)] compared to wild-type was reflected in the reaction of HN proteins to anti-HN MAbs following Western blotting of virions disrupted in SDS-containing buffer at different temperatures and separated by SDS-PAGE. 0000-7027 © 1986 SGM

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

“…Several of these bind HN in Western blots when the structure of HN has been disrupted by boiling with SDS and 2-mercaptoethanol (Le Long et al, 1986;Samson, 1986;Samson et al, 1988). These antibodies may, therefore, recognize epitopes that are continuous or are readily regenerated by refolding after denaturation.…”

“…Lane S contains phosphorylase b (M~ 92K), bovine serum albumin (68K), alcohol dehydrogenase (41K), a-chymotrypsinogen A (25K) and cytochrome c (12.4K) stained with Indian ink (Hancock & Tsang, 1983). The remaining nitrocellulose was stained with peroxidaseconjugated rabbit anti-mouse sera after incubation with MAb 14 as described by Samson (1986). Lane N contains purified NDV (Chambers & Samson, 1980) in which the HN polypeptide at 74K is prominent.…”

“…2) was to induce transcription at the lac promoter by adding IPTG to liquid cultures of bacteria, harvest cells at an appropriate optical density and resuspend cell pellets by boiling in sample buffer that contained SDS and 2-mercaptoethanol (Laemmli, 1970). Samples were run on SDSpolyacrylamide gels, blotted onto nitrocellulose (Dunn, 1986;Samson, 1986) and stained with antibodies and peroxidase as previously described (Samson, 1986).…”

“…The cell pellets were resuspended by vortexing into 100 p.1 (lanes 1 to 8) or 500 ~tl (lanes 9 and 10) SDS sample buffer (Laemmli, 1970) and immediately boiled at 100 °C for 2 min. Samples were stored at -20 °C before electrophoresis on 11% polyacrylamide gels (Laemmli, 1970), and were blotted onto nitrocellulose as described by Samson (1986) for lanes 1 to 8 or by Dunn (1986) for lanes 9 and 10. Lane S contains phosphorylase b (M~ 92K), bovine serum albumin (68K), alcohol dehydrogenase (41K), a-chymotrypsinogen A (25K) and cytochrome c (12.4K) stained with Indian ink (Hancock & Tsang, 1983).…”

Heterogeneity of the Respiratory Syncytial Virus 22K Protein Revealed by Western Blotting with Monoclonal Antibodies

“…In order to investigate the relationship between the reduced and non-reduced bands an infected cell lysate was analysed by Western blotting of two-dimensional SDS-polyacrylamide gels (Samson, 1986). The first dimension electrophoresis was carried out in non-reducing conditions, and the second either in non-reducing (Fig.…”